Method for treating microbial infections

A method for treating a microbial infection or an abscessed tissue in a subject includes administering to the subject an effective amount of a metal ion chelator. In some embodiments, the metal ion chelator can be a protein, such as a calprotectin heterodimer. In some embodiments, the metal ion chelator is a calprotectin heterodimer including an S100A8 polypeptide and an S100A9 polypeptide.

TECHNICAL FIELD

The presently-disclosed subject matter relates to treatment of microbial infections. In particular, the presently-disclosed subject matter relates to treatment of abscessed tissue caused by microbial infections.

BACKGROUND

Undesirable infections can be caused by a variety of microbes, including microbes such as fungi and antibacterial-resistant bacteria that are notoriously difficult to treat. Such microbial infections are sometimes capable of causing an abscess in a subject. An abscess is a localized accumulation of pus and/or inflammation in a tissue or organ. An abscess can form, for example, on the skin or on other tissues or organs of the subject, causing swelling and pain.

Traditional treatment for abscesses includes piercing or cutting open the abscess to remove the puss and infectious material, and treatment with antibiotics, e.g., oral, topical, injectable. Depending on the size and the location of the abscess, such traditional treatment can be successful; however, for certain abscesses, e.g., relatively large abscesses, and/or abscesses caused by antibiotic-resistant bacteria or otherwise resistant microbes, such traditional treatment can be insufficiently effective.

Accordingly, there remains a need in the art for effective methods for treating microbial infections and abscessed tissues.

SUMMARY

The presently-disclosed subject matter includes a method for treating a microbial infection or an abscessed tissue in a subject. The method includes administering to the subject an effective amount of a metal ion chelator.

In some embodiments, the metal ion chelator is a Mn2+chelator. In some embodiments, the metal ion chelator is a Mn2+and Zn2+chelator.

In some embodiments, the metal ion chelator is a protein metal ion chelator. In some embodiments, the protein metal ion chelator is a calprotectin heterodimer, or a functional fragment or a functional variant of a calprotectin heterodimer. In some embodiments, the calprotectin heterodimer is bound with Ca2+before administration.

In some embodiments, the calprotectin heterodimer can include an S100A8, or functional fragment or functional variant thereof, and an S100A9 polypeptide, or functional fragment or functional variant thereof. In some embodiments, the S100A8 polypeptide can include the amino acid sequence of SEQ ID NO: 1. In some embodiments, the S100A8 polypeptide can include the amino acid sequence of SEQ ID NO: 3. In some embodiments, the S100A9 polypeptide can include the amino acid sequence of SEQ ID NO: 2. In some embodiments, the S100A9 polypeptide can include the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the calprotectin heterodimer can include the S100A8 polypeptide including the amino acid sequence of SEQ ID NO: 1, and the S100A9 polypeptide including the amino acid sequence of SEQ ID NO: 2. In some embodiments, the calprotectin heterodimer can include the S100A8 polypeptide including the amino acid sequence of SEQ ID NO: 3, and the S100A9 polypeptide including the amino acid sequence of SEQ ID NO: 4.

In some embodiments in which the calprotectin heterodimer includes a functional fragment or functional variant of the S100A8 polypeptide and/or the S100A9 polypeptide, both the S100A8 polypeptide and the S100A9 polypeptide include a preserved metal ion binding motif.

In some embodiments, the method is for treating a microbial infection or an abscessed tissue caused by a bacterial pathogen. In some embodiments, the bacterial pathogen is an antibiotic-resistant strain of a bacterial pathogen. In some embodiments, the bacterial pathogen is selected from:Bacillus anthracis, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumonia, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andAcinetobacter baumannii. In some particular embodiments, the bacterial pathogen is aStaphylococcus. In some particular embodiments, the bacterial pathogen isStaphylococcus aureus.

In some embodiments, the method is for treating a microbial infection or an abscessed tissue caused by a fungal pathogen. In some embodiments, the fungal pathogen is selected from:Candida albicans, Toxoplasma gondii, andCryptococcus neoformans.

In some embodiments of the method for treating a microbial infection or an abscessed tissue, the metal ion chelator is administered before the microbial infection or the abscessed tissue occurs in the subject. In some embodiments, the metal ion chelator is administered after the microbial infection or the abscessed tissue occurs in the subject.

In some embodiments, the metal ion chelator is administered to the abscessed tissue. In some embodiments, the metal ion chelator is administered at or near a site of infection. In some embodiments, the metal ion chelator is administered at or near a site associated with a risk of infection. In some embodiments, the metal ion chelator is administered topically. In some embodiments, the metal ion chelator is administered by injection.

The presently-disclosed subject matter includes a kit for use in treating a microbial infection or an abscessed tissue. In some embodiments, the kit includes a vial containing a metal ion chelator, and instructions for treating a microbial infection or an abscessed tissue in a subject. In some embodiments, the metal ion chelator is a calprotectin heterodimer. In some embodiments, the calprotectin heterodimer is Ca2+-bound. In some embodiments, the kit further includes a vial containing a Ca2+-containing buffer.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a human S100A8 polypeptide.

SEQ ID NO: 2 is a human S100A9 polypeptide.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Some of the polynucleotide and polypeptide sequences disclosed herein are cross-referenced to GENBANK® accession numbers. The sequences cross-referenced in the GENBANK® database are expressly incorporated by reference as are equivalent and related sequences present in GENBANK® or other public databases. Also expressly incorporated herein by reference are all annotations present in the GENBANK® database associated with the sequences disclosed herein. Unless otherwise indicated or apparent, the references to the GENBANK® database are references to the most recent version of the database as of the filing date of this Application.

While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently-disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.

The presently-disclosed subject matter includes methods for treating a microbial infection and/or treating an abscessed tissue in a subject, including administering to the subject an effective amount of a metal ion chelator.

As used herein, the term “microbial infection” refers to an infection by a microbe. The term “microbe” refers to a non-viral pathogen, including all bacterial pathogens and all fungal pathogens. The term microbial infection further refers to antibiotic-resistant strains of bacterial pathogens.

As used herein, the term “bacterial pathogen” refers to a bacteria capable of causing infection in a subject. In some embodiments, a bacterial pathogen is capable of causing an abscessed tissue. Examples of bacterial pathogens include, but are not limited to, gram positive bacterial pathogens including,Bacillus anthracis, Enterococcus faecalis, Staphylococcus aureus, andStreptococcus pneumonia; gram negative bacterial pathogens including,Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, andAcinetobacter baumannii.

As used herein when referring to a bacterial pathogen, the term “antibiotic-resistant strain” refers to a bacterial pathogen that is capable of withstanding an effect of an antibiotic used in the art to treat the bacterial pathogen (i.e., a non-resistant strain of the bacterial pathogen). For example,Staphylococcus aureuscan be treated using methicillin; however, an antibiotic-resistant strain ofStaphylococcus aureusis methicillin-resistantStaphylococcus aureus(MRSA).

As used herein, the term “fungal pathogen” refers to a fungus capable of causing infection in a subject. In some embodiments, a fungal pathogen is capable of causing an abscessed tissue. Examples of fungal pathogens include, but are not limited to,Candida albicans, Toxoplasma gondii, andCryptococcus neoformans.

The terms “abscessed tissue” and “abscess” refers to a localized accumulation of pus and/or inflammation in a tissue or organ, e.g., skin, that is caused by a microbial infection.

As used herein, the terms “treatment” or “treating” relate to any treatment of a microbial infection, or an abscessed tissue caused by a microbial infection, including therapeutic, (i.e., post-infection), and prophylactic treatment, (i.e., pre-infection). Although most commonly therapeutic treatment will be affected, prophylactic treatment can be useful in cases that will be recognized by one of ordinary skill in the art, for example, prophylactic treatment to prevent surgical site infection. As such, the terms treatment or treating include, but are not limited to: preventing a microbial infection or an abscess or the development of a microbial infection or an abscess; inhibiting the progression of a microbial infection or an abscess; arresting or preventing the development of a microbial infection or an abscess; reducing the severity of a microbial infection or an abscess; ameliorating or relieving symptoms associated with a microbial infection or an abscess; and causing a regression of a microbial infection or an abscess or one or more of the symptoms associated with a microbial infection or an abscess. In some cases, in addition to making use of the methods described herein, it can be desirable to make use of traditional and other known treatment protocols such as surgical drainage of an abscess. Although such traditional and other known treatment protocols will not be described in any detail herein, they will be known and understood by those skilled in the art, and it is contemplated that such traditional and other known treatment protocols can be used in combination with the methods and compositions described herein, if desired.

As used herein, the term “subject” refers to humans, and other animals. Thus, veterinary treatment is provided in accordance with the presently-disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.

In accordance with the methods of the presently-disclosed subject matter, a metal ion chelator can be administered to treat the microbal infection and/or abscessed tissue of the subject.

As used herein the term “metal ion chelator” refers to an agent capable of sequestering metal ions. In some embodiments, the metal ion chelator can be a manganese ion (Mn2+) chelator, capable of sequestering Mn2+. In some embodiment the metal ion chelator is a manganese ion (Mn2+) chelator and a zinc ion (Zn2+) chelator, capable of sequestering both Mn2+and Zn2+. Without wishing to be bound by theory or mechanism, it is believed that the metal ion chelators of the presently-disclosed subject matter bind Mn2+, depriving the microbe of this growth factor; in the absence of available Mn2+the microbe becomes unable to survive.

In some embodiments, the metal ion chelator is a polypeptide capable of sequestering Mn2+(or Mn2+and Zn2+), i.e., a polypeptide or protein metal ion chelator. In some embodiments, the polypeptide is an isolated polypeptide capable of sequestering Mn2+(or Mn2+and Zn2+).

The terms “polypeptide,” “protein,” and “peptide,” which are used interchangeably herein, refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides and proteins, unless otherwise noted.

The term “isolated”, when used in the context of an isolated protein is a protein that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated protein or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell. In some embodiments, the term “isolated”, when applied to a protein, denotes that the protein is essentially free of other cellular components with which it is associated in nature. It can be in a homogeneous state although it can be in either a dry or aqueous solution. Homogeneity and whether a molecule is isolated can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially isolated. The term “isolated” denotes that a polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polypeptide is in some embodiments at least about 50% pure, 60% pure, 70% pure, 80% pure, 85% pure, 90% pure, 95% pure, or 99% pure.

In some embodiments, before calprotectin is administered it is bound with calcium (Ca2+). Without wishing to be bound by theory or mechanism, it is believed that the ability of the calprotectin to sequester Mn2+(and Zn2+) is enhanced or derived from the calprotectin being administered while bound to Ca2+. It is further believed that process by which calprotectin binds Mn2+(and Zn2+) includes the release of Ca2+. In this regard, in some embodiments, calprotectin bound to Ca2+can be lyophilized, and resuspended in an appropriate buffer prior to administration. In some embodiments, calprotectin can be lyophilized, and resuspended in an appropriate buffer containing Ca2+, wherein the calprotectin is allowed to bind to the Ca2+prior to administration. Appropriate buffers include, but are not limited to, phosphate-buffered saline (PBS) and any other physiologically-inert buffer, as can be identified by those of ordinary skill in the art.

In some embodiments, when a polypeptide metal ion chelator is used, it can be desirable to select a polypeptide, or appropriate fragment or variant thereof, having the same sequence as a polypeptide derived from the species of which the subject is a member, as will be understood by one of ordinary skill in the art. For example, when the subject is a human and the polypeptide metal ion chelator is calprotectin, it can be desirable to select human calprotectin, i.e., calprotectin heterodimer including human S100A8 (SEQ ID NO: 1), or a functional fragment or functional variant thereof, and human S100A9 (SEQ ID NO: 2), or a functional fragment or functional variant thereof.

In some embodiments, the metal ion chelator of the presently disclosed subject matter can be a functional fragment or a functional variant of calprotectin. As noted above, the calprotectin is a protein heterodimer, including an S100A8 polypeptide, and an S100A9 polypeptide. In this regard, a fragment and/or variant of calprotectin is a calprotectin heterodimer including a fragment or variant of S100A8 and/or a fragment or variant of S100A9. For example, a calprotectin fragment can include a fragment of S100A8 and a full-length S100A9. For another example, a calprotectin fragment can include a full-length S100A8 and a fragment of S100A9. For another example, a calprotectin fragment can include a fragment of S100A8 and a fragment of S100A9. For another example, a calprotectin variant can include a wild-type S100A8 and a S100A9 variant. For another example, a calprotectin variant can include a S100A8 variant and a wild-type S100A9. For another example, a calprotectin variant could include a S100A8 variant and a S100A9 variant.

The terms “polypeptide fragment” or “fragment”, when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids long, depending on the reference polypeptide. A calprotectin fragment can be a calprotectin heterodimer including a S100A8 fragment, an S100A9 fragment, or both.

A fragment can also be a “functional fragment,” in which the fragment retains some or all of the activity of the reference polypeptide as described herein. For example, in some embodiments, a functional fragment of a polypeptide metal ion chelator can retain some or all of the Mn2+-sequestering activity of the reference polypeptide. In some embodiments, a functional fragment of a polypeptide metal ion chelator can retain some or all of the Mn2+- and Zn2+-sequestering activity of the reference polypeptide. In some embodiments, the functional fragment includes a preserved metal binding motif. In some embodiments, the functional fragment can include a Mn2+binding motif, a Zn2+binding motif, or both. In some embodiments, a fragment can comprise a domain or motif, and optionally additional amino acids on one or both sides of the domain or motif, which additional amino acids can number from 5, 10, 15, 20, 30, 40, 50, or more residues. A calprotectin functional fragment can be a calprotectin heterodimer including an S100A8 functional fragment, an S100A9 functional fragment, or both, so long as the resulting calprotectin functional fragment retains some or all of the activity of calprotectin.

The terms “variant” refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., one or more amino acid substitutions. A variant of a reference polypeptide also refers to a variant of a fragment of the reference polypeptide, for example, a fragment wherein one or more amino acid substitutions have been made relative to the reference polypeptide. A variant can also be a “functional variant,” in which the variant retains some or all of the activity of the reference protein as described herein. For example, a functional variant of a polypeptide metal ion chelator retains some or all of the Mn2+-sequestering activity of the reference polypeptide. In some embodiments, a functional variant of a polypeptide metal ion chelator can retain some or all of the Mn2+- and Zn2+-sequestering activity of the reference polypeptide. In some embodiments, the functional variant includes a preserved metal binding motif. In some embodiments, the functional variant can include a Mn2+, a Zn2+binding motif, or both, which binding motifs are preserved relative to the reference polypeptide. A calprotectin functional variant can be a calprotectin heterodimer including a S100A8 functional variant, an S100A9 functional variant, or both, so long as the resulting calprotectin functional variant retains some or all of the activity of calprotectin. The term functional variant does not include variants that loose the ability to inhibit microbial growth; for example, a human calprotectin variant wherein the cysteine residue at position 3 in S100A9 (SEQ ID NOS: 2) is replaced by another amino acid residue is not considered to be a functional variant (See Examples below).

The term functional variant includes a functional variant of a functional fragment of a reference polypeptide. The term functional variant further includes conservatively substituted variants. The term “conservatively substituted variant” refers to a peptide comprising an amino acid residue sequence that differs from a reference peptide by one or more conservative amino acid substitution, and maintains some or all of the activity of the reference peptide as described herein. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. The phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

Without wishing to be bound by theory or mechanism, it is believed that the S100A8 and S100A9 polypeptides of SEQ ID NOS: 1-4 have metal binding motifs including particular amino acids identified in capitals in the sequences as set forth in Table A. In some embodiments, a calprotectin functional fragment includes a fragment of S100A8, a fragment of S100A9, or both, wherein each fragment includes a preserved metal ion binding motif. In some embodiments, a calprotectin variant includes a variant of S100A8, a variant of S100A9, or both, wherein each variant has a preserved metal ion binding motif.

Calprotectin can be made for use in the presently-disclosed methods, for example, using molecular biology techniques known to those of ordinary skill in the art. Calprotectin heterodimers self assemble from S100A8 and S100A9 polypeptides. S100A8 and S100A9 polypeptides can be made, for example, using various molecular biology techniques known to those of ordinary skill in the art. Calprotectin, or a functional fragment or functional variant thereof, can be made by expressing S100A8 and S100A9, or functional fragments or functional variants thereof, within the same cell using molecular biology techniques known to those of ordinary skill in the art. The subunits heterodimerize within the cell, and calprotectin can be purified therefrom using molecular biology techniques known to those of ordinary skill in the art. Although Ca2+is not required to successfully express and purify calprotectin, as noted above, it can be useful in some embodiments to provide calprotectin that is bound with calcium (Ca2+).

As noted above, the presently-disclosed subject matter includes methods for treating a microbial infection or an abscessed tissue in a subject, including administering an effective amount of a metal ion chelator to the subject.

The metal ion chelator can be locally administered at or near the site of infection (or site associated with a risk of infection), or the site of the abscessed tissue. For example, when the microbial infection is associated with the skin of the subject, the metal ion chelator can be administered topically. In some cases, the metal ion chelator can be injected into an abscess. Various methods of administrating the metal ion chelator can be used, as will be understood by one of ordinary skill in the art.

As used herein, an “effective amount” of the metal ion chelator is an amount sufficient to bind a desired amount of Mn2+(or Mn2+and Zn2+) accessible to the microbe that is capable of causing an infection, causing an infection, or causing an abscess. In some embodiments, the effective amount of the metal ion chelator is an amount sufficient to bind substantially all of the Mn2+(or Mn2+and Zn2+) accessible to the microbe. In some embodiments, the effective amount of the metal ion chelator is an amount sufficient to bind enough of the Mn2+(or Mn2+and Zn2+) accessible to the microbe such that a treatment is affected. In some embodiments, an effective amount of the metal ion chelator is less than about 1 mg. In some embodiments when the metal ion chelator is administered by injection into an abscess, an effective amount of the metal ion chelator can be about 100 μg, about 90 μg, about 80 μg, about 70 μg, about 60 μg, or about 50 μg. In some embodiments, the metal ion chelator can be provided in a solution of about 70 μg/ml, and an appropriate volume can be administered such that an effective amount is provided.

Injectable formulations of the metal ion chelator can include various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). Physiologically-acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. A suitable insoluble form of the compound can be prepared and administered as a suspension in an aqueous base or a pharmaceutically-acceptable oil base, such as an ester of a long chain fatty acid, (e.g., ethyl oleate).

The metal ion chelator can also be provided in a cream or ointment, or in a transdermal patch for topical administration. A topical ointment formulation can contain the metal ion chelator in a carrier such as a pharmaceutical cream base. Various formulations for topical use include drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various supports and vehicles. The optimal percentage of the metal ion chelator in each formulation varies according to the formulation itself and the therapeutic effect desired in the specific situation.

The presently-disclosed subject matter further includes a kit for use in treating a microbial infection or an abscessed tissue in a subject. In some embodiments, the kit includes a metal ion chelator, and instructions for administering the metal ion chelator to the subject.

In some embodiments, the kit includes a metal ion chelator prepared for administration by injection. In this regard, the metal ion chelator can be provided in a vial, ready to be combined with an appropriate carrier for use in administering the metal ion chelator to the subject. In some embodiments, the metal ion chelator is provided in a single-dose vial, containing an amount of the metal ion chelator that is an effective amount for use in certain instances, e.g., effective amount for treatment in common instances. In some embodiments, the metal ion chelator is provided as a lyophilate. In some embodiments, the kit additionally includes a buffer for resuspending the lyophilate for administration to the subject. In some embodiments, the kit additionally includes instructions for preparing the metal ion chelator for administration.

In some embodiments, the kit includes a metal ion chelator prepared for topical administration. In this regard, the metal ion chelator can be provided in a cream or ointment, or in a transdermal patch.

In some embodiments, the kit includes a calprotectin heterodimer, or functional fragment or functional variant thereof.

In some embodiments, the kit includes a calprotectin heterodimer, or functional fragment or functional variant thereof, prepared for administration by injection. In this regard, the calprotectin heterodimer can be provided in a vial, ready to be combined with an appropriate carrier for use in administering the metal ion chelator to the subject. In some embodiments, the calprotectin heterodimer is provided as a lyophilate. In some embodiments, the lyophilate can be a Ca2+-bound calprotectin heterodimer. In some embodiments, the kit can additionally include a buffer for resuspending the lyophilate for administration to the subject. In some embodiments, the lyophilate can be a calprotectin heterodimer that is free of Ca2+. When the calprotectin heterodimer is free of Ca2+, it can be resuspended in a buffer containing Ca2+prior to administration to the subject. In some embodiments, the kit additionally includes buffer containing Ca2+for resuspending the lyophilate for administration to the subject. In some embodiments, the kit additionally includes instructions for preparing the calprotectin heterodimer for administration.

In some embodiments, the kit includes a calprotectin heterodimer prepared for topical administration. In this regard, the calprotectin heterodimer can be provided as a Ca2+-bound calprotectin heterodimer. The calprotectin heterodimer can be provided in a cream or ointment, or in a transdermal patch for topical administration.

The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples.

EXAMPLES

Abscesses represent an immune response to infection that confines the spread of disease through the restriction of microbial growth and dissemination to neighboring tissues.Staphylococcus aureusinfection results in the formation of abscesses characterized by the extensive accumulation of host neutrophils (1). Although a role for neutrophils in abscess development is established, the specific host factors that limit microbial growth in the abscess are incompletely defined (1).

As described in these studies, to identify host factors present in abscessed tissue, Imaging Mass Spectrometry (IMS) was applied to a mouse model of staphylococcal infection. IMS utilizes matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) to profile and map the distribution of proteins present in thin tissue sections (2). This technology determines relative protein concentrations across intact tissue, simultaneously imaging at hundreds of distinct molecular weights. IMS thus provides information on the spatial distribution of individual proteins in vivo without the need for antibodies, allowing an unbiased analysis of protein distribution in healthy and diseased animals (2-4). The staphylococcal abscess is an anatomical site exhibiting macroscopic differences from healthy tissue, making it amenable to IMS-based protein detection. This work represents the first application of IMS to host-microbe interactions.

To analyze protein distribution in abscesses fromS. aureus-infected mice, 6-8 week old female C57BL/6 mice were either not exposed to staphylococci (uninfected), infected with 1×105CFU ofS. aureus(low dose; does not lead to abscesses formation), or infected with 1×106CFU ofS. aureus(high dose; leads to significant kidney and liver abscess formation). Kidneys from uninfected and infected animals were sectioned, coated with sinapinic acid, and analyzed using IMS. Representative images from these analyses are shown inFIG. 1A.FIG. 1Aincludes mass spectrometric images ofS. aureusinfected and uninfected murine kidneys. Optical images of three frozen kidney sections mounted on a gold-coated MALDI plate is shown in the far left column. Two-dimensional ion density map of representative proteins expressed in infected or uninfected murine tissue is shown in the four right panels with mass-to-charge values (m/z) for each distinct protein shown at the bottom of the panels. The ion density map is illustrated as a pseudo-color image. The presented images represent a subset of approximately 150 protein images obtained in these analyses. Among the proteins present exclusively in abscessed tissue was a protein exhibiting m/z of 10,165 that displayed the strongest mass-to-charge signal observed in these experiments (FIG. 1A). The considerable abundance of an approximately 10 kDa protein specifically inS. aureus-induced murine abscesses implies a contribution of this protein to the host-pathogen interaction.

To identify this approximately 10 kDa protein, abscessed or corresponding healthy tissue was dissected fromS. aureus-infected murine kidneys. Proteins were extracted using 50% acetonitrile and 0.2% trifluoroacetic acid and were subsequently separated using 1-dimensional SDS-PAGE.FIG. 1Bincludes SDS-PAGE analysis of material extracted from kidneys of uninfected (lane 1) or infected (lane 2) mice followed by MS/MS-based identification of proteins. As illustrated inFIG. 1B, the analysis revealed significant alterations in protein expression between infected and uninfected tissue. A colloidal blue stained protein band present exclusively in the abscessed tissue corresponding to a predicted size of approximately 10 kDa was excised from the gel, digested with trypsin, and analyzed using LC-MS/MS. These analyses revealed that the band present in the excised gel slice contained trypsin peptide cleavage fragments that match S100A8, a component of the calprotectin heterodimer (S100A8/S100A9) (5).

With reference toFIG. 1C, immunohistochemistry using anti-S100A8 antisera as a probe revealed robust immune cell recruitment to staphylococcal abscesses highlighted by a prominent polymorphonuclear leukocyte population (arrowhead denotes abscess). Moreover, S100A8 is only detectable in tissue from infected mice and localizes coordinately with the sites of staphylococcal abscesses. Furthermore, with reference toFIG. 1D, IMS revealed S100A8 and S100A9 co-localize to the staphylococcal abscesses, supporting heterodimeric calprotectin as the functional form of these proteins inside tissue abscesses.FIG. 1Dincludes mass spectrometric images ofS. aureusinfected and uninfected murine kidneys showing the distribution of S100A8 (m/z 10,165) and S100A9 (m/z 12,976) (arrowheads denote abscesses).

FIGS. 2A-2Finclude histological analyses of kidney tissue sections harvested from uninfected or infected mice 96 hr post-infection. Kidney sections from (FIG. 2A) uninfected and (FIG. 2B) infected C57BL/6 mice were stained with hematoxylin and eosin to assess inflammation.FIGS. 2C-2Finclude representative sections of tissue from uninfected or infected mice immunostained for S100A8. The arrowhead inFIG. 2Ddenotes abscess formation. The arrowhead inFIG. 2Fdenotes individual cell staining positive for S100A8. P.A. stands for proximal to the abscess, and I.A. stands for inside the abscess.

Calprotectin is an S100 EF-hand Ca2+binding protein that accounts for approximately 40% of the cytosolic protein pool of neutrophils (5), and is a minor cytoplasmic constituent of monocytes, macrophages, and keratinocytes (6-8). Approximately twenty years ago calprotectin was first shown to inhibit the growth of a variety of fungal and bacterial pathogens in vitro (9), however a mechanistic explanation for this antimicrobial activity has yet to be formulated. Calprotectin's antimicrobial activity has been proposed to be due to calprotectin-mediated chelation of nutrient Zn2+. This hypothesis is largely based on the fact that S100A8 and S100A9 encode Zn2+binding His-X-X-X-His motifs (10), and the finding that the antimicrobial activity of calprotectin againstCandida albicansandBorrelia burgdorferican be overcome by the addition of excess Zn2+(11, 12). However, it has not been established that microbial pathogens exposed to calprotectin are Zn2+starved. In addition,C. albicansandB. burgdorferihave high nutrient Zn2+requirements, making them unusually sensitive to Zn2+deprivation (13, 14). Moreover, it has been speculated that the Zn2+-reversibility of calprotectin's candidastatic and bacteriostatic effects may be due to Zn2+-dependent conformational changes in the protein and not direct sequestration of this nutrient (15). Thus, the considerable body of data on the antimicrobial mechanism of calprotectin is largely indirect and the contribution of calprotectin to the host-pathogen interaction has not been evaluated.

The first step in the approach described herein was to ascertain the cell population responsible for recruitment and/or expression of S100A8 in the staphylococcal abscess by infecting wild-type and neutropenic mice withS. aureusand applying IMS across the kidneys and livers of infected animals. To this end, mice were initially depleted of neutrophils by administering the rat IgG2b α-Gr-1 monoclonal antibody RB6-8C5 (FIG. 3) and infected with a dose ofS. aureusthat induces lesion formation without leading to lethality (FIGS. 3A and 3B).FIGS. 3A and 3Binclude results of a flow cytometry analysis of neutrophils in kidney and liver tissue of uninfected orS. aureusinfected murine tissue 96 hr post-infection. Plots are gated on live cells with large and granular forward and side scatter properties. Data represent the mean+/−the standard deviation of quadruplicate experiments. *, P<0.001.

FIG. 4includes a survival curve of neutrophil-replete mice exposed to various doses ofS. aureus. Six to eight week old female C57/BL6 mice were infected withS. aureusat the listed doses. Prior to infection, mice were either treated with an anti-neutrophil antibody (RB6) to deplete neutrophils, or an isotype matched antibody (SRF3). Infected animals were followed for 96 hours and survival was recorded. RB6-treated mice infected with 1×105CFU typically survived in spite of lesion formation in various organs.

With reference toFIG. 1E, results from an imaging mass spectrometry analyses of S100A8 expression within kidneys and livers from neutrophil replete uninfected mice (lane 1), neutrophil replete infected mice (lane 2), neutrophil-depleted uninfected mice (lane 3), and neutrophil-depleted infected mice (lane 4) are shown. The top row in each organ set shows matrix treated organs, and the bottom row in each organ set shows IMS. Asterisks denote sites of abscess formation. Neutrophils were depleted by administering the mAb RB6. An isotype matched mAb SRF3 was used as a control in neutrophil replete mice. IMS revealed that S100A8 localizes coordinately with staphylococcal kidney and liver abscesses in neutrophil replete mice. In contrast, infected neutropenic mice do not express S100A8 in the kidney or liver in spite ofS. aureus-induced lesion formation in these organs. These findings indicate that the presence of S100A8 in infected kidney and liver abscesses is dependent on an intact neutrophil compartment.

S100A8 and S100A9 were co-purified to form functional calprotectin heterodimers using published protocols (16).FIG. 5Aillustrates the effect of recombinant calprotectin onS. aureusgrowth in vitro. It was found that calprotectin inhibitsS. aureusgrowth in a dose-dependent manner with complete growth arrest observed at 75 μg/ml calprotectin. These concentrations are physiologically relevant considering the estimated concentration of calprotectin in the neutrophil cytoplasm is approximately 15 mg/ml, and greater than 1 mg/ml of calprotectin is detectable in abscess fluid of patients (17-19).

FIG. 5BdepictsS. aureusgrowth in the presence of calprotectin purified with (Ca2+) or without calcium (no Ca2+) as compared to buffer alone (buffer). The anti-staphylococcal activity of calprotectin is augmented when the protein is purified in the presence of Ca2+. This result is consistent with calprotectin's assignment as an S100 EF-hand Ca2+binding protein, and the in vitro observation that Ca2+promotes calprotectin heterotetramerization and subsequent configuration of high affinity Zn2+binding sites (20).

Although S100A9 preferentially forms heterodimers in vitro and in vivo (16), it can be expressed independently from S100A8 suggesting that homodimers retain some level of functionality (21, 22). To determine whether heterodimer formation is essential for calprotectin to inhibit staphylococcal growth, the sensitivity ofS. aureusto the S100A9 homodimer was tested.FIG. 5Cincludes an analysis ofS. aureusgrowth in the presence of S100A8/A9 heterodimers (A8/A9), S100A9 homodimers (A9), S100A8/S100A9C3S heterodimers (A8/A9*), or S100A9C3S homodimers (A9*) as compared to buffer alone (buffer). It was found that the S100A9 homodimer is unable to inhibitS. aureusgrowth at concentrations ranging from 5-500 μg/ml. The instability of S100A8 homodimers (16) precluded investigation of its activity in these assays. S100A9 contains a cysteine residue at amino acid position 3 that has been suggested to play a role in the functionality of calprotectin heterodimers (16). This supposition was tested by assaying the anti-staphylococcal properties of a previously described cysteine-to-serine mutant (S100A9C3S) (16). Neither S100A9C3S homodimer nor S100A8/S100A9C3S heterodimer were able to inhibit staphylococcal growth (FIG. 5C). Taken together, these findings indicate that heterodimerization, Ca2+binding, and the cysteine residue at position 3 in S100A9 contribute to calprotectin's ability to inhibit bacterial growth.

With reference toFIG. 5D, calprotectin's ability to bind staphylococcal cells was assessed.S. aureuswas incubated with calprotectin, centrifuged, washed, and whole cell lysates immunoblotted with an S100A8 specific antibody. In lanes 1-5S. aureuswere mixed with increasing concentrations of calprotectin. Calprotectin (15 μg/ml) was used as a control marker in lane 6. The antimicrobial activity of calprotectin is proposed to occur through metal ion chelation, suggesting that physical contact between calprotectin andS. aureusis not required for growth inhibition. Consistent with this, calprotectin-staphylococci interactions using a co-precipitation assay could not be detected, as shown inFIG. 5D.

With reference toFIG. 5E, to assess further whether calprotectin requires physical contact to mediate growth inhibition,S. aureuswas grown in medium containing calprotectin (A8/A9) but physically separated by a dialysis membrane, and compared to buffer alone (buffer). It was found that staphylococcal growth was reduced approximately 40% by calprotectin (500 μg/ml) in the absence of physical contact.

Bacterial growth medium was then treated with calprotectin followed by its removal through filtration, and the ability ofS. aureusto grow in this calprotectin-treated medium was measured. Successful removal of calprotectin from the treated medium was confirmed by immunoblot using anti-S100A8 antisera (data not shown). With reference toFIG. 5F,S. aureusgrowth was reduced in medium that had been transiently treated with calprotectin (A8/A9) as compared to medium treated with buffer alone (buffer). These data are consistent with a model whereby calprotectin inhibitsS. aureusgrowth through the sequestration of an essential growth factor from the medium.

To determine whether calprotectin chelates nutrient cations, purified calprotectin was incubated with growth medium containing known concentrations of Fe2+, Mn2+, Mg2+, Ca2+, and Zn2+. Calprotectin was subsequently removed by filtration, and the Fe2+, Mn2+, Mg2+, Ca2+, and Zn2+levels were determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). These analyses revealed no statistical difference in the number of Fe2+or Mg2+atoms remaining in the medium upon transient treatment with calprotectin (FIG. 6A). In contrast, Mn2+and Zn2+were not detected following calprotectin treatment (FIG. 6A), demonstrating that calprotectin chelates nutrient Mn2+and Zn2+from staphylococcal growth medium. A significant increase in detectable Ca2+in the growth medium following calprotectin treatment is likely due to the release of excess Ca2+ions bound to calprotectin in secondary binding sites as a by-product of purification in a Ca2+-rich buffer. The data described herein represent the first indication of a role for calprotectin in binding Mn2+.

In order to validate this hypothesis, the Mn2+-binding properties of calprotectin were examined using a standard fluorescence spectroscopy assay.FIG. 6Bincludes fluorescence emission spectra of S100A8/S100A9 in the absence (diamonds) and presence (squares) of Mn2+(250 μM). The shift in the intensity of wavelength of the peak maximum as a result of addition of Mn2+, confirmed that calprotectin does bind this metal ion.

To determine the sensitivity ofS. aureusto Zn2+and Mn2+starvation,S. aureuswas grown in media deplete in all cations (metal dep.), deplete in Zn2+(Zn2+dep.), and deplete in Mn2+(Mn2+dep.). With reference toFIG. 6C, these experiments revealed thatS. aureusis acutely sensitive to Mn2+deprivation, whereas the bacteria proliferate in media that is virtually devoid of detectable Zn2+.

With reference toFIG. 6D, addition of excess Mn2+and Zn2+to growth medium rescues calprotectin-mediated inhibition of staphylococcal proliferation, confirming that Mn2+and Zn2+chelation is responsible for the anti-staphylococcal activity of calprotectin. Presumably, the ability of individual metals to rescue staphylococcal growth is due to saturation of the Zn2+/Mn2+binding sites of calprotectin by either excess Zn2+or Mn2+.

The contribution of calprotectin to neutrophil-mediated bacterial killing was determined. Neutrophils (peritoneal PMNs) were extracted from wildtype and calprotectin-deficient mice (S100A9Δ) mice and incubated in vitro withS. aureusto measure phagocytic-dependent killing. With reference toFIG. 7, neutrophils extracted from wildtype and calprotectin-deficient mice killS. aureusin vitro with similar efficiency suggesting that calprotectin does not contribute to phagocytic killing. Data are presented as percent of bacteria that were killed after two hours of exposure to PMNs.

Next, we extracted the cytoplasmic compartment from purified neutrophils and measured its antimicrobial activity. Due to the low yield of cytoplasmic material from murine neutrophil extractions, these experiments were performed using neutrophils extracted from human blood. With reference toFIGS. 6E and 6F, these experiments revealed that the cytoplasmic fraction of neutrophils is capable of inhibiting staphylococcal growth and this activity is reversible upon the addition of excess Mn2+or immunodepletion of calprotectin.

As a further demonstration thatS. aureusexposed to calprotectin are Mn2+-starved,S. aureusstrains were created that are inactivated for the Mn2+-dependent transcriptional repressor (ΔmntR), or the Mn2+uptake system (ΔmntA, ΔmntB). MntR is a repressor of the MntABC transport system responsible for Mn2+acquisition, and its disruption rendersS. aureusacutely sensitive to Mn2+-mediated toxicity (23). It was reasoned that if calprotectin efficiently chelates nutrient Mn2+, then the addition of calprotectin to medium supplemented with toxic levels of Mn2+should protect ΔmntR from Mn-toxicity. Conversely, it was predicted that strains defective in MntABC should be more sensitive to calprotectin-dependent toxicity due to an inability to compete for available Mn2+. With reference toFIGS. 8A and 8B, the bacteriostatic effect of calprotectin was determined against either (FIG. 8A) ΔmntA and ΔmntB in Mn2+replete media or (FIG. 8B) ΔmntR in the presence of toxic levels of Mn2+. It was found that co-incubation with calprotectin alleviates the sensitivity of ΔmntR to excess Mn2+, while strains defective in mntA or mntB are more sensitive to calprotectin toxicity.

Bacteria respond to alterations in metal concentrations through changes in gene expression. These changes are metal-specific and hence represent expression signatures that can be used to determine the environmental stress placed on a bacterial population (24). Therefore, if calprotectin chelates nutrient Zn2+and Mn2+, exposingS. aureusto calprotectin should elicit a gene expression profile consistent with Zn2+and Mn2+starvation. Since the Zn2+and Mn2+regulons ofS. aureushave not been reported, the staphylococcal transcripts that change expression upon Zn2+or Mn2+starvation were first determined usingS. aureusAffymetrix GENECHIP®.S. aureusdecreased the expression of 104 genes and increased the expression of 9 genes upon Zn2+starvation (Supplemental Table 1-2). By comparison,S. aureusgrown in Mn2+deplete conditions exhibited less pronounced changes in gene expression highlighted by the down-regulation of 12 genes and the up-regulation of 2 genes (Supplemental Table 3-4).

Supplemental Table 2:S. aureustranscripts increased in the absence of Zn2+CommonFoldLocusanamecinductionDescriptiondSA0387NA3.1exotoxin 11SA0469NA2.6exotoxin 1, putativeSA1037NA4.3conserved hypothetical proteinSA1166NA3.1hypothetical proteinSA1178NA3.5exotoxin 1, putativeSA1179NA2.5exotoxin 4, putativeSA1180NA3.1exotoxin 3, putativeSA2357NA5.8bABC transporter, permease proteinSAS0388NA3.8exotoxin 3aS. aureusstrain COL locus, unless otherwise indicated (strain preceeds locus identifier).btranscript was below the lower limits of sensitivity in unstressed cells, and thus the amount of change represents an estimate.cNA = Not availableddescription of predicted function

Supplemental Table 4:S. aureustranscripts increased in the absence of Mn2+CommonFoldLocusanameinductionDescriptioncSA1166NA2.0hypothetical proteinSA0910NA2.2similar to quinol oxidasepolypeptide IV QoxDaS. aureusstrain COL locus, unless otherwise indicated (strain preceeds locus identifier).bNA = Not availablecdescription of predicted function

Transcriptome analyses ofS. aureusgrown in the presence of a sub-inhibitory concentration of calprotectin revealed 61 transcripts that change abundance (Supplemental Table 5-6). Although calprotectin treatment increased the expression of 30 genes, the large majority of these genes do not have ascribed functions (Supplemental Table 5). In contrast, calprotectin treatment decreased the expression of a variety of genes with predicted roles in metal metabolism and pathogenesis (Supplemental Table 6). More specifically, calprotectin treatment led to the down-regulation of a similar subset of genes to those that are down-regulated upon Zn2+and Mn2+starvation (Supplemental Table 5).

FIG. 6Gincludes a hierarchial clustering, where dendrogram (left) with heat map (right) illustrates the relatedness of transcription profiles ofS. aureuscells subjected to indicated stresses. Within the heat map, the normalized signal intensity value for each locus represented on theS. aureusGeneChip is shown (7775 total). Red indicates high signal intensity; green indicates low intensity in the listed condition. Manganese (Mn2+) and Zinc (Zn2+)-depleted profiles are 93.8% and 91.2% related to calprotectin treated medium (100% confidence; Pearson correlation average linkage bootstrapping). Transcripts shown in the heatmap as changing expression in any of the three conditions (Zn2+starvation, Mn2+starvation, and calprotectin treatment) are listed in the Supplemental Tables.

Comparison of the global expression changes ofS. aureusexposed to calprotectin to those observed upon Mn2+starvation, Zn2+starvation, heat shock, cold shock, alkaline shock or acid shock, as well as stringent-response and SOS-response inducing conditions (25)(unpublished Anderson and Dunman) demonstrated thatS. aureustreated with subinhibitory calprotectin elicit an expression profile most closely resembling that of staphylococci starved for Zn2+and Mn2+(FIG. 6G). Taken together, these data strongly support a model whereby calprotectin preventsS. aureusgrowth through the chelation of nutrient Mn2+and Zn2+.

To determine whether calprotectin-mediated cation chelation inhibits bacterial growth in tissue abscesses, the growth ofS. aureusin abscesses from wildtype mice and calprotectin deficient (S100A9−/−) mice were compared. S100A8 null mice are embryonic lethal (26) and therefore can not be used for these experiments. With reference toFIG. 9A, examination of the livers of wildtype and S100A9 −/− mice 96 hours following intravenous infection revealed that mice lacking calprotectin exhibit increased abscess formation as compared to infected wild-type animals. Additionally, with reference toFIG. 9B, enumeration of bacterial counts from infected livers revealed a significant increase in the number of staphylococci in S100A9-deficient animals as compared to controls.

With reference toFIG. 10, wild-type and calprotectin-deficient mice were either not infected or infected withS. aureusand the livers harvested 96 hr post-infection. Livers were subsequently homogenized and the percentage of CD11b+/Ly-6G(Gr1)+ cells determined by flow cytometry. Although the immunomodulatory functions of calprotectin (27) may contribute to the sensitivity of calprotectin-deficient animals toS. aureusinfection, a significant difference in neutrophil recruitment to the livers of these infected animals was not detected by multiparametric FACS-analyses. These data establish calprotectin as a pathophysiologically relevant component of the neutrophil armamentarium to control bacterial infection in vivo.

To assess the effect of calprotectin on nutrient metal availability inside the abscess, Laser Ablation ICP-MS mapping (LA-ICP-MS) was used to image metal distribution in infected animal tissue. LA-ICP-MS allows for two dimensional mapping of trace elements in intact soft tissue (28), and hence was used to generate an image of metal distribution in thin sections from infected murine livers.FIG. 9Cincludes LA-ICP-MS images of infected organs from wildtype and calprotectin-deficient (S100A9Δ) mice. Theop panel shows infected organs stained with hematoxylin-eosin. The bottom panels shown LA-ICP-MS maps for Ca2+(calcium-44), Mn2+(manganese 55), and Zn2+(zinc 67). Arrows denote the site of abscesses. All data are normalized to levels of carbon-13. Scales are presented in arbitrary units. LA-ICP-MS revealed that staphylococcal liver abscesses from wildtype mice are enriched in Ca2+consistent with the robust immune cell infiltrate to the infection site. In contrast, these abscesses are devoid of detectable Zn2+and Mn2+, establishing the abscess as a cation-starved environment. Abscesses from mice lacking calprotectin contain appreciable levels of Ca2+, however these levels appear diminished compared to abscesses from wildtype mice, potentially reflecting the Ca2+contribution of calprotectin to the abscess. Furthermore, calprotectin deficient abscesses contain levels of Mn2+equivalent to the surrounding healthy tissue demonstrating the in vivo requirement for calprotectin in Mn2+chelation and removal from the abscess. The absence of calprotectin did not have a significant impact on Zn2+levels in these experiments.

With reference toFIGS. 9D-9F, to confirm these results and validate the use of LA-ICP-MS to image metal distribution in infected animal tissue, abscessed material was extracted from wildtype and calprotectin-deficient animals using Laser Capture Microdissection (LCM) and Zn2+, Mn2+and Ca2+concentrations in isolated abscesses were quantitated using ICP-MS. These experiments confirmed that abscesses from mice lacking calprotectin are significantly enriched in Mn2+as compared to those from wildtype mice (FIG. 9E). Taken together, these results establish calprotectin-mediated metal chelation as an immune defense strategy to prevent bacterial outgrowth in tissue abscesses, and demonstrate the utility of LA-ICP-MS to image trace element distribution in healthy and diseased tissue. The inhibition of bacterial nutrient uptake represents a promising alternative area of research for the design of novel antimicrobials and the observed calprotectin-mediated metal chelation provides a specific direction to assess the therapeutic potential of this concept. The results reported here suggest that beyond direct treatment of abscesses with calprotectin protein, non-cytotoxic bioavailable metal ion chelators represent a promising area of investigation for the inhibition of growth of bacterial infections.

Materials and Methods

Imaging Mass Spectrometry.

IMS involves acquiring independent MALDI mass spectra on thin tissue sections mounted on a MALDI target. The intensities of all the mass-to-charge (m/z) signals present in the spectral array are then recorded and plotted in ion density maps or images. By applying IMS to a given tissue section, hundreds of images can be simultaneously recorded in two dimensions at distinct molecular weights. To perform IMS onstaphylococcus-infected murine tissue, kidneys and livers were sectioned at −20° C. in a cryostat (Leica Microsystems Inc., Bannockburn, Ill., USA) at a thickness of 12 μm. Tissue sections were thaw-mounted onto a gold-coated MALDI target. The target was vacuum desiccated for 30 minutes prior to tissue-fixation by submersion in sequential washes of 70%, 90%, 95% ethanol for 30 seconds each. The plate was returned to the desiccator for 1 hour to allow for complete drying. Matrix (20 mg/ml sinapinic acid in 50:50 acetonitrile/0.2% trifluoroacetic acid in water) was deposited over the entire plate using a glass reagent sprayer to apply uniform crystal coverage. Mass spectral images were acquired on an Autoflex II (Bruker DaltoniK GmbH, Bremen, Germany) MALDI-TOF mass spectrometer equipped with a SmartBeam laser (Nd:YAG, 355 nm) operated at 200 Hz. Data were obtained in linear mode with an accelerating voltage of 20 kV, an extraction voltage of 18.65 kV, a lens voltage of 6 kV, a delay time of 350 ns, and a detector voltage of 1588 V. Spectra were acquired at a spatial resolution of 100 μm in both x and y by rastering the laser across the tissue. Each spectrum (pixel) was a sum of 100 laser shots. Spectra were compiled into an image file which allowed for visualization of ion intensity as a function of location on the tissue using the Biomap software (Novartis, Basel, Switzerland). Images inFIG. 1Panel A were taken from BALB/c mice whereas images inFIG. 1Panel E were taken from C57BL/6 mice. These experiments revealed a similar distribution of calprotectin in both strains of mice.

Isolation and Identification of Abscess Specific Proteins.

Abscessed tissue was dissected from infected organs using a scalpel and transferred to a microcentrifuge tube. Similar areas were also cut out of control tissues for comparison. Proteins were extracted from the tissue using 100 μl of 50% acetonitrile, 0.1% TFA in water with pipette mixing. Extracts were subsequently separated using 1-dimensional SDS-PAGE. A colloidal blue stained protein band present exclusively in the abscessed tissue corresponding to a predicted size of approximately 10,165 Da was then excised from the gel, digested with trypsin, and analyzed using LC-MS/MS. A corresponding molecular weight region of the gel was excised from the uninfected lane and analyzed using LC-MS/MS as a negative control. Exclusion criteria for this analysis included proteins represented by less than three peptides, peptides corresponding to keratin, and peptides corresponding to proteins from species other thanMus musculusorStaphylococcus aureus.

Murine kidneys were sectioned into 5 μM sections and transferred to glass slides. The tissue slides were fixed as follows: 70% ethanol for 30 seconds, 95% ethanol for 1 minute, 100% ethanol for 1 minute, 100% ethanol for 1 minute, xylene for 2 minutes, xylene for 3 minutes. The slides were removed from the xylene solution and allowed to air-dry for 5 minutes. The fixed slides were loaded into a Veritas system Laser Capture Microdissection (LCM) machine (Molecular Devices). The tissue samples were microdissected at 10× magnification, and with a power range of 70 mW-100 mW and pulse range 2,500 μs-11,000 μs. The polymer sides of the LCM caps containing the captured sections were subjected to ICP-MS as described below.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). For measuring the effect of calprotectin on cation concentrations, calprotectin (75.92 μM) or a buffer control was incubated with 0.8 ml of NRPMI containing 100 μM CaCl2, 25 μM ZnCl2, 1 mM MgCl2, 25 μM MnCl2, and 5 μM FeSO2overnight at 4° C. Samples were subsequently passed through a centricon column with a 5,000 Da cutoff by spinning at 4000 RPM for 30 minutes. The flow-through was collected, mixed with high purity 15N nitric acid (SEASTAR™), and heated in sealed SAVILLEX® PFA Teflon vials at 130° C. for three hours. Samples were subsequently analyzed as described below.

For determining the concentrations of the metal in abscessed tissue, abscesses were extracted using LCM as described above. Each LCM cap contained approximately equal amounts of abscessed material. Samples were then solublized for approximately 8 hours in concentrated nitric acid (trace metal free) at 130° C. The concentration of Zn2+, Mn2+and Ca2+were subsequently analyzed as described below, and normalized to the dry weight of the sample. The values listed inFIG. 9represent the concentration of metal present in the abscessed tissue and any residual metal contaminating the LCM caps.

Sample analysis was performed by Applied Speciation (Tukwila, Wash.). A total of 10 mL of 5% HNO3(v/v) was added to each digestion vessel followed by oven digestion overnight at 80° C. The digests were allowed to cool to room temperature and were analyzed by inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS, Perkin Elmer DRC II). Aliquots of each sample are introduced into a radio frequency (RF) plasma where energy-transfer processes cause desolvation, atomization, and ionization. The ions are extracted from the plasma through a differentially-pumped vacuum interface and travel through a pressurized chamber (DRC) containing a specific reactive gas (see tables) which preferentially reacts with interfering ions of the same target mass to charge ratios (m/z). A solid-state detector detects ions transmitted through the mass analyzer, on the basis of their mass-to-charge ratio (m/z), and the resulting current is processed by a data handling system. Different reaction gases and settings are applied depending on the target analyte and projected interference. Comparison of the different isotopes, reaction gases, and reaction gas settings allow for interference monitoring and selection of optimum instrument settings depending on each sample matrix type and element.

Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS).

LA-ICP-MS was performed on 30 μM sections from murine liver tissue prepared as described below. A Nd:YAG laser system which operates at 266 nm (quadrupled wavelength) (Laser 200+, Cetac, USA) was used throughout the study with a large ablation cell from CETAC. Argon gas was used during ablation. The samples were ablated in scan mode using a 100 μm spot size with a scan speed of 25 μm/s, 20 Hz frequency and 100% energy (˜3.6 mJ). The ICP-MS system was a 7500c from Agilent (USA) used without reaction gas. The laser was directly coupled into the ICP-MS, by re-routing the nebulizer gas (argon) through the ablation cell using polyethylene tubing to connect the laser exit directly to the torch. Parameters were optimised either under wet plasma conditions before connecting the laser, or in the case of the nebulizer gas, with the laser connected to the system. The images were generated using Sigmaplot version 10.0.

Bacterial Strains, Media and Growth Conditions.

S. aureusNewman, a human clinical isolate (1), was used for all experiments in this study with the exception of the PMN lysate antimicrobial assays which employedS. aureusRN6390. This decision was made based on our observations that RN6390 exhibits increased sensitivity to neutrophil-dependent killing mechanisms (data not shown). mntR was inactivated by following a protocol described by Bae and Schneewind (2). Briefly, sequences flanking mntR were PCR amplified with primers SAV0634-5′1-AttB 1 (GGGGACAAGTTTGTACAAAAAAGCAGGCT-ACTGTACCACAAACTATCCC) (SEQ ID NO: 5) and SAV0634-3′1-Sal1 (CCCCGTCGACCCATTATTCGTAAGGATTGCC) (SEQ ID NO: 6) for the upstream fragment and primers SAV0634-3′2-AttB2 (GGGGACCACTTTGTACAAGAAAGCTGGGTAGGTAAGTCTAAAGTCTAACG) (SEQ ID NO: 7) and SAV0634-5′2-Sal1 (CCCCGTCGACGTCAGTTACGAAAATGCAATG) (SEQ ID NO: 8) for the downstream fragment. The PCR fragments were then assembled into pCR2.1 (Invitrogen) and recombined into pKOR1(2). Inactivation of mntR was achieved by allelic replacement with pKOR1ΔmntR and mutations subsequently verified by PCR. Strains defective in mntA or mntB were obtained from the Phoenix Library of definedS. aureustransposon mutations (3).S. aureusgrowth was monitored in TSB or NRPMI (Chelex-treated RPMI) supplemented with 100 μM CaCl2, 25 μM ZnCl2, 1 mM MgCl2, 25 μM MnCl2, and 5 μM FeSO4. The sensitivity ofS. aureusto calprotectin was assessed by conducting a dose response curve. Cultures were grown at 37° C. with shaking at 180 RPM in a 96-well microtiter plate and bacterial growth was monitored by measuring the increase in O.D.600over time. For microarray analyses overnight cultures of Newman were used to inoculate (1:100 dilution; flask to volume ratio of 5:1) 50 ml RPMI broth. RPMI was first incubated in the presence of calprotectin and then filtered to remove the protein and any metals it may have chelated. Cultures were incubated at 37° C. with shaking at 225 RPM until reaching O.D.600=0.25. Twenty ml of cells was added to an equal volume of ice cold acetone::ethanol (1:1) and stored at −80° C. for RNA isolation, as described below.

S. aureus(500 μl of a culture at O.D.6000.5) was mixed with purified calprotectin (0-250 μg/ml) at room temperature for 45 minutes with shaking at 180 RPM. Following incubation, cells were pelleted and the supernatant harvested. The cells were subsequently washed 3× in tryptic soy broth (TSB) medium and cells and supernatant fractions were mixed with sodium dodecyl sulfate (SDS)-loading buffer. To test for an interaction between S100A8 and whole cellS. aureus, the whole cell lysates and harvested supernatant fractions were run on a 10% SDS-polyacrylamide gel. Separated proteins were subjected to immunoblot using antisera specific to S100A8 (Santa Cruz Biotechnology).

Dialysis tubing (Spectra/Por Biotech) with a cutoff of 5,000 Da was cut into 10 mm sections and one end tied off in a knot. The tubing was prepared by first boiling for 1 hour in 1 mM EDTA, followed by boiling for 2 hours in distilled H2O to remove any bound metals. One ml of either buffer or calprotectin (2.5 mg/ml stock) was added to tubing which was tied at the open end, and placed into a 50 ml centrifuge tube containing 5 ml of TSB. The media were then inoculated withS. aureusand grown overnight at 37° C. with shaking at 180 RPM. The following morning bacterial growth was tracked by measuring changes in O.D.600.

To assess the metal binding properties of calprotectin we added an inhibitory dose of calprotectin (75 μg/ml) toS. aureuscells growing in chemically defined NRPMI medium. Mn2+and Zn2+were subsequently added to the growing staphylococcal culture in excess (1 mM) and growth monitored by measuring the increase in O.D.600over time.

Wild-type and a ΔmntR mutant strain were grown in high levels of Mn2+(1 mM) with or without calprotectin (40 μg/ml). Cell growth was monitored by measuring the increase in O.D.600over time.

PMNS were extracted from peripheral blood of human volunteers provided by the Cooperative Human Tissue Network at Vanderbilt University or from cellular filtrates from healthy human blood provided by the American Red Cross. PMNs were isolated from peripheral blood by differential migration in Ficoll-Paque PLUS and hypotonic lysis. Cells were re-suspended in lysis buffer (20 mM Tris pH 8.0, 120 mM NaCl, 3 mM CaCl2) and sonicated. The sonicated samples were subsequently centrifuged at 13,000×g for 10 minutes at 4° C. The supernatant was then transferred to an ultracentrifuge tube and centrifuged at 80,000 rpm (289,000×g) for 10 minutes at 4° C. The supernatant was subsequently filtered through a 0.2 μM filter, mixed with either an equal volume of RPMI or RPMI containing excess Mn2+(1 mM), and used for growth inhibition assays. Alternatively, cell lysates were mixed with protein G complexed with either 100 μg of monoclonal antibody to calprotectin (Calgranulin A/B (5.5)), or an isotype-matched control antibody (ebioscience) for two hours at 4° C. The preparation was then centrifuged for 1 minute at 1,000×g, the supernatant harvested, filtered through a 0.2 micron filter, and tested for anti-staphylococcal activity.

Mice were injected intraperitoneally with 1 ml of 3% thioglycollate. After 16 hours mice were sacrificed, and 10 ml of PBS injected into the peritoneal cavity. The injected wash was subsequently withdrawn while gently massaging the peritoneal wall. Peritoneal Exudate Cells (PECs) were subsequently resuspended in FACS buffer (phosphate buffered saline, pH 7.5, 0.05% NaN3, 5% fetal bovine serum) for flow cytometry analyses or RPMI/H for phagocytic killing assays. Flow cytometry analyses revealed that greater than 75% of the total PECs were neutrophils (CD11b+/Ly6G+).

PECs were counted using a hemacytometer following trypan blue staining and adjusted to a density of 1×107cells/ml in RPMI/H. Killing during a 2 hour incubation at 37° C. was assessed by combining 100 μl PECs with 100 μl ofS. aureus(2×107cells/ml) that were osponized with 50% normal mouse serum. Cells were subsequently treated with saponin (0.1% final) and incubated on ice for 15 minutes. Bacterial enumeration was performed following serial dilutions and plating on TSB media.

Protein Expression and Purification.

Recombinant human S100A8 and S100A9 protein were expressed and purified as described previously (4, 5). Briefly, competentEscherichia colistrain BL21 (DE3) cells (Novagen, Madison, Wis., USA) were transformed with the pET1120-MRP8 wt and pET1120-MRP14 wt vectors. Transformed cells were grown at 37° C. in (LB) media supplemented with 100 g/ml ampicillin. Protein was expressed into inclusion bodies and harvested by centrifugation at 7000×g at 4° C. Cells were lysed by sonication. Inclusion bodies were washed, solubilized in a refolding buffer, and purified over hydroxyapatite (Bio-Rad, Hercules, Calif., USA) and ResourceQ (GE Healthcare Bio-Sciences, Uppsala, Sweden) anion exchange resin.

Prior to experiments, S100A8/S100A9 was treated with 5 mM BAPTA and 10 mM DTT for 24 h at 25° C. to remove all divalent cations and reduce all cysteines. BAPTA and DTT were removed using a 50 mL desalting column (GE Healthcare) that had been extensively equilibrated in chelex treated 20 mM Tris at pH 7.5. All fluorescence cells were washed with a BAPTA solution followed by extensive washing with chelex treated buffer. Steady-state fluorescence experiments were performed on a SPEX FLUOROMAX spectrofluorimeter. Emission spectra were collected in 1 nm increments between 300 and 400 nm with the excitation wavelength for tryptophan set at 280 nm. S100A8/S100A9 concentration was 5 μM and data were collected at ambient temperature in a 3 mL cuvette.

Mouse Model of Infection.

Six to eight week old C57BL/6 (The Jackson Laboratory) or S100A9 −/− mice were infected with 1×106or 1×105colony forming units ofS. aureussuspended in phosphate buffered saline (PBS) by injection into the retro-orbital vein complex. Four days (96-hours) post-infection, mice were euthanized with CO2and the kidneys and liver removed and analyzed for abscess formation. Organs were subsequently prepared for FACS-based analyses as described or prepared for IMS, ICP-MS or IHC following flash freezing or fixing in 10% formalin. To partially or fully deplete PMNs, we administered a rat IgG2b anti-Gr-1 mAb RB6-8C5 as described by others (6, 7). S100A9 −/− mice represent 6thgeneration backcrossed with C57BL/6. All mice were maintained in compliance with Vanderbilt's institutional animal care and use committee regulations.

Parafin-embeded mouse tissues were stained with hematoxylin and eosin. Frozen tissue sections were stained with antiserum specific to S100A8 (Santa Cruz Biotechnology) by the Vanderbilt University Medical Center Immunohistochemistry Core Laboratory.

Flow Cytometry. Antibodies and reagents for cell surface staining were purchased from BD Pharmingen. Total erythrocyte-free kidney and liver lymphocytes and leukocytes of individual, age matched (˜7 weeks old) C57BL/6 female mice infected withS. aureus(1×106CFU) or uninfected as described above, were analyzed by four-color flow cytometric analysis. Neutrophils were gated as Ly6G+, CD11b+, B220−, F480−as described previously. Four-color flow cytometry was performed with a FACSCALIBUR® instrument (Becton Dickinson) and the data were analyzed using FlowJo software (Treestar Inc).

RNA purification. For RNA isolation, aliquots of acetone:ethanol cell suspensions were pelleted by centrifugation at 3000 RPM at 4° C. for 10 min in a tabletop centrifuge. The supernatant was discarded and cell pellet was resuspended in 500 ul TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). Cell suspensions were then transferred to BIO101 lysing matrix B tubes and were mechanically disrupted in a FastPrep 120 shaker. Cell debris was collected by centrifugation in a microcentrifuge at 4° C. for 15 min. Supernatants were used for RNA isolation using the Qiagen RNEASY® Tissue Kit following manufacturer's recommendations. RNA concentration was determined spectrophotometrically (OD 1.0=40 μg ml−1). The RNA integrity of ribosomal RNA was evaluated on 1.2% agarose-0.66M formaldehyde denaturing gels.

Nucleic acid labeling and GENECHIP® analysis. Ten micrograms of total bacterial RNA from each sample was labeled and hybridized toS. aureusGENECHIP® following the manufacturer's recommendations for antisense prokaryotic arrays (Affymetrix; Santa Clara, Calif.). Briefly, RNA was reverse transcribed in the presence of exogenous poly-A control RNAs (Affymetrix). Purified cDNA was then fragmented with DNase I (Amersham BioSciences; Piscataway, N.J.) and 3′ end-labeled with biotin using the Enzo Bioarray Terminal Labeling Kit (Enzo Life Sciences; Farmingdale, N.Y.). DNA was fragmented with DNase I and 3′ end-labeled as above. GENECHIP® were washed, stained, and scanned as previously described (8, 9). Commercially available GeneChips were used in this study representing >3,300S. aureusopen reading frames and >4,800 intergenic regions from strains N315, Mu50, NCTC 8325, and COL. GENECHIP® signal intensity values for each qualifier at each replicate time point (n≧2) were averaged and normalized to control oligo signals using GeneSpring 7.2 software (Silicon Genetics; Redwood City, Calif.).

Two-tailed t-tests were used to determine significant differences (P≦0.05); Error bars represent the standard deviation of at least three replicates, and all experiments were performed in triplicate. Pearson correlation average linkage bootstrapping was used to compareS. aureustranscriptional profiles.

Throughout this document, various references are mentioned. All such references are incorporated herein by reference, including the references set forth in the following list:

REFERENCES

MATERIALS AND METHODS REFERENCES