# Patent

## Title
Methods of treating and preventing Staphylococcus aureus infections and associated conditions

## Abstract

The present invention relates to methods of treating and preventing Staphylococcus aureus infection and/or a condition resulting from a S. aureus infection a subject that involves administering a CD11b inhibitor. The present invention further relates to a non-human transgenic animal expressing human CD11b and its use in methods of identifying novel therapeutics for the treatment and prevention of Staphylococcus aureus infection and/or a condition resulting from a S. aureus infection.

## Background
This application is a national stage application under 35 U.S.C. 371 from PCT Application No. PCT/US2013/032436, filed Mar. 15, 2013, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/641,543, filed May 2, 2012, which are hereby incorporated by reference in their entirety.


This invention was made with government support under grant number 1R56AI091856-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to methods of treating and preventing Staphylococcus aureus infections, and to methods of identifying novel therapeutics for the treatment and prevention of Staphylococcus aureus infections.
BACKGROUND OF THE INVENTION
Staphylococcus aureus (“S. aureus”) is a bacterium that commensally colonizes more than 25% of the human population. Importantly, this organism is capable of breaching its initial site of colonization, resulting in bacterial dissemination and disease. S. aureus is the leading cause of nosocomial infections, is the most common etiological agent of infectious endocarditis as well as skin and soft tissue infections, and is one of the four leading causes of food-borne illness. Altogether, S. aureus infects more than 1.2 million patients per year in U.S. hospitals. The threat of S. aureus to human health is further highlighted by the emergence of antibiotic-resistant strains (i.e., methicillin-resistant S. aureus (MRSA) strains), including strains that are resistant to vancomycin, an antibiotic considered the last line of defense against S. aureus infection. These facts highlight the importance of developing novel therapeutics against this important pathogen.
S. aureus produces a diverse array of virulence factors and toxins that enable this bacterium to neutralize and withstand attack by different kinds of immune cells, specifically subpopulations of white blood cells that make up the body's primary defense system. The production of these virulence factors and toxins allow S. aureus to maintain an infectious state (Nizet, “Understanding How Leading Bacterial Pathogens Subvert Innate Immunity to Reveal Novel Therapeutic Targets,” J. Allergy Clin. Immunol. 120(1):13 22 (2007)). Among these virulence factors, S. aureus produces several bi-component leukotoxins, which damage membranes of host defense cells and erythrocytes by the synergistic action of two non-associated proteins or subunits (see Menestrina et al., “Mode of Action of Beta-Barrel Pore-Forming Toxins of the Staphylococcal Alpha-Hemolysin Family,” Toxicol. 39(11):1661-1672 (2001)). Among these bi-component leukotoxins, gamma-hemolysin (HlgAB and HlgCB) and the Pantone-Valentine Leukocidin (PVL) are the best characterized.
The toxicity of the leukocidins towards mammalian cells involves the action of two components. The first subunit is named class S-subunit (i.e., “slow-eluted”), and the second subunit is named class F-subunit (i.e., “fast-eluted”). The S- and F-subunits act synergistically to form pores on white blood cells including monocytes, macrophages, dendritic cells and neutrophils (collectively known as phagocytes) (Menestrina et al., “Mode of Action of Beta-Barrel Pore-Forming Toxins of the Staphylococcal Alpha-Hemolysin Family,” Toxicol. 39(11):1661 1672 (2001)). The mechanism by which the bi-component toxins form pores in target cell membranes is not entirely understood. The proposed mechanism of action of these toxins involves binding of the S-subunit to the target cell membrane, most likely through a receptor, followed by binding of the F-subunit to the S-subunit, thereby forming an oligomer which in turn forms a pre-pore that inserts into the target cell membrane (Jayasinghe et al., “The Leukocidin Pore: Evidence for an Octamer With Four LukF Subunits and Four LukS Subunits Alternating Around a Central Axis,” Protein. Sci. 14(10):2550 2561 (2005)). The pores formed by the bi-component leukotoxins are typically cation-selective. Pore formation causes cell death via lysis, which in the cases of the target white blood cells, has been reported to result from an osmotic imbalance due to the influx of cations (Miles et al., “The Staphylococcal Leukocidin Bicomponent Toxin Forms Large Ionic Channels,” Biochemistry 40(29):8514 8522 (2001)).
Designing effective therapy to treat MRSA infection has been especially challenging. In addition to the resistance to methicillin and related antibiotics, MRSA has also been found to have significant levels of resistance to macrolides (e.g., erythromycin), beta-lactamase inhibitor combinations (e.g., Unasyn, Augmentin) and fluoroquinolones (e.g. ciprofloxacin), as well as to clindamycin, trimethoprim/sulfamethoxisol (Bactrim), and rifampin. In the case of serious S. aureus infection, clinicians have resorted to intravenous vancomycin. However, as noted above there have been reports of S. aureus resistance to vancomycin. Thus, there is a need to develop new antibiotic drugs that effectively combat S. aureus infection.
The present invention is directed to overcoming these and other limitations in the art.
SUMMARY OF THE INVENTION
A first aspect of the present invention is directed to a method of preventing or treating Staphylococcus aureus infection and/or a condition resulting from a S. aureus infection in a subject. This method involves selecting a subject at risk of having or having S. aureus infection and administering a CD11b inhibitor to the selected subject under conditions effective to prevent or treat S. aureus infection and/or a condition resulting from a S. aureus infection in the subject.
Another aspect of the present invention relates to a transgenic non-human animal whose genome comprises a stably integrated expression construct that comprises a polynucleotide sequence encoding human CD11b. Other aspects of the present invention relate to methods of identifying candidate compounds suitable for preventing or treating S. aureus infection and/or conditions resulting from a S. aureus infection using the transgenic non-human animal of the present invention.
Another aspect of the present invention relates to a method of identifying compounds capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection. This method involves providing a collection of candidate compounds and providing a population of cells expressing human CD11b. The method further involves treating the population of cells with an agent capable of inducing LukAB mediated cytotoxicity, and contacting the population of treated cells with one or more candidate compounds from the collection. The method further involves measuring LukAB mediated cytotoxicity level in the population of treated cells in the presence and absence of the one or more candidate compounds and comparing the measured level of LukAB mediated cytotoxicity in the presence and in the absence of the one or more candidate compound. A decrease in the level of LukAB mediated cytotoxicity in the presence of the one or more candidate compounds compared to in its absence of the one or more candidate compounds identifies a compound capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection.
Another aspect of the present invention relates to a method of identifying candidate compounds capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection. This method involves providing a collection of candidate compounds and providing an isolated CD11b receptor or a fragment thereof comprising a LukAB binding domain. The method further involves treating the isolated CD11b receptor or the fragment thereof with an agent comprising a labeled LukA, LukB, and/or labeled LukAB protein and contacting the treated, isolated CD11b receptor or the fragment thereof with one or more candidate compounds from the collection. The binding level of the labeled LukA, LukB, and/or labeled LukAB to the isolated CD11b receptor or fragment thereof is measured in the presence and in the absence of one or more candidate compounds, and the level of LukA, LukB, and/or LukAB binding to the isolated CD11b receptor or fragment thereof in the presence and absence of the one or more candidate compounds is compared. One or more candidate compounds that are capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection are identified based on this comparison.
S. aureus infects more than 1.2 million patients per year in USA hospitals, with around 40,000 deaths per year in the USA. This bacterium is the leading cause of nosocomial and community acquired infections; is the most common etiological agent of infectious endocarditis, skin, and soft tissue infections; and is one of the four leading causes of food-borne illness. The threat of S. aureus to human health is further compounded by the emergence of antibiotic-resistant strains, including methicillin-resistant S. aureus (MRSA). These facts highlight the importance of identifying new targets for the development of novel therapeutics. The present invention relates to the discovery that CD11b is the human cellular receptor for the S. aureus virulence factor leukotoxin AB (LukAB). LukAB is responsible for the cytotoxic properties of both methicillin sensitive and methicillin resistant S. aureus towards human neutrophils, and identification of its cellular receptor on human cells enables a new therapeutic approach to protect against S. aureus infection. In addition, discovery of this virulence receptor allows for the generation of improved animal models and screening assays for studying S. aureus infection and identifying novel therapeutics.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C show LukAB and S. aureus cytotoxicity. FIG. 1A is a graph of human polymorphonuclear leukocyte (PMN) viability upon intoxication with secreted proteins isolated from isogenic wildtype (WT) and ΔlukAB mutant MSSA and MRSA strains. The increased viability observed in cells treated with secreted proteins from ΔlukAB mutant strains was reversed by expressing lukAB in trans with a plasmid (ΔlukAB/plukAB). Host cell viability was monitored with CellTiter, a reagent that monitors cellular metabolic activity. FIG. 1B is a graph showing S. aureus survival in human whole blood and primary human PMNs. Colony forming units were normalized to input CFU. FIG. 1C is a graph showing bacterial burden in kidneys of mice infected with indicated strains 96-hrs post-infection. Results represent the average of PMNs/whole blood from more than 6 different donors (FIGS. 1A and 1B) and 20 mice per group (FIG. 1C)±S.E.M. * denote statistically significant difference compared to WT (ANOVA p<0.05).
FIG. 2 is a dose response of LukAB induced cytotoxicity in different human cells. The graph shows viability of indicated cells upon intoxication with purified LukAB. Host cell viability was monitored with CellTiter, a reagent that monitors cellular metabolic activity. Results represent the average of triplicate samples+S.E.M.
FIG. 3 show LukAB and S. aureus cytotoxicity towards primary neutrophils. The graph shows viability of human (hPMN) and primary murine (mPMN) neutrophils upon intoxication with secreted proteins isolated from isogenic wildtype (WT) and ΔlukAB mutant MSSA strains. Host cell viability was monitored with CellTiter, a reagent that monitors cellular metabolic activity. Results represent the average of at least six independent samples±S.E.M.
FIGS. 4A-4D show that LukAB directly interacts with the human integrin Mac-1 (CD11b/CD18). FIG. 4A is a pull-down of biotinylated PMN-HL60 lysates with his-tagged LukAB using Ni-NTA beads, where samples were transferred to a nitrocellulose membrane and probed with DyLight streptavidin. FIG. 4B is an immunoblot of a pull-down of PMN-HL60 lysates with his-tagged leukotoxins as described above using an anti-CD11b antibody. FIG. 4C is a Sypro Ruby protein stain of a pull-down of purified Mac-1 with his-tagged leukotoxins as described above, and FIG. 4D shows the corresponding immunoblot with an anti-CD11b antibody.
FIGS. 5A-5F demonstrate that CD11b is necessary and sufficient for LukAB-mediated killing of host cells. FIG. 5A is a bar graph showing cell viability after intoxication of HL60 or PMN-HL60 cells with 10 μg/ml of LukAB for 1 hour. Cell viability was measured with the metabolic dye CellTiter. FIG. 5B is a flow cytometry plot of CD18 and CD11b surface levels in PMN-HL60 cells transduced with a CD18 shRNA virus compared to a non-targeting (NT) shRNA virus. FIG. 5C is a bar graph quantifying pore formation with intoxication of the NT and CD18 shRNA PMN-HL60 cells with 10 μg/ml of LukAB or PVL for 1 hr. Pore formation was measured with the fluorescent dye Ethidium bromide (EtBr). FIG. 5D shows CD18 and CD11b surface levels in HL60 cells and PMN-HL60 cells transduced with a with a CD11b shRNA virus or a non-targeting (NT) shRNA virus measured as described for FIG. 5B. FIG. 5E is a bar graph quantifying pore formation with intoxication of the NT and CD11b shRNA PMN-HL60 cells with 10 μg/ml of LukAB or PVL for 1 hour. Pore formation was measured with EtBr. FIG. 5F shows intoxication of CD11b transfected 293T cells with 40 μg/ml of LukAB or PVL for 2 hours. Percent depletion of CD11b+ cells was determined by staining cells with an anti-CD11b antibody post intoxication and performing flow cytometry analysis. The bar graph of FIG. 5F depicts the average of two independent experiments. FACS plots are from a representative experiment. All other data is represented as the average of triplicate samples±standard deviation (SD) unless otherwise indicated. *** indicates P<0.0001 by one way analysis of variance.
FIG. 6 shows that knockdown of CD18 results in depletion of multiple β2 integrins on the PMN-HL60 cell surface. CD18, CD11b, CD11c, and CD11a cell surface levels as determined by flow cytometry with specific antibodies in NT (solid black) and CD18 shRNA (grey line) in PMN-HL60 cells compared to an isotype control antibodies (black line).
FIGS. 7A-7E demonstrate that LukAB targets the I-domain of CD11b to kill cells. FIG. 7A is a bar graph showing the viability of PMNs treated with 10 μg/ml of integrin-specific antibodies (α-CD11a, α-CD11c, and α-CD18), including three different anti-CD11b clones (α-CD11b), or no antibody (No Ab) followed by a 1-hour intoxication with 2.5 μg/ml of LukAB. Membrane damage was measured with the fluorescent dye SYTOX green. Results represent the mean from PMNs isolated from 8 donors±SEM. FIG. 7B shows the viability of PMNs treated with 10 μg/ml of LM2/1 or an isotype control then intoxicated and evaluated as described above. Results represent the mean from PMNs isolated from 4 donors±SEM. FIG. 7C shows CD11b surface levels on HL60 cells stably transduced with empty vector (EV), WT CD11b, or I-less CD11b virus compared to PMN-HL60s as determined by flow cytometry analysis with an anti-CD11b antibody. Viability of stably transduced HL60 cell lines described in FIG. 7C compared to PMN-HL60s after 1-hour intoxication with 10 μg/ml of LukAB where membrane damage (FIG. 7D) and cellular metabolism (FIG. 7E) were evaluated as described in FIG. 7A and FIG. 5A respectively. Data is represented as the average of triplicate samples±SD. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.0001 by one way analysis of variance.
FIGS. 8A-8E shows that LukAB preferentially binds to the human CD11b I-domain compared to the murine CD11b I-domain. FIG. 8A is a bar graph showing pore formation in peritoneal exudate cells (PECs) following a 1-hour intoxication with 20 μg/ml of LukAB or 10 μg/ml of LukED as measured with EtBr. Data is represented as the average of triplicate samples±SD. FIG. 8B is a flow cytometry analysis showing Ly6G and CD11b surface levels on PECs using anti-Ly6G and anti-CD11b antibodies. FIG. 8C is a phylogenetic tree of the amino acid sequence alignment of human, gorilla, rabbit and mouse I-domains constructed with DNASTAR MegAline software using the CLUSTALW method. FIG. 8D shows results of a competition dot blot assay where purified recombinant human CD11b I-domain was incubated with 5 μg/ml fluorescently labeled LukAB (FITC-LukAB) and 10-fold excess (50 μg/ml) of unlabeled LukAB or unlabeled PVL. FITC-LukAB binding was quantified by densitometry. FIG. 8E is a dot blot of purified recombinant human or murine CD11b I-domain incubated with 5 μg/ml FITC-LukAB. FITC-LukAB binding was quantified by densitometry.
FIGS. 9A-9B demonstrate that CD11b renders cells susceptible to LukAB-mediated killing by extracellular S. aureus in ex vivo infections. FIG. 9A is a graph showing viability of the non-target (NT) or CD11b shRNA PMN-HL60 cells described in FIG. 5B following a 2-hour infection with non-opsonized wild type (WT) CA-MRSA USA300 or an isogenic lukAB mutant (ΔlukAB) at the indicated multiplicity of infection (MOI). Membrane damage was measured with SYTOX green. Data is represented as the average of triplicate samples±SD. FIG. 9B is a graph showing viability of PMNs treated with 10 μg/ml of CD11b-specific antibodies (three different clones) followed by a 1 hour infection with the indicated MOI of non-opsonized WT USA300. Membrane damage was measured with SYTOX green. Results represent the mean from PMNs isolated from 8 donors±SEM. * indicates P<0.05, and *** indicates P<0.0001 by one way analysis of variance.
FIGS. 10A-10B shows that knockdown of CD11b does not affect opsonization-mediated phagocytosis of S. aureus by PMN-HL60 cells. Amount of phagocytosed bacteria in NT and CD11b shRNA PMN-HL60 cells infected with various MOI of GFP-USA300 with (FIG. 10B) or without (FIG. 10A) opsonization as determined by flow cytometry. Infections were performed in the presence of lysostaphin to eliminate GIP signal from extracellular bacteria.
FIG. 11A-11E demonstrate that LukAB-mediated cellular damage and growth rebound of phagocytosed S. aureus is dependent on CD11b. FIG. 11A is a graph showing viability of non-target (NT) or CD11b shRNA PMN-HL60 cells described in FIG. 5D following a 90 minute infection with various MOI of opsonized WT or ΔlukAB USA300. Membrane damage was measured with SYTOX green. Data is represented as the average of triplicate samples±SD. FIG. 11B is a graph showing the growth of opsonized WT or ΔlukAB USA300 upon infection of NT or CD11b shRNA PMN-HL60 cells at a MOI of 10. Bacterial colony forming units were determined by dilution plating after lysing the PMN-HL60 cells at 1, 2, or 3 hours post synchronization. To determine % growth, bacterial counts were normalized to input at time 0, which was set at 100%. Results represent the average of triplicate samples from 2 independent infections±SD. The photomicrographs of FIG. 11C show localization of CD11b in PMNs post-infection with opsonized GFP-USA300 at a MOI of 10 or in uninfected PMNs determined by staining with a fluorescently conjugated anti-CD11b antibody or an isotype control prior to infection. Cells were fixed post-synchronization and images were captured using an Applied Precision PersonalDV live-cell imaging system. A representative image for each condition is shown. FIG. 11D shows infection of PMNs pre-treated with the LM2/1 anti-CD11b antibody or an isotype control with GFP-USA300 at a MOI of 10. EtBr staining in red is indicative of pore formation. Images were captured using a fluorescent microscope at 0 and 30 minutes post-synchronization and representative images from 30 minutes are shown. FIG. 11E is a graph showing quantification of ethidium bromide positive PMNs per field of view obtained from images shown in FIG. 11D. Results represent the average of three independent counts from infections of PMNs isolated from two donors at (T0) and 30 (T30) minutes post-infection. ** indicates P<0.001 and *** indicates P<0.0001 by one way analysis of variance.
FIG. 12 shows that treatment of PMNs with the anti-CD11b LM2/1 antibody does not inhibit phagocytosis of S. aureus. Bacterial localization in PMNs infected with opsonized GFP-USA300 after pre-treatment with the α-CD11b LM2/1 antibody or an isotope control antibody. Fluorescent images were captured immediately following synchronization (T0). Background EtBr staining in red is also shown for this time point.



DETAILED DESCRIPTION OF THE INVENTION
A first aspect of the present invention is directed to a method of preventing or treating Staphylococcus aureus infection and/or a condition resulting from a S. aureus infection in a subject. This method involves selecting a subject at risk of having or having S. aureus infection and administering a CD11b inhibitor to the selected subject under conditions effective to prevent or treat S. aureus infection and/or a condition resulting from a S. aureus infection in the subject
To date, the majority of S. aureus infections are due to MRSA (Moran et al., “Methicillin-Resistant S. aureus Infections Among Patients in the Emergency Department,” The New England Journal of Medicine 355:666-674 (2006), which is hereby incorporated by reference in its entirety). Previously, the majority of MRSA infections were thought to be of nosocomial origin (HA-MRSA), however infections are now occurring in otherwise healthy individuals who have not had exposure to healthcare facilities, i.e., community-associated MRSA (CA-MRSA) (Klevens et al., “Invasive Methicillin-Resistant Staphylococcus aureus Infections in the United States,” Jama 298:1763-1771 (2007) and Klevens et al., “Changes in the Epidemiology of Methicillin-Resistant Staphylococcus aureus in Intensive Care Units in US Hospitals, 1992-2003,” Clin. Infect. Dis. 42:389-391 (2006), which are hereby incorporated by reference in their entirety). These CA-MRSA associated infections are more severe and result in higher mortality rates compared to HA-MRSA infections (Deleo et al., “Community-Associated Methicillin-Resistant Staphylococcus aureus,” Lancet 375:1557-1568 (2010), which is hereby incorporated by reference in its entirety). Recent reports have suggested that the increased virulence of strains associated with CA-MRSA infections compared to those associated with HA-MRSA infections is primarily due to the enhanced ability of CA-MRSA-associated strains to evade neutrophil (PMNs)-mediated killing (Voyich et al., “Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils,” J. Immunol. 175:3907-3919 (2005); Wang et al., “Identification of Novel Cytolytic Peptides as Key Virulence Determinants for Community-Associated MRSA,” Nat. Med. 13:1510-1514 (2007); Li et al., “Evolution of Virulence in Epidemic Community-Associated Methicillin-Resistant Staphylococcus aureus,” Proc. Nat'l Acad. Sci. U.S.A. 106:5883-5888 (2009); Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); and Alonzo III et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth in Vivo,” Mol. Microbiol. 83:423-435 (2012), which are hereby incorporated by reference in their entirety). S. aureus avoids PMN-mediated killing by targeting and killing PMNs with a collection of cytotoxins and cytolytic peptides (Wang et al., “Identification of Novel Cytolytic Peptides as Key Virulence Determinants for Community-Associated MRSA,” Nat. Med. 13:1510-1514 (2007); Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79:814-825 (2011); Alonzo III et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth in Vivo,” Mol. Microbiol. 83:423-435 (2012); Loffler et al., “Staphylococcus aureus Panton-Valentine Leukocidin is a Very Potent Cytotoxic Factor for Human Neutrophils,” PLoS Pathog. 6:e1000715 (2010); and Ventura et al., “Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics,” PLoS One 5:e11634 (2010), which are hereby incorporated by reference in their entirety). In this regard, S. aureus strains associated with human infections can produce up to four different bi-component leukotoxins. These bi-component leukotoxins are members of the β-barrel pore-forming family of toxins that exhibit marked selectivity towards host phagocytes. The cytotoxic properties of the staphylococcal leukotoxins have been attributed to the formation of octameric pores in target cell membranes in vitro, which result in cell swelling, ultimately leading to cell death (Ferreras et al., “The Interaction of Staphylococcus aureus Bi-Component Gamma-Hemolysins and Leucocidins With Cells and Lipid Membranes,” Biochim. Biophys. Acta 1414:108-126 (1998); Jayasinghe & Bayley, “The Leukocidin Pore: Evidence for an Octamer With Four LukF Subunits and Four LukS Subunits Alternating Around a Central Axis,” Protein Sci. 14:2550-2561 (2005); Sugawara-Tomita et al., “Stochastic Assembly of Two-Component Staphylococcal Gamma-Hemolysin into Heteroheptameric Transmembrane Pores With Alternate Subunit Arrangements in Ratios of 3:4 and 4:3,” J. Bacteriol. 184:4747-4756 (2002); Menestrina et al., “Mode of Action of Beta-Barrel Pore-Forming Toxins of the Staphylococcal Alpha-Hemolysin Family,” Toxicon 39:1661-1672 (2001), which are hereby incorporated by reference in their entirety). Among the four different bicomponent leukotoxins, Leukotoxin AB (LukAB) is primarily responsible for the cytotoxic properties of both MSSA and MRSA respectively, towards human neutrophils (see Examples infra and U.S. Patent Publication No. 2011/0274693 to Torres, which is hereby incorporated by reference in its entirety).
Given the large number of individual who contract MRSA annually, it is likely that a substantial proportion of these infections will be refractory to traditional courses of antibiotic treatment. An innovative approach to treat such infections is to inhibit S. aureus virulence factors, such as LukAB, which are responsible for killing PMNs, the most critical innate immune cell involved in defense against S. aureus infection. As described herein, applicants have identified CD11b as the cellular receptor for LukAB on human PMNs. Binding of LukAB to CD11b is the first step in LukAB cytotoxicity, which is followed by LukAB oligomerization and pore formation leading to cell death. Therefore, agents which inhibit the LukAB/CD11b interaction, such as CD11b inhibitors, are clinically useful for blocking LukAB cytotoxicity, in turn preventing depletion of PMNs, and promoting the natural clearance of S. aureus by the innate immune system. In a preferred embodiment of the present invention, the CD11b inhibitor selectively inhibits the CD11b/LukAB interaction without interfering with CD11b binding to its physiological ligands.
In accordance with this aspect of the present invention, suitable CD11b inhibitors include, without limitation, protein or peptide inhibitors, antibodies, and small molecules, many of which are known in the art as described below.
An exemplary peptide inhibitor of CD11b comprises a recombinant Neutrophil Inhibitory Factor (rNIF), also known as UK-279276. NIF is a 41-kDa glycoprotein isolated and cloned from the canine hookworm Ancylostoma caninum (Moyle et al., “A Hookworm Glycoprotein That Inhibits Neutrophil Function is a Ligand for the Integrin CD11b/CD18,” J. Biol. Chem. 209(13):10008-10015(1994), which is hereby incorporated by reference in its entirety). NIF binds with high affinity to the CD11b/CD18 receptor complex (also known as Mac-1, Mo1, αMβ2, and CR3), thereby blocking CD11b/CD18 receptor binding to its physiological ligand on endothelial cells. In accordance with the present invention, therapeutic compositions comprising rNIF (UK-279276) will readily inhibit LukAB interaction with CD11b and prevent its subsequently induced cytotoxicity.
Another exemplary protein or peptide inhibitor suitable for use in the methods of the present invention is a recombinant soluble protein comprising the LukAB receptor binding domain. In a preferred embodiment of this aspect of the invention, the soluble protein comprises a recombinant human CD11b protein or a CD11b LukAB binding domain. An exemplary soluble protein comprising the LukAB binding domain is a soluble protein comprising the I-domain of CD11b or a fragment thereof. The I-domain of CD11b spans amino acid residues 147-337 of SEQ ID NO: 2 (NCBI Accession No. NP_000632) and residues 147-337 of SEQ ID NO: 4 (NCBI Accession No. NP_001139280). Another exemplary soluble protein comprising a CD11b protein is the soluble human CD11b/CD18 receptor described by Dana et al., “Expression of a Soluble and Functional Form of the Human β2 Integrin CD11b/CD18,” Proc. Natl. Acad. Sci. USA 88:3106-3110 (1991), which is hereby incorporated by reference in its entirety. In accordance with this aspect of the present invention, therapeutic compositions of the present invention comprising the soluble LukAB receptor binding protein will bind the S. aureus LukAB virulence factor, preventing its interaction with CD11b expressing target cells (e.g. phagocytes) and its subsequently induced cytotoxicity.
In another embodiment of this aspect of the invention, the CD11b inhibitor is a CD11b or CD11b/CD18 specific antibody. As used herein, the term “antibody” is meant to include intact immunoglobulins derived from natural sources or from recombinant sources, as well as immunoreactive portions (i.e. antigen binding portions) of intact immunoglobulins. Antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, antibody fragments, diabodies, tribodies, pentabodies, nanobodies, genetically engineered forms of the antibodies, and combinations thereof. Suitable antibodies includes full length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecules (e.g., an IgG antibody) and immunologically active fragments thereof (i.e., including the specific binding portion of the full-length immunoglobulin molecule), which again may be naturally occurring or synthetic in nature. Accordingly, the term “antibody fragment” includes a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody, and, in the context of the present invention, specifically binds CD11b or CD11b/CD18 and prevents LukAB binding. In a preferred embodiment, an antibody of the present invention, binds to the LukAB binding domain of CD11b, i.e., the I-domain of CD11b, but does not bind to other domains of CD11b so as to allow other physiological ligands of the CD11b/CD18 receptor to bind to the receptor while specifically blocking S. aureus LukAB binding. Methods of making and screening antibodies and antibody fragments are well-known in the art.
Monoclonal antibodies of the present invention may be derived from any mammalian animal, for example, and without limitation, a rodent, rabbit, dog, goat, horse, camel, llama, chicken, human.
Methods for monoclonal antibody production may be carried out using techniques well-known in the art (MONOCLONAL ANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of a mammal which has been previously immunized with the antigen of interest (i.e., Cd11b or fragment thereof) either in vivo or in vitro.
The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is achieved by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation of Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated by reference in its entirety). The immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and have good fusion capability. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody.
In another embodiment of the present invention, monoclonal CD11b antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990), which is hereby incorporated by reference in its entirety. Clackson et al., “Making Antibody Fragments using Phage Display Libraries,” Nature 352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporated by reference in their entirety, describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., BioTechnology 10:779-783 (1992), which is hereby incorporated by reference in its entirety), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993), which is hereby incorporated by reference in its entirety). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
Alternatively monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, which is hereby incorporated by reference in its entirety. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, generate monoclonal antibodies.
The CD11b antibody can also be a humanized or chimeric antibody. “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), which are hereby incorporated by reference in their entirety.
Chimeric antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human. The chimerization process can be made more effective by also replacing the variable regions—other than the hyper-variable regions or the CDRs, of a murine (or other non-human mammalian) antibody with the corresponding human sequences. The variable regions other than the CDRs are also known as the variable framework regions (FRs). Yet other monoclonal antibodies of the present invention are bi-specific, in that they have specificity for both LukA and/or LukB and CD11b. Bispecific antibodies are preferably human or humanized.
As described in the Examples herein, CD11b specific antibodies are known in the art (see also Dana et al., “Two Functional Domains in the Phagocyte Membrane Glycoprotein Mo1 Identified with Monoclonal Antibodies,” J. Immunol. 137: 3259-3263 (1986) and Jaeschke et al., “Functional Inactivation of Neutrophils with Mac-1 (CD11b/CD18) Monoclonal Antibody Protects Against Ischemia-Reperfusion Injury in Rat Liver,” Hepatology 17(5) 915-923 (1993), which are hereby incorporated by reference in their entirety). A particularly suitable antibody is the murine LM2/1 CD11b antibody (Santa Cruz) that binds the human I-domain of CD11b. Similar antibodies, i.e., human or humanized antibodies, have the same antigen binding domain as the LM2/1 CD11b antibody are also suitable for use in the methods of the present invention. A number of other human CD11b and CD11b/CD18 antibodies are also commercially available, see e.g., anti-CR3 (CD11b/CD18) antibodies and 2LPM19c (anti-CD11b antibody) from DAKO (Carpinteria, Calif.) and αM-44 antibody (CD11b) from Santa Cruz Biotechnology (Santa Cruz, Calif.).
In another embodiment of this aspect of the present invention, a suitable CD11b inhibitor is a small molecule inhibitor. Suitable small molecule CD11b inhibitors are known in the art and include 2-[4-(3,4-dihydro-2H-quinolin-1-yl)-buta-1,3-dienyl]-1-thylnaptho[1,2-d]thiazol-1-ium; chloride (Compound 1) and derivative thereof, and 1-ethyl-2-/3-/1-ethylbenzothiazolin-2-ylidiene/propenyl/-thiazolium; iodide (Compound 2) and derivatives thereof (Bansal et al., “Small Molecule Antagonists of Complement Receptor Type 3 Bock Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes,” J. Pharm. Exp. Therap. 304(3):1016-1024 (2003), which is hereby incorporated by reference in its entirety). Suitable derivatives of these small molecule inhibitors (i.e., Compounds 1 and 2) include any derivative compounds that maintain the ability to block ligand binding to the CD11b/CD18 receptor complex, measured using an in vitro ligand binding assay or cellular adhesion assay. Exemplary derivative small molecule inhibitors that are also suitable for use in the methods of the present invention are described by Bansal et al., “Small Molecule Antagonists of Complement Receptor Type 3 Bock Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes,” J. Pharm. Exp. Therap. 304(3):1016-1024 (2003), which is hereby incorporated by reference in its entirety.
Another small molecule inhibitor of CD11b that is suitable for use in the methods of the present invention comprises N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine (NPC 15669) (see Bator et al., “N-[9H-(2,7-dimethylfluorenyl-9-methoxy)carbonyl]-L-leucine, NPC 15669, Prevents Neutrophil Adherence to Endothelium and Inhibits CD11b/CD18 Upregulation,” Immunopharmacology 23(2):139-49 (1992), which is hereby incorporated by reference in its entirety).
A suitable subject for treatment in accordance with the methods of the present invention includes, without limitation, any animal, preferably, a mammal, more preferably a human. Suitable subjects include both immunocompromised and non-immunocompromised infants, juveniles, and adults. In one embodiment of the present invention the subject has or is at risk of having a methicillin-resistant S. aureus (MRSA) infection. In another embodiment of the present invention, the subject has or is at risk of having a methicillin sensitive S. aureus (MSSA) infection. Other suitable subjects include those subjects which may have or are at risk for developing a condition resulting from a S. aureus infection, i.e., a S. aureus associated condition, such as, for example, skin wounds and infections, tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skin syndrome, septicemia, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.
In one embodiment of the present invention, the CD11b inhibitor is administered prophylactically to prevent, delay, or inhibit the development of S. aureus infection in a subject at risk of getting a S. aureus infection or associated condition. In some embodiments of the present invention, prophylactic administration of a CD11b inhibitor is effective to fully prevent S. aureus infection in an individual. In other embodiments, prophylactic administration is effective to prevent the full extent of infection that would otherwise develop in the absence of such administration, i.e., substantially prevent or inhibit S. aureus infection in an individual.
In another embodiment of the present invention, the CD11b inhibitor is administered therapeutically to an individual having a S. aureus infection to inhibit further development of the infection, i.e., to inhibit the spread of the infection to other cells in an individual.
The therapeutic compositions of the present invention can be administered as part of a combination therapy in conjunction with another active agent, depending upon the nature of the S. aureus infection that is being treated. Such additional active agents include anti-infective agents, antibiotic agents, and antimicrobial agents.
Representative anti-infective agents that may be useful in the present invention include vancomycin and lysostaphin. Other anti-infective agents include a LukAB inhibitor as described in U.S. Patent Application Publication No. 2011/0274693 to Torres et al., which is hereby incorporated by reference in its entirety; a LukED inhibitor or antibody as described in U.S. Patent Publication No. 2013/0017203 to Torres et al., which is hereby incorporated by reference in its entirety; and a CCR5 inhibitor 2013/0039885 to Torres et al., which is hereby incorporated by reference in its entirety.
Representative antibiotic agents and antimicrobial agents that may be useful in the present invention include penicillinase-resistant penicillins, cephalosporins and carbapenems, including vancomycin, lysostaphin, penicillin G, ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin, cephalothin, cefazolin, cephalexin, cephradine, cefamandole, cefoxitin, imipenem, meropenem, gentamycin, teicoplanin, lincomycin and clindamycin. Dosages of these antibiotics are well known in the art (see, e.g., MERCK MANUAL OF DIAGNOSIS AND THERAPY (Beers & Berkow eds., 2004), which is hereby incorporated by reference in its entirety). The anti-infective, antibiotic and/or antimicrobial agents may be combined prior to administration, or administered concurrently (as part of the same composition or by way of a different composition) or sequentially with the CD11b inhibitor compositions of the present invention. In certain embodiments, the administering is repeated.
Therapeutic compositions of the present invention may be administered in a single dose, or in accordance with a multi-dosing protocol. For example, in one embodiment of the present invention, relatively few doses of the therapeutic composition are administered, such as one or two doses. In another embodiment of the present invention, the therapeutic composition is administered more frequently, e.g., daily until the level of infection decreases or is gone. In embodiments that include conventional antibiotic therapy, which generally involves multiple doses over a period of days or weeks, the antibiotics can be taken one, two or three or more times daily for a period of time, such as for at least 5 days, 10 days or even 14 or more days, while the CD11b inhibitor composition is administered only once or twice. However, the different dosages, timing of dosages, and relative amounts of the therapeutic composition and antibiotics can and should be selected and adjusted by one of ordinary skill in the art based on the subject and infection being treated.
In the context of using CD11b inhibitory compositions of the present invention to prevent a S. aureus infection, the concentration of the inhibitory CD11b compositions must be adequate to achieve the prevention or substantial prevention of S. aureus infection, particularly the prevention of S. aureus in susceptible populations (i.e., an infant, juvenile, adult, or an immunocompromised infant, juvenile, or adult). In the context of using therapeutic compositions to treat a S. aureus infection, the dosage of a CD11b inhibitory composition is one that is adequate to inhibit LukAB mediated cytotoxicity and is capable of achieving a reduction in a number of symptoms, a decrease in the severity of at least one symptom, or a delay in the further progression of at least one symptom, or even a total alleviation of the infection.
A therapeutically effective amount of a CD11b inhibitor for inhibiting LukAB mediated cytotoxicity can be determined in accordance with standard procedures, which take numerous factors into account, including, for example, the concentrations of these active agents in the composition, the mode and frequency of administration, the severity of the S. aureus infection to be treated (or prevented), and subject details, such as age, weight and overall health and immune condition. General guidance can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990), which is hereby incorporated by reference in its entirety. A clinician may administer a CD11b inhibitory composition, until a dosage is reached that provides the desired or required prophylactic or therapeutic effect. The progress of this therapy can be easily monitored by conventional assays.
The agents of the present invention can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment.
The pharmaceutical agents of the present invention may be formulated for parenteral administration. Solutions or suspensions of the agent can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
When it is desirable to deliver the pharmaceutical agents of the present invention systemically, they may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Intraperitoneal or intrathecal administration of the agents of the present invention can also be achieved using infusion pump devices such as those described by Medtronic, Northridge, Calif. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations.
In addition to the formulations described previously, the agents may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Another aspect of the present invention relates to a transgenic non-human animal whose genome comprises a stably integrated expression construct that comprises a polynucleotide sequence encoding human CD11b.
Suitable nucleotide sequences encoding human CD11b are known in the art and are shown below as SEQ ID NO: 1 (NCBI Accession No. NM_000632) and SEQ ID NO: 3 (NCBI Accession No. NM_001145808). The corresponding CD11b amino acid sequences are also shown below as SEQ ID NO: 2 (NCBI Accession No. NP_000632) and SEQ ID NO: 4 (NM_00001139280), respectively.








SEQ ID NO: 1



tggcttcctt gtggttcctc agtggtgcct gcaacccctg gttcacctcc ttccaggttc   60



tggctccttc cagccatggc tctcagagtc cttctgttaa cagccttgac cttatgtcat  120


gggttcaact tggacactga aaacgcaatg accttccaag agaacgcaag gggcttcggg  180


cagagcgtgg tccagcttca gggatccagg gtggtggttg gagcccccca ggagatagtg  240


gctgccaacc aaaggggcag cctctaccag tgcgactaca gcacaggctc atgcgagccc  300


atccgcctgc aggtccccgt ggaggccgtg aacatgtccc tgggcctgtc cctggcagcc  360


accaccagcc cccctcagct gctggcctgt ggtcccaccg tgcaccagac ttgcagtgag  420


aacacgtatg tgaaagggct ctgcttcctg tttggatcca acctacggca gcagccccag  480


aagttcccag aggccctccg agggtgtcct caagaggata gtgacattgc cttcttgatt  540


gatggctctg gtagcatcat cccacatgac tttcggcgga tgaaggagtt tgtctcaact  600


gtgatggagc aattaaaaaa gtccaaaacc ttgttctctt tgatgcagta ctctgaagaa  660


ttccggattc actttacctt caaagagttc cagaacaacc ctaacccaag atcactggtg  720


aagccaataa cgcagctgct tgggcggaca cacacggcca cgggcatccg caaagtggta  780


cgagagctgt ttaacatcac caacggagcc cgaaagaatg cctttaagat cctagttgtc  840


atcacggatg gagaaaagtt tggcgatccc ttgggatatg aggatgtcat ccctgaggca  900


gacagagagg gagtcattcg ctacgtcatt ggggtgggag atgccttccg cagtgagaaa  960


tcccgccaag agcttaatac catcgcatcc aagccgcctc gtgatcacgt gttccaggtg 1020


aataactttg aggctctgaa gaccattcag aaccagcttc gggagaagat ctttgcgatc 1080


gagggtactc agacaggaag tagcagctcc tttgagcatg agatgtctca ggaaggcttc 1140


agcgctgcca tcacctctaa tggccccttg ctgagcactg tggggagcta tgactgggct 1200


ggtggagtct ttctatatac atcaaaggag aaaagcacct tcatcaacat gaccagagtg 1260


gattcagaca tgaatgatgc ttacttgggt tatgctgccg ccatcatctt acggaaccgg 1320


gtgcaaagcc tggttctggg ggcacctcga tatcagcaca tcggcctggt agcgatgttc 1380


aggcagaaca ctggcatgtg ggagtccaac gctaatgtca agggcaccca gatcggcgcc 1440


tacttcgggg cctccctctg ctccgtggac gtggacagca acggcagcac cgacctggtc 1500


ctcatcgggg ccccccatta ctacgagcag acccgagggg gccaggtgtc cgtgtgcccc 1560


ttgcccaggg ggagggctcg gtggcagtgt gatgctgttc tctacgggga gcagggccaa 1620


ccctggggcc gctttggggc agccctaaca gtgctggggg acgtaaatgg ggacaagctg 1680


acggacgtgg ccattggggc cccaggagag gaggacaacc ggggtgctgt ttacctgttt 1740


cacggaacct caggatctgg catcagcccc tcccatagcc agcggatagc aggctccaag 1800


ctctctccca ggctccagta ttttggtcag tcactgagtg ggggccagga cctcacaatg 1860


gatggactgg tagacctgac tgtaggagcc caggggcacg tgctgctgct caggtcccag 1920


ccagtactga gagtcaaggc aatcatggag ttcaatccca gggaagtggc aaggaatgta 1980


tttgagtgta atgatcaggt ggtgaaaggc aaggaagccg gagaggtcag agtctgcctc 2040


catgtccaga agagcacacg ggatcggcta agagaaggac agatccagag tgttgtgact 2100


tatgacctgg ctctggactc cggccgccca cattcccgcg ccgtcttcaa tgagacaaag 2160


aacagcacac gcagacagac acaggtcttg gggctgaccc agacttgtga gaccctgaaa 2220


ctacagttgc cgaattgcat cgaggaccca gtgagcccca ttgtgctgcg cctgaacttc 2280


tctctggtgg gaacgccatt gtctgctttc gggaacctcc ggccagtgct ggcggaggat 2340


gctcagagac tcttcacagc cttgtttccc tttgagaaga attgtggcaa tgacaacatc 2400


tgccaggatg acctcagcat caccttcagt ttcatgagcc tggactgcct cgtggtgggt 2460


gggccccggg agttcaacgt gacagtgact gtgagaaatg atggtgagga ctcctacagg 2520


acacaggtca ccttcttctt cccgcttgac ctgtcctacc ggaaggtgtc cacactccag 2580


aaccagcgct cacagcgatc ctggcgcctg gcctgtgagt ctgcctcctc caccgaagtg 2640


tctggggcct tgaagagcac cagctgcagc ataaaccacc ccatcttccc ggaaaactca 2700


gaggtcacct ttaatatcac gtttgatgta gactctaagg cttcccttgg aaacaaactg 2760


ctcctcaagg ccaatgtgac cagtgagaac aacatgccca gaaccaacaa aaccgaattc 2820


caactggagc tgccggtgaa atatgctgtc tacatggtgg tcaccagcca tggggtctcc 2880


actaaatatc tcaacttcac ggcctcagag aataccagtc gggtcatgca gcatcaatat 2940


caggtcagca acctggggca gaggagcccc cccatcagcc tggtgttctt ggtgcccgtc 3000


cggctgaacc agactgtcat atgggaccgc ccccaggtca ccttctccga gaacctctcg 3060


agtacgtgcc acaccaagga gcgcttgccc tctcactccg actttctggc tgagcttcgg 3120


aaggcccccg tggtgaactg ctccatcgct gtctgccaga gaatccagtg tgacatcccg 3180


ttctttggca tccaggaaga attcaatgct accctcaaag gcaacctctc gtttgactgg 3240


tacatcaaga cctcgcataa ccacctcctg atcgtgagca cagctgagat cttgtttaac 3300


gattccgtgt tcaccctgct gccgggacag ggggcgtttg tgaggtccca gacggagacc 3360


aaagtggagc cgttcgaggt ccccaacccc ctgccgctca tcgtgggcag ctctgtcggg 3420


ggactgctgc tcctggccct catcaccgcc gcgctgtaca agctcggctt cttcaagcgg 3480


caatacaagg acatgatgag tgaagggggt cccccggggg ccgaacccca gtagcggctc 3540


cttcccgaca gagctgcctc tcggtggcca gcaggactct gcccagacca cacgagcccc 3600


caggctgctg gacacgtcgg acagcgaagt atccccgaca ggacgggctt gggcttccat 3660


ttgtgtgtgt gcaagtgtgt atgtgcgtgt gtgcgagtgt gtgcaagtgt ctgtgtgcaa 3720


gtgtgtgcac gtgtgcgtgt gcgtgcatgt gcactcgcac gcccatgtgt gagtgtgtgc 3780


aagtatgtga gtgtgtccag tgtgtgtgcg tgtgtccatg tgtgtgcagt gtgtgcatgt 3840


gtgcgagtgt gtgcatgtgt gtgctcaggg gctgtggctc acgtgtgtga ctcagagtgt 3900


ctctggcgtg tgggtaggtg acggcagcgt agcctctccg gcagaaggga actgcctggg 3960


ctcccttgtg cgtgggtaag ccgctgctgg gttttcctcc gggagagggg acggtcaatc 4020


ctgtgggtga agagagaggg aaacacagca gcatctctcc actgaaagaa gtgggacttc 4080


ccgtcgcctg cgagcctgcg gcctgctgga gcctgcgcag cttggatgga tactccatga 4140


gaaaagccgt gggtggaacc aggagcctcc tccacaccag cgctgatgcc caataaagat 4200


gcccactgag gaatcatgaa gcttcctttc tggattcatt tattatttca atgtgacttt 4260


aattttttgg atggataagc ctgtctatgg tacaaaaatc acaaggcatt caagtgtaca 4320


gtgaaaagtc tccctttcca gatattcaag tcacctcctt aaaggtagtc aagattgtgt 4380


tttgaggttt ccttcagaca gattccaggc gatgtgcaag tgtatgcacg tgtgcacaca 4440


ccacacacat acacacacac aagctttttt acacaaatgg tagcatactt tatattggtc 4500


tgtatcttgc tttttttcac caatatttct cagacatcgg ttcatattaa gacataaatt 4560


actttttcat tcttttatac cgctgcatag tattccattg tgtgagtgta ccataatgta 4620


tttaaccagt cttcttttga tatactattt tcatctcttg ttattgcatc tgctgagtta 4680


ataaatcaaa tatatgtcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaat 4740


 


SEQ ID NO: 2



Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His Gly



1               5                   10                  15


Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gln Glu Asn Ala Arg


            20                  25                  30


Gly Phe Gly Gln Ser Val Val Gln Leu Gln Gly Ser Arg Val Val Val


        35                  40                  45


Gly Ala Pro Gln Glu Ile Val Ala Ala Asn Gln Arg Gly Ser Leu Tyr


    50                  55                  60


Gln Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro Ile Arg Leu Gln Val


65                  70                  75                  80


Pro Val Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Thr


                85                  90                  95


Thr Ser Pro Pro Gln Leu Leu Ala Cys Gly Pro Thr Val His Gln Thr


            100                 105                 110


Cys Ser Glu Asn Thr Tyr Val Lys Gly Leu Cys Phe Leu Phe Gly Ser


        115                 120                 125


Asn Leu Arg Gln Gln Pro Gln Lys Phe Pro Glu Ala Leu Arg Gly Cys


    130                 135                 140


Pro Gln Glu Asp Ser Asp Ile Ala Phe Leu Ile Asp Gly Ser Gly Ser


145                 150                 155                 160


Ile Ile Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val


                165                 170                 175


Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gln Tyr


            180                 185                 190


Ser Glu Glu Phe Arg Ile His Phe Thr Phe Lys Glu Phe Gln Asn Asn


        195                 200                 205


Pro Asn Pro Arg Ser Leu Val Lys Pro Ile Thr Gln Leu Leu Gly Arg


    210                 215                 220


Thr His Thr Ala Thr Gly Ile Arg Lys Val Val Arg Glu Leu Phe Asn


225                 230                 235                 240


Ile Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys Ile Leu Val Val Ile


                245                 250                 255


Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val Ile


            260                 265                 270


Pro Glu Ala Asp Arg Glu Gly Val Ile Arg Tyr Val Ile Gly Val Gly


        275                 280                 285


Asp Ala Phe Arg Ser Glu Lys Ser Arg Gln Glu Leu Asn Thr Ile Ala


    290                 295                 300


Ser Lys Pro Pro Arg Asp His Val Phe Gln Val Asn Asn Phe Glu Ala


305                310                  315                 320


Leu Lys Thr Ile Gln Asn Gln Leu Arg Glu Lys Ile Phe Ala Ile Glu


                325                 330                 335


Gly Thr Gln Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gln


            340                 345                 350


Glu Gly Phe Ser Ala Ala Ile Thr Ser Asn Gly Pro Leu Leu Ser Thr


        355                 360                 365


Val Gly Ser Tyr Asp Trp Ala Gly Gly Val Phe Leu Tyr Thr Ser Lys


    370                 375                 380


Glu Lys Ser Thr Phe Ile Asn Met Thr Arg Val Asp Ser Asp Met Asn


385                 390                 395                 400


Asp Ala Tyr Leu Gly Tyr Ala Ala Ala Ile Ile Leu Arg Asn Arg Val


                405                 410                 415


Gln Ser Leu Val Leu Gly Ala Pro Arg Tyr Gln His Ile Gly Leu Val


            420                 425                 430


Ala Met Phe Arg Gln Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val


        435                 440                 445


Lys Gly Thr Gln Ile Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val


    450                 455                 460


Asp Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu Ile Gly Ala Pro


465                 470                 475                 480


His Tyr Tyr Glu Gln Thr Arg Gly Gly Gln Val Ser Val Cys Pro Leu


                485                 490                 495


Pro Arg Gly Gln Arg Ala Arg Trp Gln Cys Asp Ala Val Leu Tyr Gly 


            500                 505                 510


Glu Gln Gly Gln Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu 


        515                 520                 525


Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala Ile Gly Ala Pro 


    530                 535                 540


Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe His Gly Thr Ser 


545                 550                 555                 560


Gly Ser Gly Ile Ser Pro Ser His Ser Gln Arg Ile Ala Gly Ser Lys 


                565                 570                 575


Leu Ser Pro Arg Leu Gln Tyr Phe Gly Gln Ser Leu Ser Gly Gly Gln 


            580                 585                 590


Asp Leu Thr Met Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gln Gly 


        595                 600                 605


His Val Leu Leu Leu Arg Ser Gln Pro Val Leu Arg Val Lys Ala Ile 


    610                 615                 620


Met Glu Phe Asn Pro Arg Glu Val Ala Arg Asn Val Phe Glu Cys Asn 


625                 630                 635                 640


Asp Gln Val Val Lys Gly Lys Glu Ala Gly Glu Val Arg Val Cys Leu 


                645                 650                 655


His Val Gln Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gln Ile Gln 


            660                 665                 670


Ser Val Val Thr Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser 


        675                 680                 685


Arg Ala Val Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gln Thr Gln 


    690                 695                 700


Val Leu Gly Leu Thr Gln Thr Cys Glu Thr Leu Lys Leu Gln Leu Pro 


705                 710                 715                 720


Asn Cys Ile Glu Asp Pro Val Ser Pro Ile Val Leu Arg Leu Asn Phe 


                725                 730                 735


Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro Val 


            740                 745                 750


Leu Ala Glu Asp Ala Gln Arg Leu Phe Thr Ala Leu Phe Pro Phe Glu 


        755                 760                 765


Lys Asn Cys Gly Asn Asp Asn Ile Cys Gln Asp Asp Leu Ser Ile Thr 


    770                 775                 780


Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly Gly Pro Arg Glu 


785                 790                 795                 800


Phe Asn Val Thr Val Thr Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg 


                805                 810                 815


Thr Gln Val Thr Phe Phe Phe Pro Leu Asp Leu Ser Tyr Arg Lys Val 


            820                 825                 830


Ser Thr Leu Gln Asn Gln Arg Ser Gln Arg Ser Trp Arg Leu Ala Cys 


        835                 840                 845


Glu Ser Ala Ser Ser Thr Glu Val Ser Gly Ala Leu Lys Ser Thr Ser 


    850                 855                 860


Cys Ser Ile Asn His Pro Ile Phe Pro Glu Asn Ser Glu Val Thr Phe 


865                 870                 875                 880


Asn Ile Thr Phe Asp Val Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu 


                885                 890                 895


Leu Leu Lys Ala Asn Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn 


            900                 905                 910


Lys Thr Glu Phe Gln Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met 


        915                 920                 925


Val Val Thr Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala 


    930                 935                 940


Ser Glu Asn Thr Ser Arg Val Met Gln His Gln Tyr Gln Val Ser Asn 


945                 950                 955                 960


Leu Gly Gln Arg Ser Pro Pro Ile Ser Leu Val Phe Leu Val Pro Val 


                965                 970                 975


Arg Leu Asn Gln Thr Val Ile Trp Asp Arg Pro Gln Val Thr Phe Ser 


            980                 985                 990


Glu Asn Leu Ser Ser Thr Cys His Thr  Lys Glu Arg Leu Pro  Ser His 


        995                 1000                 1005


Ser Asp Phe  Leu Ala Glu Leu Arg  Lys Ala Pro Val Val  Asn Cys 


    1010                 1015                 1020


Ser Ile Ala  Val Cys Gln Arg Ile  Gln Cys Asp Ile Pro  Phe Phe 


    1025                 1030                 1035


Gly Ile Gln  Glu Glu Phe Asn Ala  Thr Leu Lys Gly Asn  Leu Ser 


    1040                 1045                 1050


Phe Asp Trp  Tyr Ile Lys Thr Ser  His Asn His Leu Leu  Ile Val 


    1055                 1060                 1065


Ser Thr Ala  Glu Ile Leu Phe Asn  Asp Ser Val Phe Thr  Leu Leu 


    1070                 1075                 1080


Pro Gly Gln  Gly Ala Phe Val Arg  Ser Gln Thr Glu Thr  Lys Val 


    1085                 1090                 1095


Glu Pro Phe  Glu Val Pro Asn Pro  Leu Pro Leu Ile Val  Gly Ser 


    1100                 1105                 1110


Ser Val Gly  Gly Leu Leu Leu Leu  Ala Leu Ile Thr Ala  Ala Leu 


    1115                 1120                 1125


Tyr Lys Leu  Gly Phe Phe Lys Arg  Gln Tyr Lys Asp Met  Met Ser 


    1130                 1135                 1140


Glu Gly Gly  Pro Pro Gly Ala Glu  Pro Gln


    1145                 1150


 


SEQ ID NO: 3



ttttctgccc ttctttgctt tggtggcttc cttgtggttc ctcagtggtg cctgcaaccc   60



ctggttcacc tccttccagg ttctggctcc ttccagccat ggctctcaga gtccttctgt  120


taacagcctt gaccttatgt catgggttca acttggacac tgaaaacgca atgaccttcc  180


aagagaacgc aaggggcttc gggcagagcg tggtccagct tcagggatcc agggtggtgg  240


ttggagcccc ccaggagata gtggctgcca accaaagggg cagcctctac cagtgcgact  300


acagcacagg ctcatgcgag cccatccgcc tgcaggtccc cgtggaggcc gtgaacatgt  360


ccctgggcct gtccctggca gccaccacca gcccccctca gctgctggcc tgtggtccca  420


ccgtgcacca gacttgcagt gagaacacgt atgtgaaagg gctctgcttc ctgtttggat  480


ccaacctacg gcagcagccc cagaagttcc cagaggccct ccgagggtgt cctcaagagg  540


atagtgacat tgccttcttg attgatggct ctggtagcat catcccacat gactttcggc  600


ggatgaagga gtttgtctca actgtgatgg agcaattaaa aaagtccaaa accttgttct  660


ctttgatgca gtactctgaa gaattccgga ttcactttac cttcaaagag ttccagaaca  720


accctaaccc aagatcactg gtgaagccaa taacgcagct gcttgggcgg acacacacgg  780


ccacgggcat ccgcaaagtg gtacgagagc tgtttaacat caccaacgga gcccgaaaga  840


atgcctttaa gatcctagtt gtcatcacgg atggagaaaa gtttggcgat cccttgggat  900


atgaggatgt catccctgag gcagacagag agggagtcat tcgctacgtc attggggtgg  960


gagatgcctt ccgcagtgag aaatcccgcc aagagcttaa taccatcgca tccaagccgc 1020


ctcgtgatca cgtgttccag gtgaataact ttgaggctct gaagaccatt cagaaccagc 1080


ttcgggagaa gatctttgcg atcgagggta ctcagacagg aagtagcagc tcctttgagc 1140


atgagatgtc tcaggaaggc ttcagcgctg ccatcacctc taatggcccc ttgctgagca 1200


ctgtggggag ctatgactgg gctggtggag tctttctata tacatcaaag gagaaaagca 1260


ccttcatcaa catgaccaga gtggattcag acatgaatga tgcttacttg ggttatgctg 1320


ccgccatcat cttacggaac cgggtgcaaa gcctggttct gggggcacct cgatatcagc 1380


acatcggcct ggtagcgatg ttcaggcaga acactggcat gtgggagtcc aacgctaatg 1440


tcaagggcac ccagatcggc gcctacttcg gggcctccct ctgctccgtg gacgtggaca 1500


gcaacggcag caccgacctg gtcctcatcg gggcccccca ttactacgag cagacccgag 1560


ggggccaggt gtccgtgtgc cccttgccca gggggcagag ggctcggtgg cagtgtgatg 1620


ctgttctcta cggggagcag ggccaaccct ggggccgctt tggggcagcc ctaacagtgc 1680


tgggggacgt aaatggggac aagctgacgg acgtggccat tggggcccca ggagaggagg 1740


acaaccgggg tgctgtttac ctgtttcacg gaacctcagg atctggcatc agcccctccc 1800


atagccagcg gatagcaggc tccaagctct ctcccaggct ccagtatttt ggtcagtcac 1860


tgagtggggg ccaggacctc acaatggatg gactggtaga cctgactgta ggagcccagg 1920


ggcacgtgct gctgctcagg tcccagccag tactgagagt caaggcaatc atggagttca 1980


atcccaggga agtggcaagg aatgtatttg agtgtaatga tcaggtggtg aaaggcaagg 2040


aagccggaga ggtcagagtc tgcctccatg tccagaagag cacacgggat cggctaagag 2100


aaggacagat ccagagtgtt gtgacttatg acctggctct ggactccggc cgcccacatt 2160


cccgcgccgt cttcaatgag acaaagaaca gcacacgcag acagacacag gtcttggggc 2220


tgacccagac ttgtgagacc ctgaaactac agttgccgaa ttgcatcgag gacccagtga 2280


gccccattgt gctgcgcctg aacttctctc tggtgggaac gccattgtct gctttcggga 2340


acctccggcc agtgctggcg gaggatgctc agagactctt cacagccttg tttccctttg 2400


agaagaattg tggcaatgac aacatctgcc aggatgacct cagcatcacc ttcagtttca 2460


tgagcctgga ctgcctcgtg gtgggtgggc cccgggagtt caacgtgaca gtgactgtga 2520


gaaatgatgg tgaggactcc tacaggacac aggtcacctt cttcttcccg cttgacctgt 2580


cctaccggaa ggtgtccacg ctccagaacc agcgctcaca gcgatcctgg cgcctggcct 2640


gtgagtctgc ctcctccacc gaagtgtctg gggccttgaa gagcaccagc tgcagcataa 2700


accaccccat cttcccggaa aactcagagg tcacctttaa tatcacgttt gatgtagact 2760


ctaaggcttc ccttggaaac aaactgctcc tcaaggccaa tgtgaccagt gagaacaaca 2820


tgcccagaac caacaaaacc gaattccaac tggagctgcc ggtgaaatat gctgtctaca 2880


tggtggtcac cagccatggg gtctccacta aatatctcaa cttcacggcc tcagagaata 2940


ccagtcgggt catgcagcat caatatcagg tcagcaacct ggggcagagg agcctcccca 3000


tcagcctggt gttcttggtg cccgtccggc tgaaccagac tgtcatatgg gaccgccccc 3060


aggtcacctt ctccgagaac ctctcgagta cgtgccacac caaggagcgc ttgccctctc 3120


actccgactt tctggctgag cttcggaagg cccccgtggt gaactgctcc atcgctgtct 3180


gccagagaat ccagtgtgac atcccgttct ttggcatcca ggaagaattc aatgctaccc 3240


tcaaaggcaa cctctcgttt gactggtaca tcaagacctc gcataaccac ctcctgatcg 3300


tgagcacagc tgagatcttg tttaacgatt ccgtgttcac cctgctgccg ggacaggggg 3360


cgtttgtgag gtcccagacg gagaccaaag tggagccgtt cgaggtcccc aaccccctgc 3420


cgctcatcgt gggcagctct gtcgggggac tgctgctcct ggccctcatc accgccgcgc 3480


tgtacaagct cggcttcttc aagcggcaat acaaggacat gatgagtgaa gggggtcccc 3540


cgggggccga accccagtag cggctccttc ccgacagagc tgcctctcgg tggccagcag 3600


gactctgccc agaccacacg tagcccccag gctgctggac acgtcggaca gcgaagtatc 3660


cccgacagga cgggcttggg cttccatttg tgtgtgtgca agtgtgtatg tgcgtgtgtg 3720


caagtgtctg tgtgcaagtg tgtgcacatg tgtgcgtgtg cgtgcatgtg cacttgcacg 3780


cccatgtgtg agtgtgtgca agtatgtgag tgtgtccaag tgtgtgtgcg tgtgtccatg 3840


tgtgtgcaag tgtgtgcatg tgtgcgagtg tgtgcatgtg tgtgctcagg ggcgtgtggc 3900


tcacgtgtgt gactcagatg tctctggcgt gtgggtaggt gacggcagcg tagcctctcc 3960


ggcagaaggg aactgcctgg gctcccttgt gcgtgggtga agccgctgct gggttttcct 4020


ccgggagagg ggacggtcaa tcctgtgggt gaagacagag ggaaacacag cagcttctct 4080


ccactgaaag aagtgggact tcccgtcgcc tgcgagcctg cggcctgctg gagcctgcgc 4140


agcttggatg gagactccat gagaagccgt gggtggaacc aggaacctcc tccacaccag 4200


cgctgatgcc caataaagat gcccactgag gaatgatgaa gcttcctttc tggattcatt 4260


tattatttca atgtgacttt aattttttgg atggataagc ttgtctatgg tacaaaaatc 4320


acaaggcatt caagtgtaca gtgaaaagtc tccctttcca gatattcaag tcacctcctt 4380


aaaggtagtc aagattgtgt tttgaggttt ccttcagaca gattccaggc gatgtgcaag 4440


tgtatgcacg tgtgcacaca caccacacat acacacacac aagctttttt acacaaatgg 4500


tagcatactt tatattggtc tgtatcttgc tttttttcac caatatttct cagacatcgg 4560


ttcatattaa gacataaatt actttttcat tcttttatac cgctgcatag tattccattg 4620


tgtgagtgta ccataatgta tttaaccagt cttcttttga tatactattt tcattctctt 4680


gttattgcat caatgctgag ttaataaatc aaatatatgt catttttgca tatatgtaag 4740


gataa                                                             4745


 


SEQ ID NO: 4



Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His Gly



1               5                   10                  15


Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gln Glu Asn Ala Arg


            20                  25                  30


Gly Phe Gly Gln Ser Val Val Gln Leu Gln Gly Ser Arg Val Val Val


        35                  40                  45


Gly Ala Pro Gln Glu Ile Val Ala Ala Asn Gln Arg Gly Ser Leu Tyr


    50                  55                  60


Gln Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro Ile Arg Leu Gln Val


65                  70                  75                  80


Pro Val Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Thr


                85                  90                  95


Thr Ser Pro Pro Gln Leu Leu Ala Cys Gly Pro Thr Val His Gln Thr


            100                 105                 110


Cys Ser Glu Asn Thr Tyr Val Lys Gly Leu Cys Phe Leu Phe Gly Ser


        115                 120                 125


Asn Leu Arg Gln Gln Pro Gln Lys Phe Pro Glu Ala Leu Arg Gly Cys


    130                 135                 140


Pro Gln Glu Asp Ser Asp Ile Ala Phe Leu Ile Asp Gly Ser Gly Ser


145                 150                 155                 160


Ile Ile Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val


                165                 170                 175


Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gln Tyr


            180                 185                 190


Ser Glu Glu Phe Arg Ile His Phe Thr Phe Lys Glu Phe Gln Asn Asn


        195                 200                 205


Pro Asn Pro Arg Ser Leu Val Lys Pro Ile Thr Gln Leu Leu Gly Arg


    210                 215                 220


Thr His Thr Ala Thr Gly Ile Arg Lys Val Val Arg Glu Leu Phe Asn


225                 230                 235                 240


Ile Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys Ile Leu Val Val Ile


                245                 250                 255


Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val Ile


            260                 265                 270


Pro Glu Ala Asp Arg Glu Gly Val Ile Arg Tyr Val Ile Gly Val Gly


        275                 280                 285


Asp Ala Phe Arg Ser Glu Lys Ser Arg Gln Glu Leu Asn Thr Ile Ala


    290                 295                 300


Ser Lys Pro Pro Arg Asp His Val Phe Gln Val Asn Asn Phe Glu Ala


305                 310                 315                 320


Leu Lys Thr Ile Gln Asn Gln Leu Arg Glu Lys Ile Phe Ala Ile Glu


                325                 330                 335


Gly Thr Gln Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gln


            340                 345                 350


Glu Gly Phe Ser Ala Ala Ile Thr Ser Asn Gly Pro Leu Leu Ser Thr


        355                 360                 365


Val Gly Ser Tyr Asp Trp Ala Gly Gly Val Phe Leu Tyr Thr Ser Lys


    370                 375                 380


Glu Lys Ser Thr Phe Ile Asn Met Thr Arg Val Asp Ser Asp Met Asn


385                 390                 395                 400


Asp Ala Tyr Leu Gly Tyr Ala Ala Ala Ile Ile Leu Arg Asn Arg Val


                405                 410                 415


Gln Ser Leu Val Leu Gly Ala Pro Arg Tyr Gln His Ile Gly Leu Val


            420                 425                 430


Ala Met Phe Arg Gln Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val


        435                 440                 445


Lys Gly Thr Gln Ile Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val


    450                 455                 460


Asp Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu Ile Gly Ala Pro


465                 470                 475                 480


His Tyr Tyr Glu Gln Thr Arg Gly Gly Gln Val Ser Val Cys Pro Leu


                485                 490                 495


Pro Arg Gly Gln Arg Ala Arg Trp Gln Cys Asp Ala Val Leu Tyr Gly


            500                 505                 510


Glu Gln Gly Gln Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu


        515                 520                 525


Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala Ile Gly Ala Pro


    530                 535                 540


Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe His Gly Thr Ser


545                 550                 555                 560


Gly Ser Gly Ile Ser Pro Ser His Ser Gln Arg Ile Ala Gly Ser Lys


                565                 570                 575


Leu Ser Pro Arg Leu Gln Tyr Phe Gly Gln Ser Leu Ser Gly Gly Gln


            580                 585                 590


Asp Leu Thr Met Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gln Gly


        595                 600                 605


His Val Leu Leu Leu Arg Ser Gln Pro Val Leu Arg Val Lys Ala Ile


    610                 615                 620


Met Glu Phe Asn Pro Arg Glu Val Ala Arg Asn Val Phe Glu Cys Asn


625                 630                 635                 640


Asp Gln Val Val Lys Gly Lys Glu Ala Gly Glu Val Arg Val Cys Leu


                645                 650                 655


His Val Gln Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gln Ile Gln


            660                 665                 670


Ser Val Val Thr Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser


        675                 680                 685


Arg Ala Val Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gln Thr Gln


    690                 695                 700


Val Leu Gly Leu Thr Gln Thr Cys Glu Thr Leu Lys Leu Gln Leu Pro


705                 710                 715                 720


Asn Cys Ile Glu Asp Pro Val Ser Pro Ile Val Leu Arg Leu Asn Phe


                725                 730                 735


Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro Val


            740                 745                 750


Leu Ala Glu Asp Ala Gln Arg Leu Phe Thr Ala Leu Phe Pro Phe Glu


        755                 760                 765


Lys Asn Cys Gly Asn Asp Asn Ile Cys Gln Asp Asp Leu Ser Ile Thr


    770                 775                 780


Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly Gly Pro Arg Glu


785                 790                 795                 800


Phe Asn Val Thr Val Thr Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg


                805                 810                 815


Thr Gln Val Thr Phe Phe Phe Pro Leu Asp Leu Ser Tyr Arg Lys Val


            820                 825                 830


Ser Thr Leu Gln Asn Gln Arg Ser Gln Arg Ser Trp Arg Leu Ala Cys


        835                 840                 845


Glu Ser Ala Ser Ser Thr Glu Val Ser Gly Ala Leu Lys Ser Thr Ser


    850                 855                 860


Cys Ser Ile Asn His Pro Ile Phe Pro Glu Asn Ser Glu Val Thr Phe


865                 870                 875                 880


Asn Ile Thr Phe Asp Val Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu


                885                 890                 895


Leu Leu Lys Ala Asn Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn


            900                 905                 910


Lys Thr Glu Phe Gln Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met


        915                 920                 925


Val Val Thr Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala


    930                 935                 940


Ser Glu Asn Thr Ser Arg Val Met Gln His Gln Tyr Gln Val Ser Asn


945                 950                 955                 960


Leu Gly Gln Arg Ser Leu Pro Ile Ser Leu Val Phe Leu Val Pro Val


                965                 970                 975


Arg Leu Asn Gln Thr Val Ile Trp Asp Arg Pro Gln Val Thr Phe Ser


            980                 985                 990


Glu Asn Leu Ser Ser Thr Cys His  Thr Lys Glu Arg Leu  Pro Ser His


        995                 1000                 1005


Ser Asp  Phe Leu Ala Glu Leu  Arg Lys Ala Pro Val  Val Asn Cys


    1010                 1015                 1020


Ser Ile  Ala Val Cys Gln Arg  Ile Gln Cys Asp Ile  Pro Phe Phe


    1025                 1030                 1035


Gly Ile  Gln Glu Glu Phe Asn  Ala Thr Leu Lys Gly  Asn Leu Ser


    1040                 1045                 1050


Phe Asp  Trp Tyr Ile Lys Thr  Ser His Asn His Leu  Leu Ile Val


    1055                 1060                 1065


Ser Thr  Ala Glu Ile Leu Phe  Asn Asp Ser Val Phe  Thr Leu Leu


    1070                 1075                 1080


Pro Gly  Gln Gly Ala Phe Val  Arg Ser Gln Thr Glu  Thr Lys Val


    1085                 1090                 1095


Glu Pro  Phe Glu Val Pro Asn  Pro Leu Pro Leu Ile  Val Gly Ser


    1100                 1105                 1110


Ser Val  Gly Gly Leu Leu Leu  Leu Ala Leu Ile Thr  Ala Ala Leu


    1115                 1120                 1125


Tyr Lys  Leu Gly Phe Phe Lys  Arg Gln Tyr Lys Asp  Met Met Ser


    1130                 1135                 1140


Glu Gly  Gly Pro Pro Gly Ala  Glu Pro Gln


    1145                 1150






A polynucleotide sequence encoding a human CD11b protein or polypeptide can be integrated into the genome of the transgenic mouse by any standard method well known to those skilled in the art. Any of a variety of techniques known in the art can be used to introduce the transgene into an animal to produce the founder line of transgenic animals (see e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, 1986); Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, 1994), and U.S. Pat. No. 5,602,299 to Lazzarini; U.S. Pat. No. 5,175,384 to Krimpenfort; U.S. Pat. No. 6,066,778 to Ginsburg; and U.S. Pat. No. 6,037,521 to Sato et al, which are hereby incorporated by reference in their entirety). Such techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191 to Wagner et al., which is hereby incorporated by reference in its entirety); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA 82:6148-6152 (1985), which is hereby incorporated by reference in its entirety); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989), which is hereby incorporated by reference in its entirety); electroporation of embryos (Lo et al., Mol. Cell. Biol. 3:1803-1814 (1983), which is hereby incorporated by reference in its entirety); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989), which is hereby incorporated by reference in its entirety).
For example, embryonic cells at various developmental stages can be used to introduce transgenes for the production of transgenic animals. Different methods are used depending on the stage of development of the embryonic cell. The zygote is a good target for micro-injection, and methods of microinjecting zygotes are well known to (see U.S. Pat. No. 4,873,191 to Wagner et al., which is hereby incorporated by reference in its entirety). In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 picoliters (pl) of DNA solution. The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985), which is hereby incorporated by reference in its entirety). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
The transgenic animals of the present invention can also be generated by introduction of the targeting vectors into embryonic stem (ES) cells. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans et al., Nature 292:154-156 (1981); Bradley et al., Nature 309:255-258 (1984); Gossler et al., Proc. Natl. Acad. Sci. USA 83:9065-9069 (1986); and Robertson et al., Nature 322:445-448 (1986), which are hereby incorporated by reference in their entirety). Transgenes can be efficiently introduced into the ES cells by DNA transfection using a variety of methods known to the art including electroporation, calcium phosphate co-precipitation, protoplast or spheroplast fusion, lipofection and DEAE-dextran-mediated transfection. Transgenes can also be introduced into ES cells by retrovirus-mediated transduction or by micro-injection. Such transfected ES cells can thereafter colonize an embryo following their introduction into the blastocoel of a blastocyst-stage embryo and contribute to the germ line of the resulting chimeric animal (reviewed in Jaenisch, Science 240:1468-1474 (1988), which is hereby incorporated by reference in its entirety). Prior to the introduction of transfected ES cells into the blastocoel, the transfected ES cells can be subjected to various selection protocols to enrich for ES cells that have integrated the transgene if the transgene provides a means for such selection. Alternatively, PCR can be used to screen for ES cells that have integrated the transgene. This technique obviates the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.
In addition, retroviral infection can also be used to introduce transgenes into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260-1264 (1976), which is hereby incorporated by reference in its entirety). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927-6931 (1985); Van der Putten et al. Proc. Natl. Acad. Sci. USA 82:6148-6152 (1985)). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells. Alternatively, infection can be performed at a later stage. Additional means of using retroviruses or retroviral vectors to create transgenic animals known to the art involves the micro-injection of retroviral particles or mitomycin C-treated cells producing retrovirus into the perivitelline space of fertilized eggs or early embryos (WO 90/08832 to Onions, which is hereby incorporated by reference in its entirety).
The present invention provides transgenic non-human animals that carry the transgene in all their cells, as well as animals that carry the transgene in some, but not all their cells, i.e., expression of the transgene is controlled by a cell specific promoter and/or enhancer elements placed upstream of the transgene. In one embodiment of the present invention, the transgenic animal expressing human CD11b, expresses the CD11b transgene in leukocytes only. In accordance with this embodiment of the invention, a leukocyte specific promoter sequence is operably linked to the polynucleotide sequence encoding human CD11b. Suitable leukocyte specific promoters include, without limitation, the LSP1 promoter (Malone et al, “Leukocyte-Specific Expression of the pp52 (LSP1) Promoter is Controlled by the cis-acting pp52 Silencer and Anti-Silencer Elements,” Gene 268:9-16 (2001), which is hereby incorporated by reference in its entirety), macrosialin promoter (Li et al., “The Macrosialin Promoter Directs High Levels of Transcriptional Activity in Macrophages Dependent on Combinatorial Interactions Between Pu.1 and c-Jun,” J. Biol. Chem. 273:5389-5399 (1998), which is hereby incorporated by reference in its entirety, lysozyme promoter (Bonifer et al., “Tissue Specific and Position Independent Expression of the Complete Gene Domain for the Chicken Lysozyme in Transgenic Mice,” EMBO J. 9:2843-48 (1990), which is hereby incorporated by reference in its entirety), and the myeloid specific CD11b promoter to promote the expression of the human CD11b only in cells that normally express CD11b (e.g., granulocytes, monocytes, macrophages and Natural Killer cells) (Pahl et al., “Characterization of the Myeloid-Specific CD11b Promoter,” Blood 79:865-870 (1992) and Hickstein et al., “Identification of the Promoter of the Myelomonocytic Leukocyte Integrin CD11b,” Proc. Natl. Acad. Sci. USA 89:2105-09 (1992), which are hereby incorporated by reference in their entirety). Expression or cloning constructs suitable for driving transgene expression in a transgenic animal are well known in the art. Other components of the expression construct include a strong polyadenylation site, appropriate restriction endonuclease sites, and introns to ensure the transcript is spliced.
The polynucleotides encoding human CD11b can be inserted into any non-human animal. In one embodiment the animal is a rodent, for example, a mouse. Suitable strains of mice commonly used in the generation of transgenic models include, without limitation, CD-1® Nude mice, NU/NU mice, BALB/C Nude mice, BALB/C mice, NIH-III mice, SCID® mice, outbred SCID® mice, SCID Beige mice, C3H mice, C57BL/6 mice, DBA/2 mice, FVB mice, CB17 mice, 129 mice, SJL mice, B6C3F1 mice, BDF1 mice, CDF1 mice, CB6F1 mice, CF-1 mice, Swiss Webster mice, SKH1 mice, PGP mice, and B6SJL mice.
The transgenic animals are screened and evaluated to select those animals having a phenotype wherein human CD11b is expressed on all cells or on leukocytes specifically. Initial screening can be performed using, for example, Southern blot analysis or PCR techniques to analyze animal cells to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the cells of the transgenic animals can also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). In addition, surface expression of human CD11b can be evaluated by flow cytometry using human-specific anti-CD11b antibodies conjugated with fluorescent molecules. The transgenic non-human mammals can be further characterized to identify those animals having a phenotype useful in methods of the invention. In particular, the transgenic non-human animal can be exposed to S. aureus and leukocyte cell death can be examined.
Another aspect of the present invention relates to methods of identifying candidate compounds suitable for preventing or treating S. aureus infection and/or conditions resulting from a S. aureus infection using the transgenic non-human animal of the present invention. In one embodiment of this aspect of the invention, the method of identifying candidate compounds involves providing a collection of candidate compounds. The method further involves exposing the transgenic animal expressing human CD11b to an agent capable of inducing LukAB mediated leukocyte death and administering the one or more candidate compounds to the transgenic animal. The method further involves measuring LukAB mediated leukocyte death level in the transgenic animal to which the one or more candidate compounds are administered and comparing that level of LukAB mediated leukocyte death in the transgenic animal to which the one or more candidate compounds are administered to a control level of LukAB mediated leukocyte death in a transgenic animal to which the one or more candidate compounds was not administered. A control level of LukAB mediated cell death is the level of LukAB mediated cell death in a transgenic animal administered the LukAB agent but not the candidate compound. A candidate compound that reduces the level of LukAB mediated leukocyte death in the transgenic animal compared to the control level is identified as a compound suitable for preventing or treating S. aureus and/or conditions resulting from a S. aureus infection.
In accordance with this method of the present invention, agents capable of inducing LukAB mediated leukocyte death, or cell death of any cell expressing the human CD11b protein, include, without limitation, S. aureus particularly a MRSA or MSSA strain, a composition comprising an isolated LukA, LukB or LukAB protein complex, a composition comprising a recombinantly produced LukA, LukB, or LukAB protein complex, or a prokaryotic and/or eukaryotic cells engineered to produced LukA, LukB or LukAB protein complex.
In one embodiment of this aspect of the invention, the candidate compound is administered prior to exposing the transgenic animal to the agent capable of inducing LukAB cytotoxicity as a means for identifying a suitable prophylactic agent. Alternatively, the candidate compound is administered after exposure of the transgenic animal to the LukAB agent as a means for identifying a suitable therapeutic agent.
Another method of the present invention for identifying candidate compounds suitable for preventing or treating S. aureus infection and/a condition resulting from a S. aureus infection using the transgenic rodent involves the steps of providing a collection of candidate compounds exposing the transgenic animal expressing human CD11b to S. aureus and administering a one or more candidate compounds from the collection to the transgenic animal. The method further involves measuring S. aureus infection level in the transgenic animal to which the one or more candidate compounds was administered, comparing the S. aureus infection level in the transgenic animal to which the one or more candidate compounds was administered to a control S. aureus infection level in a transgenic animal that was exposed to S. aureus but not administered the one or more candidate compounds, and identifying a candidate compound that reduces S. aureus infection level in the transgenic animal compared to the control S. aureus infection level as a compound suitable for preventing or treating S. aureus and/or conditions resulting from a S. aureus infection.
Measuring S. aureus infection level encompasses evaluation or measurement of any one or more indicators of S. aureus infection, including, without limitation, animal survival, cell viability, inflammatory response, bacterial burden, and infection related pathology. A candidate compound that increases animal survival and/or cell viability, reduces the inflammatory response or bacterial burden in the animal, and improves pathology of infection is a compound that is suitable for preventing or treating S. aureus and/or a condition resulting from a S. aureus infection.
In one embodiment of this aspect of the invention, the candidate compound is administered prior to exposing the transgenic animal to S. aureus as a means for identifying suitable prophylactic agents. Alternatively, the candidate compound is administered after exposure of the transgenic animal to S. aureus as a means for identifying suitable therapeutic agents.
Another aspect of the present invention relates to a method of identifying a compound capable of preventing or treating S. aureus infection and/or conditions resulting from a S. aureus infection. This method is typically carried out in vitro, i.e., in cell culture. This method involves providing a collection of candidate compounds and providing a population of cells expressing human CD11b. The method further involves treating the population of cells with an agent capable of inducing LukAB mediated cytotoxicity, and contacting the population of treated cells with one or more candidate compounds from the collection. The method further involves measuring LukAB mediated cytotoxicity level in the population of treated cells in the presence and absence of the one or more candidate compounds and comparing the measured level of LukAB mediated cytotoxicity in the presence and in the absence of the one or more candidate compound. A decrease in the level of LukAB mediated cytotoxicity in the presence of the one or more candidate compounds compared to in its absence of the one or more candidate compounds identifies a compound capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection.
Cells expressing human CD11b that are suitable for use in accordance with this aspect of the invention include human leukocytes, such as monocytes, granulocytes, macrophages, and natural killer cells. Other suitable cells include any nucleated cell that has been engineered to express CD11b, e.g., cells stably or transiently transfected with an expression construct containing a human CD11b polynucleotide sequence (e.g., an expression construct comprising the nucleotide sequence of SEQ ID NOs: 1 or 3).
As described herein, this method of the present invention is designed to identify agents that inhibit some aspect of the cascade of events that leads to LukAB-mediated cytotoxicity and lysis of human phagocytes. The targeted events that are part of the cascade include for example, binding of LukA and/or LukB to the CD11b receptor on phagocytes, binding of LukB to LukA (LukAB oligomerization), and blockage of the membrane pore formed by the LukAB oligomer. The assay utilizes any mammalian or non-mammalian cell expressing the human CD11b protein or a fragment thereof that comprises the LukAB binding domain, suitable culture medium, and isolated or recombinant LukA and/or LukB, or S. aureus. The assay further includes a labeled marker of cytotoxicity that is exposed to the cells before, during, or after the cells expressing human CD11b are contacted with an agent capable of inducing LukAB cytotoxicity. The labeled marker of cytotoxicity may comprise a cell viability dye, a cell impermeable dye, and/or an indicator of cell lysis.
The person of skill will appreciate that the following protocols are merely illustrative and that various operating parameters such as reaction conditions, choice of detectable label and apparati (e.g., instrumentation for detection and quantification) may be varied as deemed appropriate. The following methods are generally directed to identifying agents that inhibit LukAB cytotoxicity, without necessarily revealing the exact event in the cascade that is affected.
To identify inhibitors of CD11b-LukAB cytotoxicity, cells expressing human CD11b (e.g., human phagocytes or murine phagocytes transfected with human CD11b) are plated in 384-well clear-bottom black tissue culture treated plate (Corning) at 5×103 cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS). Cells may then be contacted/mixed/reacted/treated with the test compound/molecule ( ̃5 μl/different concentrations) and then intoxicated with LukA and LukB, which in preferred embodiments are substantially purified (5 ul of a  ̃0.001-2 μM solution), preferably added together, under culture conditions to allow for intoxication of the phagocytes by LukA and LukB, e.g., for 1 hr at 37° C., 5% CO2, As controls, cells may be treated with culture medium (100% viable) and with 0.1% v/v Triton X100 (100% death).
In these embodiments, cells treated as described above may then be incubated with a dye to monitor cell viability such as CellTiter (Promega) (which enables determination of cell viability via absorbance by measuring the number of viable cells in a culture by quantification of the metabolic activity of the cells) and incubated for an additional time period (e.g., about 2 hrs at 37° C., 5% CO2). Cell viability may then be determined such as by measuring the colorimetric reaction at 492 nm using a plate reader e.g., Envision 2103 Multi-label Reader (Perkin-Elmer). Percent viable cells may be calculated such as by using the following equation: % Viability=100×[(Ab492Sample-Ab492Triton X)/(Ab492Tissue culture media)]. An increase in the percent viability suggests inhibition of LukAB cytotoxicity.
A variation of this assay is referred to as a membrane damage assay. In these embodiments, cells treated as described above (e.g., up to and including treating of the cells with test compound/molecule and then intoxicating the cells with purified LukA or LukAB may then be incubated with a cell-impermeable fluorescent dye such as SYTOX green (0.1 μM; Invitrogen) (in accordance with manufacturer's instructions) and incubated e.g., for an additional 15 minutes at room temperature in the dark. Fluorescence, as an indicator of membrane damage, may then be measured using a plate reader such as Envision 2103 Multilabel Reader (Perkin-Elmer) at Excitation 485 nm, Emission 535 nm. A decrease in fluorescence suggests inhibition of LukAB cytotoxicity.
Together these assays facilitate the identification of compounds that inhibit or reduce LukAB cytotoxic effects towards cells expressing human CD11b. Additional methods may be used, independently or in conjunction with the methods described above, particularly if the above methods reveal inhibitory activity, that will enable a person skilled in the field to determine more precisely what event in the biochemical cascade is being affected or targeted by the agent. These events include binding of LukA, LukB or LukAB to the CD11b receptor, binding of LukB to LukA (LukAB oligomerization), and blockage of the membrane pore formed by the LukAB oligomer.
To screen for inhibitors that block or reduce LukA, LukB, or LukAB binding to target cells, which is believed to be the first step in the intoxication process, cells expressing human CD11b (e.g., PMN-HL60 cells) may be plated in 384-well flat-bottom tissue culture treated plates (Corning) at 2.5×103 cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS). Cells may then be treated with the test compound/molecule ( ̃5 μl/different concentrations) and intoxicated with purified, fluorescently labeled LukA, LukB, or LukAB (e.g., FITC, Cy3, Cy5, APC, PE) 5 ul of a  ̃0.01-2 μM solution for 1 hr at 37° C., 5% CO2. To evaluate the efficacy of the tested compounds/molecules, the cell-associated fluorescence may be measured as an indicator of LukA, LukB, or LukAB binding to CD11b, e.g., using an automated fluorescence microscopic imaging system designed for high content screening and high content analysis (e.g., Cellomics ArrayScan ECS Reader (Thermo Scientific) (Excitation 485 nm, Emission 535 nm)).
To screen for inhibitors that block or reduce LukA/LukB interaction, which is believed to be the second step in the intoxication process, cells expressing human CD11b (e.g., PMN-HL60 cells) may be plated in 384-well flat-bottom tissue culture treated plates (Corning) at 2.5×103 cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS). Cells may then be treated with the test compound/molecule and then intoxicated with a mixture of purified LukA and purified LukB where LukB is fluorescently-labeled with a fluorescence molecule such as FITC, Cy3, Cy5, APC, and PE, and allowed to stand to complete the intoxication process (e.g., for 1 hr at 37° C., 5% CO2). To evaluate the efficacy of the tested compounds/molecules, cell-associated LukB-FITC fluorescence may be measured as an indicator of LukA/LukB-FITC interaction, using for example, an automated fluorescence microscopic imaging system designed for high content screening and high content analysis (e.g., a Cellomics ArrayScan ECS Reader (Thermo Scientific) (Excitation 485 nm, Emission 535 nm)).
To screen for inhibitors that block or inhibit formation of the LukAB pore, the effector molecule that leads to cell lysis, cells expressing human CD11b (e.g., PMN-HL60 cells) may be plated in 384-well clear-bottom black tissue culture treated plate (Corning) at 2.5×103 cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS) and 50 μM of the ethidium bromide cation dye. LukAB pores facilitate the uptake of this dye. Cells may then be treated with the test compound/molecule ( ̃5 μl containing different concentrations) and then intoxicated with purified LukAB (0.001-2 μM) for 10-20 minutes at 37° C., 5% CO2. Fluorescence, as an indicator of membrane damage, may then be measured using a plate reader such as Envision 2103 Multilabel Reader (Perkin-Elmer). A decrease in fluorescence suggests inhibition of LukAB pores. As controls, PMN-HL60 cells may be treated with culture medium (negative control) and with 0.01% v/v Triton X100 (positive control).
Another aspect of the present invention relates to a method of identifying candidate compounds capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection. This method involves providing a collection of candidate compounds and providing an isolated CD11b receptor or a fragment thereof comprising a LukAB binding domain. The method further involves treating the isolated CD11b receptor or the fragment thereof with an agent comprising a labeled LukA, LukB, and/or labeled LukAB protein and contacting the treated, isolated CD11b receptor or the fragment thereof with one or more candidate compounds from the collection. The binding level of the labeled LukA, LukB, and/or labeled LukAB to the isolated CD11b receptor or fragment thereof is measured in the presence and in the absence of one or more candidate compounds, and the level of LukA, LukB, and/or LukAB binding to the isolated CD11b receptor or fragment thereof in the presence and absence of the one or more candidate compounds is compared. One or more candidate compounds that are capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection are identified based on this comparison.
In accordance with this aspect of the present invention, a decrease in LukA, LukB, and/or LukAB binding to the isolated CD11b receptor or fragment thereof in the presence of the candidate compound compared to in its absence identifies a compound capable of preventing or treating S. aureus infection and/or a condition resulting from a S. aureus infection.
In accordance with this aspect of the present invention, methods of carrying out in vitro ligand binding assays in the presence and in the absence of candidate CD11b inhibitor agents are well known in the art (see e.g., Bansal et al., “Small Molecule Antagonists of Complement Receptor Type 3 Block Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes,” J. Pharm. Exp. Therap. 304(3):1016-24 (2003), which is hereby incorporated by reference in its entirety). These methods typically involve isolation and purification of CD11b or CD11b/CD18 receptor complex from suitable cells, e.g., human PMNs using the method described by Cai et al., “Energetics of Leukocyte Integrin Activation,” J. Biol. Chem. 270:14358-65 (1995) and modified by, Bansal et al., “Small Molecule Antagonists of Complement Receptor Type 3 Block Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes,” J. Pharm. Exp. Therap. 304(3):1016-24 (2003) both of which are hereby incorporated by reference in their entirety. Alternatively, CD11b, a fragment thereof, or CD11b/CD18 can be recombinantly produced. When a peptide or polypeptide of CD11b comprising the LukAB binding domain is utilized in the method of the present invention, the desired peptide or polypeptide can be synthetically produced. This aspect of the present invention further involves purification and labeling of isolated or recombinant LukA, LukB and LukAB proteins. The polynucleotides sequences encoding LukA and LukB and methods of synthesizing or isolating LukA and LukB are described in detail in U.S. Patent Publication No. 2011/0274693 to Torres et al., which is hereby incorporated by reference in its entirety. Finally, methods of measuring labeled LukA, LukB, and/or LukAB binding to the isolated CD11b receptor, fragment thereof, or CD11b/CD18 receptor complex in the presence and absence of a candidate CD inhibitor are fully described in Bansal et al., “Small Molecule Antagonists of Complement Receptor Type 3 Block Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes,” J. Pharm. Exp. Therap. 304(3):1016-24 (2003), which is hereby incorporated by reference in its entirety.
EXAMPLES
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Materials and Methods for Examples 1-7
Cell Culture.
HL60 and HEK293T cells were maintained at 37° C. with 5% CO2 in RPMI and DMEM, respectively, both supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (Mediatech) unless stated otherwise. HL60 cells were differentiated into PMN-HL60 cells with 1.5% dimethyl sulfoxide (DMSO; Sigma Aldrich) for 72 hours at  ̃2.5×105. Transduced HL60 cells were maintained in 2 μg/ml puromycin.
Isolation of Primary Human PMNs.
Blood samples were obtained from anonymous healthy donors as buffy coats (New York Blood Center). The New York City Blood Center obtained written informed consent from all participants involved in the study. PMNs were isolated by Dextran gradient.
His-LukAB Purification from S. aureus. 
To co-purify recombinant LukAB from S. aureus a construct was generated where LukA was fused to an N-terminal 6×-Histidine (His) tag. The construct was generated through multiple cloning steps by first PCR-amplifying the lukAB promoter region and lukA signal sequence from S. aureus Newman genomic DNA where nucleotides encoding a 6×-His tag were added after the lukA signal sequence (ss) using the following primers:









(SEQ ID NO: 5)



5′-CCC GTGTTATTTGATTTCGTTCTATG-3′ 



and


 



(SEQ ID NO: 6)



5′-CCC GTGGTGGTGGTGGTGGTGAGCTGAAT



TTGCTTGAGTCGTTG-3′.






The amplified sequences were cloned into the pOS1 plasmid (Schneewind et al., “Sorting of Protein A to the Staphylococcal Cell Wall,” Cell 70(2):267-281 (1992), which is hereby incorporated by reference in its entirety) using XmaI and BamHI. Then lukB with the lukAB intergenic region was PCR-amplified from S. aureus Newman genomic DNA with the following primers: 5′-CCCGGATCCTCTAGAAAGGGCGGATTACTAATGATTAAAC-3′ (SEQ ID NO: 7) and 5′-CCCCTGCAGTTATTTCTTTTCATTATCATTAAGTAC-3′ (SEQ ID NO: 8). This sequence was cloned into the pOS1 PlukAB-sslukA-6His vector with BamHI and PstI. Finally mature lukA was PCR-amplified with the following primers: 5′-CCCGGATCCCATAAAGACTCTCAAGACCAAAAT-3′ (SEQ ID NO: 9) and 5′-CCCTCTAGATTATCCTTCTTTATAAGGTTTATTG-3′ (SEQ ID NO: 10). This sequence was cloned into the pOS1 PlukAB-sslukA-6His-lukB vector with BamHI and XbaI to yield PlukAB-sslukA-6His-lukA-lukB. Recombinant plasmids were transformed into Escherichia coli CH5α and transformants selected by ampicillin resistance. Positive clones were transformed into S. aureus Newman ΔlukAB (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety).

The protein was purified from S. aureus by growing the strain in tryptic soy broth (TSB) with 10 μg/ml chloramphenicol for 5 hrs at 37° C., 180 rpm to an OD600 of  ̃1.5. The bacteria were then pelleted at 4000 rpm, 4° C. for 15 minutes and the supernatant was collected and filtered through 0.2 μm filters. The culture supernatant was incubated with nickel-NTA resin (Qiagen) in the presence of 10 mM immidazole for 30 minutes at 4° C. with agitation. The sample was applied to a column and washed with tris buffered saline (TBS: 50 mM Tris, 150 mM NaCl, pH 7.5) supplemented with 25 mM imidazole, and eluted with 500 mM imidazole. The protein was dialyzed in 1×TBS+10% glycerol at 4° C. overnight.
Biochemical Studies to Detect the Interaction of LukAB with Mac-1.
For detection of pull-down products with streptavidin, PMN-HL60 cells were incubated with EZ-link sulfo-NHS-LC-Biotin (Thermo Scientific) in cold PBS for 30 minutes at 4° C. with rotation. To quench the reaction cells were then washed with cold 100 mM glycine in cold PBS. The cells were resuspended in cold TBS with EDTA-free protease inhibitor cocktail (Thermo Scientific) and solubilized with 1% N-octyl-β-D-glucopyranoside (Affimetrix) for 30 minutes at 4° C. with rotation. The samples were centrifuged at 15000 rpm, 4° C. for 30 minutes and the supernatant containing the solubilized portion was collected. The solubilized portion (from approximately 2×106 cells) was incubated with 10 μg (5 μg/million cells) of His-LukAB or mock incubated with TBS for 30 minutes at 4° C. with rotation. The samples were incubated with 50 μl of nickel resin in the presence of 10 mM immidazole for 1 hour at 4° C. with rotation. The resin was washed with 1× PBS+50 mM Immidazole and the proteins were eluted with 1×PBS+500 mM Immidazole. The samples were boiled in 4×SDS boiling buffer and run on a 4-15% SDS-PAGE gradient (BioRad) at 80 V, followed by transfer to a nitrocellulose membrane at 30 V for 1 hour. The membrane was blocked with 0.01% tween in PBS for 1 hour, and then incubated with Streptavidin-Dylight 680 (Thermo Scientific) at 1:1000 for 1 hour. The membrane was dried and scanned using an Odyssey infrared imaging system (LI-COR Biosciences).
Pull-downs with PMN-HL60s lysates were also performed with His-tagged LukAB, His-tagged LukED (Alonzo et al., “Staphylococcus aureus Leucocidin ED Contributes to Systemic Infection by Targeting Neutrophils and Promoting Bacterial Growth In Vivo,” Mol. Microbiol. 83(2):423-435 (2012), which is hereby incorporated by reference in its entirety) or His-tagged PVL (Alonzo et al., “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2013), which is hereby incorporated by reference in its entirety) as described above without biotinylation where the samples were run on a 15% SDS-PAGE and transferred to a nitrocellulose membrane at 1 amp for 1 hour. The membrane was probed with an anti-CD11b antibody (clone 23843, R&D Systems), which was detected using an AlexaFluor-680-conjugated anti-rabbit secondary (Invitrogen) antibody diluted 1:25,000, and the Odyssey imaging system.
For the pull-down with purified LukAB and purified Mac-1, 4 μg recombinant Mac-1 (R&D Systems) was incubated with 4 μg of purified recombinant His-LukAB, His-LukED, His-PVL, or PBS in the presence of 0.1% N-octyl-β-D-glucopyranoside for 30 minutes at 4° C. with rotation. The samples were incubated with 100 μl nickel resin, washed, and eluted as described above. The boiled samples in 4× SDS buffer were run on 4-15% gradient gels. One set of samples was processed by immunoblot with an anti-CD11b antibody as described above. For the other set of samples, the gel was stained with the total protein stain Sypro Ruby (Invitrogen) at the manufactures instructions.
PMNs (2×107) were solubilized with 1% N-octyl-β-D-glucopyranoside, the soluble portion was incubated with 20 μg His-LukAB, and complexes were purified with nickel resin as described above. The samples were run on a 4-15% gradient gel and stained with Sypro Ruby. The entire lane was excised from the gel and subjected to mass spectrometry analysis.
Fluorescence Activated Cell Sorting (FACS) Analysis.
Cells were stained with fluorescently-conjugated antibodies for 30 minutes on ice, then washed with 1× PBS+2% FBS+0.05% sodium azide (FACS buffer). For unconjugated anitbodies, cells were stained with primary antibodies antibodies for 30 minutes on ice, washed with FACS buffer, stained for 30 min on ice with fluorescently-conjugated secondary antibody, then washed with FACS buffer. All FACS data were acquired on an LSRII flow cytometer (BD Biosciences) using FACSDiva software. Data were analysed using Flowjo software (Treestar).
Antibodies for FACS Analysis.
Antibodies used for surface staining of primary human cells and human cell lines included the following: anti-CD11b-APC (clone ICRF44), anti-CD18-PE/Cy5 (clone TS1/18), anti-CD11a-FITC (clone HI111), and anti-CD11c-PerCP/Cy5.5 (clone Bu15) (Biolegend). Antibodies for mapping the interaction between LukAB and CD11b included the un-conjugated versions of the human specific antibodies listed above as well as the LM2/1 (Santa Cruz) and CBRM1/5 (BioLegend) anti-CD11b clones. To detect the I-less CD11b, goat anti-CD11b (polyclonal) with anti-goat IgG-APC (R&D Systems) was used.
Antibodies used for surface staining of primary murine cells 293T cells expressing murine Mac-1 included the following: anti-CD11b-APC (clone M1/70), and anti-Ly-6G-FITC (clone 1A8) (BD Pharmingen).
Transfection of HEK293T Cells with CD11b cDNA.
HEK293T cells were incubated with the pCMV6-XL5 plasmid containing full-length human CD11b cDNA (OriGene) or empty vector using Lipofectamine 2000 (Invitrogen) at the manufacturers instructions. Transfection efficiency was between 70-80% as determined with a GFP-producing control vector, and CD11b surface levels were determined 48 hours later by flow cytometry. At this time susceptibility to LukAB or PVL was determined by adding 40 μg/ml of each toxin or PBS to the cells for 2 hours at 37° C., 5% CO2. The cells were then washed and stained with α-CD11b-APC (clone ICRF44). Depletion of CD11b+ cells was measured by flow cytometry where the % of CD11b+ cells with PBS treatment was normalized to 100%.
Generation of the hCD11b I-Less Mutant by Overlap PCR.
Deletion of the I domain from human CD11b was achieved by overlap PCR where a 5′ segment upstream of the I-domain and a 3′ segment downstream of the I-domain were amplified from the pCMV6-XL5 vector containing human CD11b cDNA (OriGene). For amplification of the 5′ segment of CD11b without the 5′UTR but with a Kozak sequence the following primers were used:







(SEQ ID NO: 11)


5′-TGAC CCACCATGGCTCTCAGAGTCCTTCTG-3′ 


and


 


(SEQ ID NO: 12)


5′-GCTGCTACTTCCTGTCTGAGTTTGAGGACACCCTCGGAGG-3′.






For amplification of the 3′ segment of CD11b the following primers were used:










(SEQ ID NO: 13)



5′-CCTCCGAGGGTGTCCTCAAACTCAGACAGGAAGTAGCAGC-3′ 



and


 



(SEQ ID NO: 14)



5′-TTT AGCCCAAGCCCGTCCTGTC-3′.






The two segments were joined by overlap PCR using the following primers: 5′-TGACTCTAGACCACCATGGCTCTCAGAGTCCTTCTG-3′ (SEQ ID NO: 15) and 5′-TTTGCGGCCGCAGCCCAAGCCCGTCCTGTC-3′ (SEQ ID NO: 16). Wild type (WT) human CD11b was also amplified from the OriGene plasmid with this last set of primers. The amplified sequences were cloned into pLenti-CMV-GFP-Puro (Addgene) using XbaI and NotI resulting in the pLenti-CMV-hCD11b-puro and pLenti-CMV-I-less.hCD11b-puro constructs. Recombinant plasmids were transformed into E. coli RecA− 5α (New England BioLabs) and transformants were selected by ampicillin resistance.

Lentivirus-Based Knockdown of Human CD11b and CD18 and Overexpression of CD11b.
Lentiviral shRNA expression vector stocks were produced as described previously (Unutmaz et al., “Cytokine Signals are Sufficient for HIV-1 Infection of Resting Human T Lymphocytes,” J. Exp. Med. 189(11):1735-1746 (1999), which is hereby incorporated by reference in its entirety) by calcium phosphate co-transfection of HEK293T cells with the following plasmids: pMDG gag-pol, pRSV-Rev, pVSV-G Env, and pLKO.1 CD11b or CD18 shRNA constructs purchased from SIGMA MISSION TRC 1.5 library. The following shRNA sequences were used: 5′-CCGGCGCAATGACCTTCCAAGAGAACTCGAGTTCTCTTGGAAGGTCATTGCG TTTTT-3′ (SEQ ID NO: 17) for CD11b and 5′-CCGGGAAACCCAGGAAGACCACAATCTCGAGATTGTGGTCTTCCTGGGTTTC TTTTT-3′ (SEQ ID NO: 18) for CD18. Supernatants were collected 48 hrs later, centrifuged, filtered to remove cell debris, and titered on Jurkat cells as described previously (Unutmaz et al., “Cytokine Signals are Sufficient for HIV-1 Infection of Resting Human T Lymphocytes,” J. Exp. Med. 189(11):1735-1746 (1999), which is hereby incorporated by reference in its entirety). HL60 cells were transduced with the respective viruses or empty vector control virus for 72 hours followed by selection with 2 μg/ml puromycin, which was determined to kill  ̃95-99% of untransduced cells. Surviving cells were expanded knockdown was confirmed by flow cytometry.
Lentiviral expression vector stocks were generated by co-transfecting HEK293T cells with the following plasmids: pMDG gag-pol, pRSV-Rev, pVSV-G Env, and pLenti-CMV-hCD11b-puro or pLenti-CMV-I-less.hCD11b-puro as previously described (Hofmann et al., “The Vpx Lentiviral Accessory Protein Targets SAMHD1 for Degradation in the Nucleus,” J. Virol. 86(23):12552-12560 (2012), which is hereby incorporated by reference in its entirety) using Lipofectamine 2000. Virus was collected and HL60 cells were transduced as described above. Surviving cells were expanded and WT and I-less CD11b surface levels were confirmed by flow cytometry. Cells were sorted using the BD Biosciences FACSAria cell sorter to collect the top 25% of cells staining with an α-CD11b antibody.
Elicitation of Peritoneal Exudate Cells (PECs).
Murine PECs were elicited with heat killed S. aureus as described previously (Alonzo et al., “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2013), which is hereby incorporated by reference in its entirety).
Generation of FITC-LukAB.
To generate recombinant N-terminal fluorescein labeled LukAB, the mature protein coding sequence of LukA from S. aureus Newman genomic DNA was PCR-amplified where a cysteine was added to the N-terminus after the signal sequence using the following primers: 5′-CCCCGGATCCTGTAATTCAGCTCATAAAGACTCTCAAG-3′ (SEQ ID NO: 19) and 5′-CCCTCTAGATTATCCTTCTTTATAAGGTTTATTG-3′ (SEQ ID NO: 20). Amplified sequences were cloned into the PlukAB-sslukA-6His-lukB using BamH1 and Xbal as described above. Recombinant plasmids were transformed into E. coli CH5α and transformants were selected by ampicillin resistance. Positive clones were transformed into S. aureus Newman ΔlukAB (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety). The protein was purified from S. aureus as described above and labeled with 20 fold molar excess Alexa Fluor-488 C5 maleimide (Invitrogen) overnight at 4° C. with agitation. Excess dye was removed through dialysis with 10 kDa molecular weight cutoff dialysis cassettes in TBS with 10% glycerol. Activity of the labeled protein was confirmed by cytotoxicity assays.
Purification of Flag-Tagged CD11b I-Domains from E. coli. 
To generate recombinant human and mouse CD11b I domain with a C-terminal 3× Flag tag and N-terminal 6×-His tag, human and mouse I domain was amplified from the pCMV6-XL5 and pCMV-Entry human and mouse CD11b cDNA constructs (OriGene) respectively. For human I-domain amplification with a C-terminal 6×-glycine linker followed by a 3×-Flag tag the following primers were used:







(SEQ ID NO: 21)


5′-TTT GGATCCAACCTACGGCAGCAG-3′ 


and


 


(SEQ ID NO: 22)


5′-TTT TTACTTGTCATCGTCATCCTTGTAATCGATATCAT


GATCTTTATAATCACCGTCATGGTCTTTGTAGTCTCCTCCTCCTCCTCC


TCCCGCAAAGATCTTCTCCCGAAG-3′.






For murine I-domain amplification with a C-terminal 6×-glycine linker followed by a 3×-Flag tag the following primers were used:








(SEQ ID NO: 23)


5′-TTT GGCTCCAACCTGCTGAGGCC-3′ 


and


 


(SEQ ID NO: 24)


5′-TTT TTACTTGTCATCGTCATCCTTGTAATCGATATCATG


ATCTTTATAATCACCGTCATGGTCTTTGTAGTCTCCTCCTCCTCCTCCTC


CTGCAAAGATCTTTTCCTGAAGCTG-3′.






Amplified sequences were cloned into the pET15b vector (Novagen) with NdeI and XhoI so that the vector-encoded 6×-His tag is at the N-terminus of the I domains. Recombinant plasmids were transformed into E. coli T7 LysY lacQ and transformants and were selected by ampicillin resistance.

To purify the proteins from E. coli, the strains were grown at 37° C., 180 rpm in Luria-Bertani (LB) broth supplemented with 100 μg/ml ampicillin to an OD600 of 0.5, and then induced with 1 mM IPTG for 3 hours at 37° C., 180 rpm. Bacteria were lysed through and lysates were incubated with nickel resin. His-tagged I domains were eluted with 500 mM imidazole.
Dot Blot Analysis to Determine LukAB-CD11b I-Domain Interactions.
5-0.156 μg of purified recombinant human and mouse CD11b I domain were absorbed to PVDF membranes using a dot blot vacuum (BioRad). The membranes were blocked with 2% BSA in 1×TBS for 1 hour followed by incubation with 5 μg/ml purified FITC-LukAB in TBS+2% BSA for 1 hour. For competition assays, 10-fold excess (50 μg/ml) unlabeled purified LukAB or PVL was also incubated with the membranes. Binding of FITC-LukAB was detected using the Odyssey infrared imaging system and quantified by densitometry using the AlphaImager software.
Surface Plasmon Resonance Analysis of LukAB Binding to Mac-1 and CD11b I-Domains.
Surface Plasmon resonance (SPR) was run using the Biacore T100 system (GE) as described previously (Huergo et al., “The Campylobacter Jejuni Dps Protein Binds DNA in the Presence of Iron or Hydrogen Peroxide,” J. Bacteriol. (2013), which is hereby incorporated by reference in its entirety). Briefly, recombinant MAC-1 (R&D Systems), or recombinant I-Domain (mouse and human) were immobilized onto flow cell 2-4 of a series S sensor chip CM5 (GE) using the NHS capture kit, and flow cell 1 was run as a blank immobilization. LukAB and its mutants were run at concentrations ranging from 0.625-25 ug/mL using multi cycle kinetics with at least three experiments performed for each interaction. Single cycle kinetics was utilized to optimize concentrations prior to completion of multi cycle kinetics. The running buffer for all SPR experiments was 1×PBS at pH 6.8.
Cytotoxicity Assays.
Cells were intoxicated as described previously (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety). Briefly, 1×105 cells/well were intoxicated for 1-2 hours at 37° C., 5% CO2 with the indicated concentrations of purified recombinant LukAB. Cell membrane damage, toxin pore formation, or cellular metabolism was evaluated with SYTOX green (Invitrogen), ethidium bromide (MP biomedicals), or CellTiter (Promega) respectively. For experiments with anti-Integrin antibodies, the antibodies were added 30 minutes prior to intoxication at room temperature and were present during the intoxication.
In Vitro and Ex Vivo Infections with S. Aureus. 
These infections were performed as described previously with ΔlukAB, ΔlukAB chromosomally complimented with lukAB (ΔlukAB::lukAB) or the wild type (WT) USA300 clonal type LAC strains. Briefly, to determine killing of PMNs or PMN-HL60s by extracellular S. aureus, normalized USA300 was incubated with 1×105 cells/well at multiplicity of infections (MOIs) of 100, 50, 10, or 1, at 37° C., 5% CO2 for 1-2 hours. For experiments with anti-Integrin antibodies, the antibodies were added 30 minutes prior to infection at room temperature and were present during the infection. Membrane disruption was evaluated using SYTOX green.
To determine growth rebound of phagocytosed S. aureus upon infection with PMN-HL60s, opsonized USA300 was synchronized with 1×105 PMN-HL60s/well at an MOI of 10 through centrifugation. At 30, 60, 120, and 180 min post-synchronization the PMN-H160s were lysed with saponin and serially diluted. Recovered bacteria were determined by counting colony-forming units CFUs.
PMN or PMN-HL60 membrane damage following infection with opsonized S. aureus was also determined by preparing the PMNs and bacteria as described above, where SYTOX green was added at 1-2 hours post synchronization.
Fluorescence Microscopy.
PMNs were infected with opsonized LAC WT, ΔlukAB, and ΔlukAB::lukAB strains transformed with pOS1-PsarA-sodRBS-sgfp to constitutively express GFP.
To determine the location of CD11b in PMNs phagocytosing S. aureus, PMNs were pre-stained with the anti-CD11b-APC (ICRF44) antibody or respective isotype control (mouse IgG1κ-APC, clone MOPC-21, BioLegend) for 30 min on ice. PMNs were then plated at 3×106 cells in 35 mm glass bottom microwell dishes (20 mm microwell, 1.5 thickness, uncoated, MatTek) and synchronized with GFP-USA300 at a MOI of 10. A plate of PMNs was mock infected to detect CD11b staining in the absence of infection. Polyclonal anti-LukA antibody affinity purified from rabbit sera and Lysostaphin (Ambi Products LLC) were added to eliminate the effect of extracellular bacteria. After a 10-minute incubation with lysostaphin at 37° C., 5% CO2 the cells were fixed with 2% paraformaldehyde and 0.1 M lysine in 1×PBS for 30 minutes on ice. The plates were washed with 1×PBS and stored in 1×PBS at 4° C. until imaging. Images were captured using a 60× oil objective on an Applied Precision PersonalDV live-cell imaging system comprised of am Olympus IX-71 inverted microscope, a CoolSnap HQ2 CCD camera, and SoftWorx suite with z-stack capabilities. Images were processed using ImageJ software.
To image GFP-USA300 and ethidium bromide incorporation in the presence of neutralizing antibody, PMNs were pre-treated with anti-CD11b (LM2/1, Santa Cruz) antibody or the respective isotype control (mouse IgG1, Santa Cruz) for 30 minutes at room temperature. PMNs were then infected as described above and images were captured at 0 and 30 minutes post-synchronization using a 40× objective on a Axiovert 40 CFL fluorescent microscope (Zeiss), Axiocam ICc 1 (Zeiss), and the Zen software from Zeiss.
Statistics. Data were analyzed using a one-way ANOVA and Tukey's multiple comparisons post-test (GraphPad Prism version 5.0; GraphPad Software) unless indicated otherwise. Data presented here are from one of at least three independent experiments that gave similar results unless otherwise indicated
Example 1
LukAB Mediates the Cytotoxic Properties of S. Aureus in Human Neutrophils
Human polymorphonuclear cells were exposed to secreted proteins isolated from isogenic wildtype and lukAB mutant (ΔlukAB) methicillin sensitive S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Exposure of the PMNs to secreted proteins from wildtype S. aureus strains resulted in potent cell death as examined by the CellTiter assay (FIG. 1A; black bars). In contrast, exposure of PMNs to secreted proteins from the lukAB mutant strains resulted in significantly reduced cell death. (FIG. 1A; grey bars). The lack of cytotoxic activity exerted by the ΔlukAB strain was reversed by transforming the strain with a plasmid expressing lukAB (ΔlukAB/pLukAB) as shown in FIG. 1A (white bars). These data indicate that LukAB is responsible for the cytotoxic properties of both MSSA and MRSA strains towards human neutrophils.
The importance of LukAB is further supported by the findings that LukAB is critical for S. aureus survival during ex vivo infection of human whole blood and primary human PMNs (FIG. 1B; compare black (WT) and white (ΔlukAB/pLukAB) bars to grey bars (ΔlukAB). Additionally, the contribution of LukAB to S. aureus pathogenesis in vivo using a mouse model of kidney abscess formation has been determined. In this model, mice are challenged with a retro-orbital injection containing S. aureus and then euthanized 96 hours post-infection. Bacterial burden in kidney homogenates are used as a measure of S. aureus pathogenesis. Using this model, an isogenic mutant lacking lukAB exhibits a  ̃100-fold decrease in bacterial burden in renal tissue compared to WT CA-MRSA (FIG. 1C; grey bar). The phenotype of the lukAB mutant was partially complemented by expressing lukAB in trans with a plasmid (FIG. 1C; white bar). Collectively, these data indicate that LukAB is a virulence factor critical for S. aureus pathogenesis.
Experiments with purified recombinant toxin have revealed that LukAB is necessary and sufficient for targeting and killing a variety of human cells including human PMNs, monocytes (both primary and THPI cells), macrophages, and dendritic cells (FIG. 2; see also Dumont et al., “Characterization of a New Cytotoxin that Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-25, which is hereby incorporated by reference in its entirety). In contrast, mast cells (RBL3H3), lymphocytes (Jurkats), epithelial cells (HepG2 and 293T), and red blood cells are highly refractory to LukAB (FIG. 2). In addition, differentiation of the human myeloid cell line HL60 into PMN-like cells (“PMN-HL60”) renders these cells highly susceptible to LukAB cytotoxicity (FIG. 2).
The data presented above indicate that LukAB targets and kills human phagocytes (FIG. 2). Importantly, experiments employing primary murine neutrophils have revealed that LukAB appears to be human specific, since no or little cytotoxicity is observed when secreted proteins isolated from isogenic wildtype S. aureus are incubated with murine cells (FIG. 3). These data have tremendous implications because they suggest that murine models of infection do not fully measure the contribution of LukAB, which based on the studies with human cell lines, is the most important leukotoxin produced by S. aureus. Thus, identification of the cellular determinant(s) that render human phagocytes susceptible to LukAB would enable the generation of transgenic animals that would better represent the pathobiology of S. aureus infection in humans.
Example 2
LukAB Directly Interacts with Integrin αM/β2 (Mac-1 or CR3)
To identify host proteins that interact with LukAB, a pull-down assay was performed with PMN-HL60 cells, which are short-lived neutrophil-like cells differentiated from the HL60 myeloid cell line that are extremely sensitive to LukAB (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety). The lysates were incubated with a His-tagged LukAB and a nickel column was used to isolate toxin-host protein complexes. The surface proteins on the PMN-HL60 cells were biotinylated prior to incubation with LukAB so that the host proteins could be visualized using fluorescently conjugated streptavidin (FIG. 4A). By employing this technique, a large number of host proteins were observed to be associated with LukAB (FIG. 4A). The pull-down was repeated with primary human PMNs isolated from human blood without biotinylation, and the identity and quantity of the cellular factors enriched in the pull-down with LukAB were determined by mass spectrometry. The most abundant LukAB-interacting cellular surface proteins were CD18 and CD11b (Table 1), which are respectively the α and β components of the integrin complex known as integrin αM/β2, CR3, or Mac-1, herein referred to as Mac-1. The association of LukAB, but not LukED or PVL, with CD11b was confirmed by immunoblot with a CD11b specific antibody (FIG. 4B). A specific and direct interaction between LukAB and Mac-1 was established when a pull-down was conducted with purified recombinant toxin and purified receptor. A total protein stain revealed that LukAB, but not LukED or PVL, could pull down both the CD11b and CD18 subunits of the purified Mac-1 complex which are about 150 and 95 kDa respectively (FIG. 4C). Immunoblot further validated the presence of CD11b in the pull-down with LukAB but not the other toxins (FIG. 4D).







TABLE 1







Mass spectometry analysis of LukAB-interacting cellular factors












Molecular
# of


Identified Proteins (≧3 peptides)
Accession Number
Weight
peptides













Integrin alpha-M
ITAM_HUMAN
127 kDa 
17


Serum albumin
ALBU_HUMAN (+2)
69 kDa
13


Integrin beta 2
B4E0R1_HUMAN (+1)
77 kDa
12


Cntm_P13645
Cntm_P13645
24 kDa
10


Pyruvate kinase isozymes M1/M2
KPYM_HUMAN
58 kDa
9


cDNA FLJ78440
A8K494_HUMAN (+2)
78 kDa
8


Plastin-2
PLSL_HUMAN (+2)
70 kDa
8


Myeloperoxidase
PERM_HUMAN
84 kDa
7


Actin
ACTG_HUMAN (+1)
42 kDa
7


cDNA FLJ55635
B4E0S6_HUMAN (+1)
90 kDa
7


Glyceraldehyde-3-phosphate dehydrogenase
G3P_HUMAN (+2)
36 kDa
6


Coronin-1A
COR1A_HUMAN
51 kDa
6


Bactericidal permeability-increasing protein
BPI_HUMAN
54 kDa
6


cDNA FLJ55493
B4DMF5_HUMAN (+3)
57 kDa
5


Protein S100-A8
S10A8_HUMAN
11 kDa
5


Mitogen-activated protein kinase 1
MK01_HUMAN
41 kDa
5


cDNA FLJ75120
A8K345_HUMAN (+1)
42 kDa
5


Hemoglobin subunit beta
HBB_HUMAN
16 kDa
5


Adipocyte plasma membrane-associated
APMAP_HUMAN
46 kDa
5


protein


ATP synthase subunit beta
A8K4X0_HUMAN (+2)
57 kDa
5


Putative uncharacterized protein LCN2
A6NII8_HUMAN (+7)
23 kDa
5


Heat shock 70kDa protein 1A
A8K5I0_HUMAN (+1)
70 kDa
5


Malic enzyme
B2R8J2_HUMAN (+1)
65 kDa
5


Endoplasmic reticulum resident protein
ERP44_HUMAN
47 kDa
4


ERp44


Peptidoglycan recognition protein
PGRP_HUMAN
22 kDa
4


SFPQ protein
Q6PIX2_HUMAN (+3)
55 kDa
4


Hemoglobin subunit alpha
HBA_HUMAN (+1)
15 kDa
4


Adenylyl cyclase-associated protein
B2RDY9_HUMAN (+4)
52 kDa
4


Transketolase
A8K089_HUMAN (+6)
68 kDa
4


cDNA, FLJ92148
B2R4M6_HUMAN (+1)
13 kDa
4


cDNA FLJ76817
A8K525_HUMAN (+2)
54 kDa
3


CD63 antigen
CD63_HUMAN (+1)
26 kDa
3


Alpha-enolase
ENOA_HUMAN
47 kDa
3


E3 ubiquitin-protein ligase CBL
CBL_HUMAN
100 kDa 
3


Guanine nucleotide-binding protein G(i)
GNAI2_HUMAN (+1)
40 kDa
3


cDNA FLJ53963
B4E3A8_HUMAN (+1)
39 kDa
3


6-phosphogluconate dehydrogenase
6PGD_HUMAN (+3)
53 kDa
3


Catalase
CATA_HUMAN
60 kDa
3


Glucose-6-phosphate isomerase
G6PI_HUMAN (+3)
63 kDa
3


Myosin-9
MYH9_HUMAN (+1)
227 kDa 
3


Beta-adrenergic receptor kinase 1
ARBK1_HUMAN
80 kDa
3


Protein tyrosine phosphatase
B1ALS2_HUMAN (+2)
147 kDa 
3


Alpha-actinin-1
ACTN1_HUMAN (+5)
103 kDa 
3









In order to better characterize the direct interaction of LukAB with Mac-1 surface plasmon resonance (SPR) analysis was performed, which indicated that LukAB binds to Mac-1 in a dose-dependent and saturable manner resulting in a dissociation constant (Kd) of approximately 38.4 nM (Table 2).







TABLE 2







SPR analysis of LukAB/Mac-1 interactions










Protein + LukAB
Disassociation constant (Kd)







human recombinant Mac-1
3.84 × 10−8M (±2.61 × 10−8)



human recombinant I domain
1.92 × 10−9M (±1.13 × 10−9)



murine recombinant I domain
1.06M (±0.89)










Example 3
The CD11b Subunit of Mac-1 is Necessary and Sufficient to Render Cells Susceptible to LukAB
In order to provide a link between the susceptibility of cells to LukAB and Mac-1, HL60 cells were transduced with viruses containing non-targeting shRNA (NT shRNA) or with CD18 shRNA. To enhance the susceptibility of these cells to LukAB, the stably-transduced HL60 cell lines were differentiated to PMN-HL60s (FIG. 5A), and the effect of the shRNAs on the cell surface levels of CD18 and CD11b were confirmed by flow cytometry (FIG. 5B). Compared to NT shRNA cells, the CD18 shRNA cells were markedly depleted of CD18 (FIG. 5B). Because CD18 is required for the stability and surface localization of all the integrin a subunits (Weber et al., “Characterization of Lymphocyte Function-Associated Antigen 1 (LFA-1)-Deficient T Cell Lines: The AlphaL and Beta2 Subunits are Interdependent for Cell Surface Expression,” J. Immunol. 158(1):273-279 (1997); Springer et al., “Inherited Deficiency of the Mac-1, LFA-1, p150,95 Glycoprotein Family and Its Molecular Basis,” J. Exp. Med. 160(6):1901-1918 (1984), which are hereby incorporated by reference in their entirety), CD11b was also depleted in the CD18 shRNA cells (FIG. 5B). Thus, by targeting CD18 a Mac-1 depleted cell line was generated. Intoxication of the CD18 shRNA cells with purified LukAB revealed that Mac-1 is required for the formation of LukAB pores (FIG. 5C). In contrast, PVL formed pores in a Mac-1 independent manner, indicating that LukAB and PVL exploit different cellular determinants to exert their cytotoxicity (FIG. 5C).
In addition to Mac-1, PMN-HL60s are decorated with CD11a/CD18 (LFA) and CD11c/CD18 (p150/95), and depletion of CD18 resulted in a reduction in the surface levels of these β2 integrins as well (FIG. 6). To ensure that the knockdown of the Mac-1 complex was responsible for the increased resistance to LukAB and not the general knockdown of β2 integrins, HL60 cells were stably transduced with CD11b-targeting shRNA. This strategy resulted in marked depletion of CD11b with no notable effect on CD18 levels (FIG. 5D). In fact, the Mac-1 levels observed on the cells transduced with the CD11b-targeting shRNA resembled those of the parental HL60 cells (FIG. 5D). Depletion of CD11b rendered the cells resistant to LukAB pores, but not to PVL pores (FIG. 5E). These findings demonstrate that CD11b is crucial for rendering cells susceptible to LukAB.
To determine whether CD11b is sufficient to render cells susceptible to LukAB, a gain of function experiment was performed. It has been shown that HEK293T cells can support CD11b surface localization in the absence of CD18 (Solovjov et al., “Distinct Roles for the Alpha and Beta Subunits in the Functions of Integrin AlphaMbeta2,” J. Biol. Chem. 280(2):1336-1345 (2005), which is hereby incorporated by reference in its entirety). Therefore, these cells were transiently transfected with either a plasmid encoding CD11b or an empty plasmid, and CD11b surface levels were determined via flow cytometry (FIG. 5F). Intoxication of these cells with LukAB, but not PVL, resulted in depletion of the majority (80-90%) of the CD11b+ HEK293T cells, confirming that CD11b is necessary and sufficient to render cells susceptible to LukAB (FIG. 5F).
Example 4
The I-domain of CD11b is Required for LukAB-Mediated Toxicity Towards Target Cells
Whether LukAB cytotoxicity could be blocked with CD11b specific antibodies was examined. Prior to intoxication with LukAB, primary PMNs were pre-treated with three different antibodies targeting CD11b, as well as antibodies against CD18, CD11a, and CD11c. Although all three CD11b antibodies and the CD18 antibody displayed some degree of blocking LukAB toxicity, only the LM2/1 CD11b antibody significantly inhibited LukAB activity when compared to an untreated cells or an isotype control (FIGS. 7A-7B).
The LM2/1 antibody recognizes the CD11b I-domain (or A-domain), which is where most endogenous Mac-1 ligands bind through a metal ion-dependent adhesion site (MIDAS) (Arnaout et al., “Integrin Structure, Allostery, and Bidirectional Signaling,” Annu. Rev. Cell Dev. Biol. 21:381-410 (2005), which is hereby incorporated by reference in its entirety). Based on the LM2/1 blocking data, it was hypothesized that the I-domain of CD11b was required for LukAB-mediated killing of target cells. To address this possibility, a mutated CD11b was constructed where the I-domain was deleted using overlap PCR as previously described (Yalamanchili et al., “Folding and Function of I Domain-Deleted Mac-1 and Lymphocyte Function-Associated Antigen-1,” J. Biol. Chem. 275(29):21877-21882 (2000), which is hereby incorporated by reference in its entirety). It has been established that the deletion of the I-domain does not affect the interaction of CD11b with CD18 or the interaction between Mac-1 and endogenous ligands that do not require the I-domain (Yalamanchili et al., “Folding and Function of I Domain-Deleted Mac-1 and Lymphocyte Function-Associated Antigen-1,” J. Biol. Chem. 275(29):21877-21882 (2000), which is hereby incorporated by reference in its entirety). HL60 cells were transduced with virus made from constructs containing wild type (WT) CD11b, I-less CD11b, or an empty vector control. These cells were chosen because they are highly resistant to LukAB and have low levels of CD11b (FIGS. 5A and 5D). If the I-domain is necessary for cytotoxicity, exogenous WT CD11b would render these cells as susceptible as PMN-HL60 cells, where as an I-less version of CD11b would not. Following transduction and stable integration, the levels of CD11b on the surface of the HL60 cell lines was evaluated by flow cytometry with an α-CD11b antibody that recognizes both the WT and I-less versions of CD11b (FIG. 7C). Both WT and I-less CD11b were surface exposed at levels comparable to or higher than that of PMN-HL60 cells (FIG. 7C). Exogenous WT CD11b rendered HL60 cells susceptible to LukAB as evidenced by increased membrane damage and cell death compared to the empty vector control HL60 cells (FIGS. 7D-7E). The level of susceptibility exhibited by the HL60 cells with exogenous WT CD11b was comparable to differentiated PMN-HL60 cells. In contrast, HL60 cells with exogenous I-less CD11b were highly resistant to LukAB-mediated cytotoxicity despite having equivalent levels of surface CD11b to PMN-HL60 cells (FIG. 7C-7E).
Example 5
LukAB Displays Higher Affinity for Human CD11b I-Domain Compared to Murine CD11b I-Domain
Purified LukAB has been shown to be highly cytotoxic towards human and monkey PMNs, intermediately toxic towards rabbit PMNs, and least toxic towards murine PMNs (FIG. 3) (Malachowa et al., “Staphylococcus aureus Leukotoxin GH Promotes Inflammation,” J. Infect. Dis. 206(8):1185-1193 (2012), which is hereby incorporated by reference in its entirety). These findings suggest that LukAB targets blood-purified PMNs in a species-specific manner. Murine peritoneal exudate cells (PECs), which are highly susceptible to LukED, are resistant to LukAB (FIG. 8A). PECs mostly consist of recruited PMNs (Ly6G+/CD11b+), and monocytes and macrophages (Ly6G−/CD11b+), all of which have high levels of surface CD11b (FIG. 8B).
In view of the species specificity of LukAB together with the necessity of the CD11b I-domain for toxin activity (FIG. 7C-7D), the conservation of this domain from different species was examined. Alignment of the amino acid sequences of the human, gorilla, rabbit, and mouse CD11b I-domains revealed that as expected, gorilla is the most similar to human (98.6% identity), followed by rabbit (79.1% identity), and then murine (78.1% identity) (FIG. 8C). These data correlate with the tropism of LukAB towards PMNs from these species (Malachowa et al., “Staphylococcus aureus Leukotoxin GH Promotes Inflammation,” J. Infect. Dis. 206(8):1185-1193 (2012), which is hereby incorporated by reference in its entirety). To investigate if these differences could influence LukAB binding to the CD11b I-domain, a dot blot assay to detect LukAB-CD11b I-domain interaction was developed. A dose-dependent interaction between fluorescently labeled LukAB and the human CD11b I-domain was observed, which was competed off with excess unlabeled LukAB but not with unlabeled PVL (FIG. 8D). Comparison of LukAB binding to human versus murine CD11b I-domain using this assay revealed that LukAB preferentially binds to the human CD11b I domain (FIG. 8E). SPR analysis revealed that LukAB binds to the human CD11b I-domain with an approximate Kd of 1.92 nM,  ̃8-9 logs lower than that of the LukAB-murine CD11b I-domain interaction at 1.06 M (Table 1).
Example 6
Extracellular S. Aureus Utilize CD11b to Cause LukAB-Mediated Cell Damage During Infection
To establish a role for CD11b in S. aureus infections the NT or CD11b shRNA PMN-HL60 cells were infected with the CA-MRSA USA300 strain LAC or an isogenic mutant lacking LukAB (ΔlukAB). WT USA300 killed the NT PMN-HL60 cells in a LukAB-dependent manner (FIG. 9A). In contrast, when CD11b surface levels are reduced in these cells by shRNA (CD11b), the WT USA300 no longer causes cell damage and instead resembles the lukAB mutant strain (FIG. 9A).
Ex vivo infection of purified human PMNs with the USA300 strain was performed, and whether LukAB-mediated cell damage could be blocked through pre-treatment with anti-CD11b antibodies prior to infection was tested. These experiments revealed that the anti-I-domain LM2/1 antibody successfully neutralized USA300-mediated cell damage (FIG. 9B), thus establishing a role for LukAB-mediated targeting of the CD11b I-domain during S. aureus-PMN interaction.
Example 7
Phagocytosed S. Aureus Exploits LukAB-Mediated Targeting of CD11b to Cause Cell Damage and Promote Escape from within
It was recently established that LukAB-mediated cell damage post-phagocytosis promotes the early escape of USA300 from within PMNs and subsequent USA300 outgrowth. To determine if CD11b contributes to the intracellular cytotoxic activity of LukAB, the NT and CD11b shRNA PMN-HL60 cells were infected with opsonized USA300 and synchronized to promote phagocytosis. Importantly, depletion of CD11b did not influence phagocytosis of USA300 (FIGS. 10A-10B). Under these conditions, knockdown of CD11b abolished cell damage caused by the WT USA300 (FIG. 11A).
These experiment revealed that, phagocytosed USA300 employs LukAB to prevent PMN-HL60-mediated growth restriction (FIG. 11B). However, knockdown of CD11b eliminated the growth advantage of WT USA300 compared to the ΔlukAB mutant strain (FIG. 11B).
In order for CD11b to be utilized by phagocytosed S. aureus to escape from within PMNs, CD11b must be present in the phagosomal membrane surrounding S. aureus. To determine the location of CD11b during phagocytosis of S. aureus, human PMNs were pre-stained with a fluorescently labeled α-CD11b antibody or a fluorescently labeled isotype control, followed by infection with GFP-USA300. Infected cells were fixed post synchronization and imaged using an Applied Precision Personal DV live-cell imaging system with z-stack capability. In uninfected human PMNs the CD11b staining is dispersed across the plasma membrane of the cell (FIG. 11C). However, upon infection with USA300, CD11b was found to be associated with the phagocytosed GFP-USA300 (FIG. 11C).
Neutralizing LM2/1 anti-CD11b antibody was used in an attempt to block the LukAB-mediated PMN damage caused by phagocytosed USA300. For these experiments, PMNs were pretreated with the LM2/1 antibody or an isotype control prior to infection with GFP-USA300 WT, isogenic ΔlukAB, or isogenic ΔlukAB chromosomally complemented with lukAB. These experiments were performed in the presence of lysostaphin and anti-LukA to eliminate extracellular bacteria and the potential contribution of extracellular LukAB, as well as the fluorescent dye ethidum bromide to measure pore-formation. Of note, pre-treatment with LM2/1 prior to infection does not block phagocytosis of S. aureus as the amount of GFP-USA300 observed within PMNs was similar regardless of LM2/1 treatment (FIG. 12). Phagocytosed USA300 causes LukAB-mediated pore formation at 30 minutes post synchronization when PMNs are pre-treated with isotype control antibody (FIG. 11D). In contrast, LM2/1 pre-treatment resulted in decreased LukAB-mediated pore formation (FIGS. 11D-11E), mimicking the phenotype observed with the lukAB mutant strain.
Discussion of Examples 1-7
This study describes the identification of CD11b of the Mac-1 integrin as a cellular molecule exploited by the staphylococcal leukotoxin LukAB to specifically target and kill cells. This conclusion is supported by the findings that LukAB directly interacts with the Mac-1 complex (specifically the I-domain of CD11b), and CD11b is necessary and sufficient to render cells susceptible to LukAB as evidenced by knockdown and gain of function analyses.
The identification of a cellular target that is specifically utilized by LukAB and not other bi-component toxins such as LukED and PVL highlights that the staphylococcal leukotoxins possess non-redundant mechanisms for targeting specific cell types. CCR5 was recently identified as a cellular receptor utilized by LukED to target and kill lymphocytes, macrophages and dendritic cells (Alonzo et al., “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2013), which is hereby incorporated by reference in its entirety). However, monocytes and PMNs are killed by LukED in a CCR5-independent manner suggesting that additional cellular receptors may be utilized by LukED to target these cells (Alonzo et al., “CCR5 is a Receptor for Staphylococcus aureus Leukotoxin ED,” Nature 493(7430):51-55 (2013), which is hereby incorporated by reference in its entirety). The fact that a single staphylococcal toxin may target multiple receptors and that each toxin may utilize distinct non-redundant receptors vastly increases the number of cell types that S. aureus can eliminate with an already extensive repertoire of toxins.
The targeted killing of innate immune cells such as PMNs is crucial to the pathogenesis of S. aureus as well as a number of other human pathogens. Mac-1 is expressed on all of the cells targeted by LukAB (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety) including PMNs, macrophages, monocytes, and dendritic cells (Ho & Springer, “Mac-1 Antigen: Quantitative Expression in Macrophage Populations and Tissues, and Immunofluorescent Localization in Spleen,” J. Immunol. 128(5):2281-2286 (1982), which is hereby incorporated by reference in its entirety), and is involved in multiple cellular functions such as phagocytosis, cellular activation, cell-mediated killing and chemotaxis (Solovjov et al., “Distinct Roles for the Alpha and Beta Subunits in the Functions of Integrin AlphaMbeta2,” J. Biol. Chem. 280(2):1336-1345 (2005); Hynes R. O., “Integrins: Bidirectional, Allosteric Signaling Machines,” Cell 110(6):673-687 (2002), which are hereby incorporated by reference in their entirety). The present study demonstrates that both extracellular S. aureus and phagocytosed S. aureus employ LukAB to cause PMN damage during infection by targeting CD11b. The finding that CD11b surrounds phagocytosed S. aureus, links CD11b to the LukAB-mediated escape of S. aureus from the phagosome.
The identification of human CD11b I-domain as a cellular target of LukAB provides an explanation for the observed species specificity exhibited by this toxin. The affinity of LukAB toward the murine CD11b I-domain is  ̃8-9 logs less than that observed towards the human CD11b I-domain, which correlates to the previously reported susceptibility of murine PMNs (Malachowa et al., “Staphylococcus aureus Leukotoxin GH Promotes Inflammation,” J. Infect. Dis. 206(8):1185-1193 (2012), which is hereby incorporated by reference in its entirety). The difference in binding affinity is most likely explained by the divergent sequence homology between the I-domains from these two species based on amino acid sequence alignments, which yielded a 78.1% identity between the two I-domains. Of note, it was observed that USA300 expresses lukAB in vivo in murine abscess, and that the toxin contributes to both the infection process and the bacterial burden in a murine renal abscess model (Dumont et al., “Characterization of a New Cytotoxin That Contributes to Staphylococcus aureus Pathogenesis,” Mol. Microbiol. 79(3):814-825 (2011), which is hereby incorporated by reference in its entirety). Even though LukAB plays a role in this murine model of renal abscess formation, the marked resistance of mouse PMNs to this toxin compared to human PMNs suggests that mouse models underestimate the true contribution of LukAB to S. aureus pathobiology in humans. The species-specific activities of an expanding number of virulence factors produced by S. aureus (e.g. superantigens, CHIPS, PVL, LukAB) (Vandenesch et al., “Staphylococcus aureus Hemolysins, Bi-Component Leukocidins, and Cytolytic Peptides: A Redundant Arsenal of Membrane-Damaging Virulence Factors?” Front Cell Infect. Microbiol. 2:12 (2012); Rooijakkers et al., “Staphylococcal Innate Immune Evasion,” Trends Microbiol. 13(12):596-601 (2005), which are hereby incorporated by reference in their entirety) highlight the limitations of the animal models currently employed to study S. aureus pathogenesis. Thus, improved animal models are paramount for understanding the full virulence potential of S. aureus, which is a prerequisite for the development of effective drugs that can combat this important human pathogen.
Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

## Claims

1. A method of treating Staphylococcus aureus infection and/or a condition resulting from a S. aureus infection in a subject comprising:
selecting a subject having or at risk of having S. aureus infection and
administering a CD11b inhibitor to the selected subject under conditions effective to treat S. aureus infection and/or a condition resulting from a S. aureus infection in the subject, wherein the CD11b inhibitor is selected from (i) a recombinant soluble polypeptide comprising the I-domain of CD11b, and (ii) an antibody that binds to the I-domain of CD11b.

2. The method of claim 1, wherein the S. aureus infection is a methicillin-resistant S. aureus (MRSA) infection or a methicillin sensitive S. aureus (MSSA) infection.

3. The method of claim 1, wherein the recombinant soluble polypeptide comprises an amino acid sequence corresponding to residues 147-337 of SEQ ID NO: 2.

4. The method of claim 1 further comprising
administering, to the selected subject in conjunction with the CD11b inhibitor, an agent selected from the group consisting of an anti-infective agent, an antibiotic agent, and an antimicrobial agent.

5. The method of claim 1, wherein a condition resulting from S. aureus infection is treated, said condition being selected from the group consisting of skin wounds and infections, tissue abscesses, folliculitis, osteomyelitis, pneumonia, scalded skin syndrome, septicemia, septic arthritis, myocarditis, endocarditis, and toxic shock syndrome.

6. The method of claim 1, wherein said administering is carried out orally, by inhalation, by intranasal instillation, topically, transdermally, parenterally, subcutaneously, intravenous injection, intra-arterial injection, intramuscular injection, intraplurally, intraperitoneally, or by application to a mucous membrane.

7. The method of claim 1 further comprising:
repeating said administering.

8. The method of claim 1, wherein the subject is an infant, a juvenile, or an adult.

9. The method of claim 1, wherein the subject is an immunocompromised infant, juvenile, or adult.