bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Contact Lens Wear, Bacterial Flora, and Mannose-Induced Pathogenic Protease in the Pathogenesis of Amoebic Keratitis
Authors: Alizadeh Hassan , Neelam Sudha , Hurt Michael , Niederkorn Jerry Y.
Published: January 2005

The ocular surface is continuously exposed to potential pathogens, including free-living amoebae.
Acanthamoeba
species are among the most ubiquitous amoebae, yet
Acanthamoeba
keratitis is remarkably rare. The pathogenesis of
Acanthamoeba
keratitis is a complex, sequential process. Here we show that
Acanthamoeba
keratitis is profoundly affected by mannosylated proteins on the ocular surface, which stimulate the amoebae to elaborate a 133-kDa pathogenic protease. The mannose-induced protease (MIP133) mediates apoptosis of the corneal epithelium, facilitates corneal invasion, and degrades the corneal stroma. We show that contact lens wear upregulates mannosylated proteins on the corneal epithelium, stimulates MIP133 secretion, and exacerbates corneal disease.
Corynebacterium xerosis
, a constituent of the ocular flora, contains large amounts of mannose and is associated with
Acanthamoeba
keratitis. The present results show that amoebae exposed to
C. xerosis
produce increased amounts of MIP133 and more severe corneal disease. Oral immunization with MIP133 mitigates
Acanthamoeba
keratitis and demonstrates the feasibility of antidisease vaccines for pathogens that resist immune elimination.
Murine Macrophage Transcriptional Responses to
Bacillus anthracis
Infection and Intoxication
Authors: Bergman Nicholas H. , Passalacqua Karla D. , Gaspard Renee , Shetron-Rama Lynne M. , Quackenbush John , Hanna Philip C.
Published: January 2005

Interactions between
Bacillus anthracis
and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with
B. anthracis
and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to
B. anthracis
but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying
B. anthracis
-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in
B. anthracis
-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into
B. anthracis
pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.
Murein Lipoprotein Is a Critical Outer Membrane Component Involved inSalmonella entericaSerovar Typhimurium Systemic Infection
Authors: Fadl A. A. , Sha J. , Klimpel G. R. , Olano J. P. , Niesel D. W. , Chopra A. K.
Published: January 2005

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppAandlppB) located on the chromosome ofSalmonella entericaserovar Typhimurium contributed to bacterial virulence. In this study, we characterizedlppAandlppBsingle-knockout (SKO) mutants and compared them with anlppdouble-knockout (DKO) mutant using in vitro and in vivo models. Compared to thelppDKO mutant, which was nonmotile, the motility of thelppSKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT)S. entericaserovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with thelppSKO mutants compared to the cytotoxicity when cells were infected with thelppDKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with thelppBSKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WTS. entericaserovar Typhimurium, than the level in host cells infected with thelppASKO mutant. ThelppDKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with thelppBSKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-α and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in thelppASKO mutant-infected mice than in animals challenged with thelppDKO mutant. The increased cytokine levels observed with thelppBSKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with thelppASKO andlppDKO mutants. Moreover, thelppBSKO mutant-infected mice had increased susceptibility to death. Since thelppDKO mutant retained intact LPS, we constructed anS. entericaserovar Typhimurium triple-knockout (TKO) mutant in which thelppAandlppBgenes were deleted from an existingmsbBmutant (msbBencodes an enzyme required for the acylation of lipid A). Compared to thelppDKO andmsbBSKO mutants, thelpp-msbBTKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation toSalmonellapathogenesis.
Phagosomal Processing ofMycobacterium tuberculosisAntigen 85B Is Modulated Independently of Mycobacterial Viability and Phagosome Maturation
Authors: Ramachandra Lakshmi , Smialek Jamie L. , Shank Sam S. , Convery Marilyn , Boom W. Henry , Harding Clifford V.
Published: January 2005

Control ofMycobacterium tuberculosisinfection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing ofM. tuberculosisantigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK)M. tuberculosismore efficiently than liveM. tuberculosis. These observations suggested that liveM. tuberculosismay inhibit Ag processing by inhibiting phagosome maturation or that HKM. tuberculosismay be less resistant to Ag processing. In the present study we examined the correlation betweenM. tuberculosisviability and phagosome maturation and efficiency of Ag processing. Since heat treatment could renderM. tuberculosisAgs more accessible to proteolysis,M. tuberculosiswas additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK)M. tuberculosisin activated murine bone marrow macrophages was examined by using an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HKM. tuberculosismore rapidly and efficiently than they processed live, RadK, or RifKM. tuberculosis. Live, RadK, and RifKM. tuberculosiscells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadKM. tuberculosisexpressed fewerM. tuberculosispeptide-MHC-II complexes than phagosomes containing HKM. tuberculosisexpressed. Since only liveM. tuberculosiswas able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms ofM. tuberculosis. These findings suggest that the mechanisms used byM. tuberculosisto inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
The Capsular Polysaccharide ofBurkholderia pseudomalleiContributes to Survival in Serum by Reducing Complement Factor C3b Deposition
Authors: Reckseidler-Zenteno Shauna L. , DeVinney Rebekah , Woods Donald E.
Published: January 2005

Burkholderia pseudomalleiproduces an extracellular polysaccharide capsule -3)-2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(1- which has been shown to be an essential virulence determinant. The addition of purified capsule was shown to increase the virulence of a capsule mutant strain in the Syrian hamster model of acute melioidosis. An increase in the number of wild-typeB. pseudomalleicells in the blood was seen by 48 h, while the number of capsule mutant cells in the blood declined by 48 h. Capsule expression was shown to be induced in the presence of serum using aluxreporter fusion to the capsule genewcbB. The addition of purifiedB. pseudomalleicapsule to serum bactericidal assays increased the survival ofB. pseudomalleiSLR5, a serum-sensitive strain, by 1,000-fold in normal human serum. Capsule production byB. pseudomalleicontributed to reduced activation of the complement cascade by reducing the levels of complement factor C3b deposition. An increase in phagocytosis of the capsule mutant compared to the wild type was observed in the presence of normal human serum. These results suggest that the production of this capsule contributes to resistance to phagocytosis by reducing C3b deposition on the surface of the bacterium, thereby contributing to the persistence of bacteria in the blood of the infected host. Continued studies to characterize this capsule are essential for understanding the pathogenesis ofB. pseudomalleiinfections and the development of preventive strategies for treatment of this disease.
Localized Multigene Expression Patterns Support an Evolving Th1/Th2-Like Paradigm in Response to Infections withToxoplasma gondiiandAscaris suum
Authors: Dawson Harry D. , Beshah Ethiopia , Nishi Sandra , Solano-Aguilar Gloria , Morimoto Motoko , Zhao Aiping , Madden Kathleen B. , Ledbetter Tonya K. , Dubey J. P. , Shea-Donohue Terez , Lunney Joan K. , Urban Joseph F.
Published: January 2005

Human infectious diseases have been studied in pigs because the two species have common microbial, parasitic, and zoonotic organisms, but there has been no systematic evaluation of cytokine gene expression in response to infectious agents in porcine species. In this study, pigs were inoculated with two clinically and economically important parasites,Toxoplasma gondiiandAscaris suum, and gene expression in 11 different tissues for 20 different swine Th1/Th2-related cytokines, cytokine receptors, and markers of immune activation were evaluated by real-time PCR. A generalized Th1-like pattern of gene expression was evident in pigs infected withT. gondii, along with an increased anti-inflammatory gene expression pattern during the recovery phase of the infection. In contrast, an elevated Th2-like pattern was expressed during the period of expulsion ofA. suumfourth-stage larvae from the small intestine of pigs, along with low-level Th1-like and anti-inflammatory cytokine gene expression. Prototypical immune and physiological markers of infection were observed in bronchial alveolar lavage cells, small intestinal smooth muscle, and epithelial cells. This study validated the use of a robust quantitative gene expression assay to detect immune and inflammatory markers at multiple host tissue sites, enhanced the definition of two important swine diseases, and supported the use of swine as an experimental model for the study of immunity to infectious agents relevant to humans.
The Region Comprising Amino Acids 100 to 255 ofNeisseria meningitidisLipoprotein GNA 1870 Elicits Bactericidal Antibodies
Authors: Giuliani Marzia Monica , Santini Laura , Brunelli Brunella , Biolchi Alessia , Aricò Beatrice , Di Marcello Federica , Cartocci Elena , Comanducci Maurizio , Masignani Vega , Lozzi Luisa , Savino Silvana , Scarselli Maria , Rappuoli Rino , Pizza Mariagrazia
Published: January 2005

GNA 1870 is a novel surface-exposed lipoprotein, identified by genome analysis ofNeisseria meningitidisstrain MC58, which induces bactericidal antibodies. Three sequence variants of the protein were shown to be sufficient to induce bactericidal antibodies against a panel of strains representative of the diversity of serogroup B meningococci. Here, we studied the antigenic and immunogenic properties of GNA 1870, which for convenience was divided into domains A, B, and C. The immune responses of mice immunized with each of the three variants were tested using overlapping peptides scanning the entire protein length and using recombinant fragments. We found that while most of the linear epitopes are located in the A domain, the bactericidal antibodies are directed against conformational epitopes located in the BC domain. This was also confirmed by the isolation of a bactericidal murine monoclonal antibody, which failed to recognize linear peptides on the A, B, and C domains separately but recognized a conformational epitope formed only by the combination of the B and C domains. Arginine in position 204 was identified as important for binding of the monoclonal antibody. The identification of the region containing bactericidal epitopes is an important step in the design of new vaccines against meningococci.
Mouse Model of Enteropathogenic
Escherichia coli
Infection
Authors: Savkovic Suzana D. , Villanueva Jennilee , Turner Jerrold R. , Matkowskyj Kristina A. , Hecht Gail
Published: January 2005

Enteropathogenic
Escherichia coli
(EPEC) is an important cause of diarrhea in humans. EPEC infection of cultured intestinal epithelial cells induces attaching and effacing (A/E) lesions, alters intestinal ion transport, increases paracellular permeability, and stimulates inflammation. The lack of a small-animal model has restricted in vivo studies examining EPEC-host interactions. The aim of this study was to characterize the C57BL/6J mouse as a model of EPEC infection. We have shown that EPEC can adhere to and colonize the intestinal epithelium of C57BL/6J mice. Animal weight and water intake were not altered during 10 days of EPEC infection. The proximal colon of infected mice contained semisolid stool, with stool pellets forming only in the distal colon. In contrast, the entire colon of control mice contained formed stool. Microvillous effacement and actin rearrangement, characteristic of A/E lesions, were seen in the intestine of infected mice but not control mice. Histological assessment revealed increased numbers of lamina propria neutrophils with occasional crypt abscesses, intraepithelial lymphocytes, and goblet cells in the intestine of EPEC-infected mice. Altogether, these data suggest that the C57BL/6J mouse is susceptible to infection by EPEC and will provide a suitable in vivo model for studying the consequences of EPEC infection.
Long-Term Survival and Intracellular Replication of
Mycoplasma hominis
in
Trichomonas vaginalis
Cells: Potential Role of the Protozoon in Transmitting Bacterial Infection
Authors: Dessì Daniele , Delogu Giuseppe , Emonte Eleonora , Catania Maria Rosaria , Fiori Pier Luigi , Rappelli Paola
Published: January 2005

The existence of a symbiotic relationship between
Trichomonas vaginalis
and
Mycoplasma hominis
, which is the first reported example of symbiosis between two obligate human pathogens, has been recently reported by our research group. In this work, we examined the cellular location of
M. hominis
in respect to
T. vaginalis
. By using gentamicin protection assays, double immunofluorescence, and confocal microscopy, we obtained strong evidence that
M. hominis
is located within protozoan cells. 5-Bromodeoxyuridine incorporation assays showed that intracellularly located mycoplasmas actively synthesize DNA. Our results demonstrate that
M. hominis
has the capability of entering trichomonad cells and of replicating inside the protozoon. These findings suggest that symbiosis might provide the bacteria, during human infection, with the capability to resist to environmental stresses, such as host defense mechanisms and pharmacological therapies.
Defective Phagocytosis in
Anaplasma phagocytophilum
- Infected Neutrophils
Authors: Garyu Justin W. A. , Choi Kyoung-seong , Grab Dennis J. , Dumler J. Stephen
Published: January 2005

Anaplasma phagocytophilum
infection induces functional neutrophil changes. Using both
Candida albicans
and fluorescent-aggregate phagocytosis assays, we examined whether neutrophil and dimethyl sulfoxide-differentiated HL-60 cell infection impairs internalization.
A. phagocytophilum
infection significantly decreased phagocytosis compared to that of controls (
P
< 0.05). This further impairment of neutrophil function may promote opportunistic infections and exacerbate disease.
Long-Term Protection against Tuberculosis following Vaccination with a Severely Attenuated Double Lysine and Pantothenate Auxotroph ofMycobacterium tuberculosis
Authors: Sambandamurthy Vasan K. , Derrick Steven C. , Jalapathy Kripa V. , Chen Bing , Russell Robert G. , Morris Sheldon L. , Jacobs William R.
Published: January 2005

We report the safety and immunogenicity of a double lysine and pantothenate auxotroph ofMycobacterium tuberculosisin mice. The ΔlysAΔpanCDmutant is completely attenuated in immunocompromised SCID and gamma interferon knockout mice yet induces short-term and long-term protection in immunocompetent and CD4-deficient mice following single-dose subcutaneous vaccination.
Salmonella
-Induced Filament Formation Is a Dynamic Phenotype Induced by Rapidly Replicating
Salmonella enterica
Serovar Typhimurium in Epithelial Cells
Authors: Birmingham Cheryl L. , Jiang Xiuju , Ohlson Maikke B. , Miller Samuel I. , Brumell John H.
Published: January 2005

Salmonella enterica
serovar Typhimurium has the fascinating ability to form tubular structures known as
Salmonella
-induced filaments (Sifs) in host cells. Here, we show that the prevalence of the Sif phenotype in HeLa cells is affected by host cell density, growth, and the multiplicity of infection. Sif formation was observed in cells that displayed rapid intracellular bacterial replication and was found to be dynamic, being maximal 8 to 10 h postinfection and declining thereafter. The virulence factors SpvB and SseJ were found to negatively modulate Sif formation. Our findings demonstrate the complex and dynamic nature of the Sif phenotype.
Colonization of Gnotobiotic Piglets by a
luxS
Mutant Strain of
Escherichia coli
O157:H7
Authors: Jordan Dianna M. , Sperandio Vanessa , Kaper James B. , Dean-Nystrom Evelyn A. , Moon Harley W.
Published: January 2005

Gnotobiotic piglets inoculated with
Escherichia coli
O157:H7, its
luxS
mutant derivative, or nonpathogenic
E. coli
were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the
luxS
mutant, the
luxS
mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.
Modulation of an Outer Membrane Protease Contributes to the Virulence Defect of
Shigella flexneri
Strains Carrying a Mutation in the
virK
Locus
Authors: Wing Helen J. , Goldman Seth R. , Ally Shabeen , Goldberg Marcia B.
Published: January 2005

The
Shigella
actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of
virK
::Tn
10
mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of
Shigella
virulence.
Identification of New
hmwA
Alleles from Nontypeable
Haemophilus influenzae
Authors: Ecevit I. Zafer , McCrea Kirk W. , Marrs Carl F. , Gilsdorf Janet R.
Published: January 2005

High-molecular-weight proteins of
Haemophilus influenzae
mediate attachment to epithelial cells. Previous reports describe several allelic versions of
hmwA
genes that have different adherence properties. Here we report three new alleles of
hmwA
(
hmwA
from strain AAr96,
hmwA
from strain AAr105, and
hmwA
from strain G822), demonstrating the high degree of DNA variation of these genes among different strains.
Down-Regulation of the
kps
Region 1 Capsular Assembly Operon following Attachment of
Escherichia coli
Type 1 Fimbriae to
d
-Mannose Receptors
Authors: Schwan William R. , Beck Michael T. , Hultgren Scott J. , Pinkner Jerry , Woolever Nathan L. , Larson Thomas
Published: January 2005

A differential-display PCR procedure identified the capsular assembly gene
kpsD
after
Escherichia coli
type 1 fimbrial binding to mannose-coated Sepharose beads. Limiting-dilution reverse-transcribed PCRs confirmed down-regulation of the
kpsD
gene, and Northern blot and
lacZ
fusion analyses showed down-regulation of the
kpsFEDUCS
region 1 operon. KpsD protein levels fell, and an agglutination test showed less K capsular antigen on the surface following the bacterial ligand-receptor interaction. These data show that binding of type 1 fimbriae (pili) to
d
-mannose receptors triggers a cross talk that leads to down-regulation of the capsule assembly region 1 operon in uropathogenic
E. coli
.
Plasmodium falciparum
Merozoite Surface Protein 6 Displays Multiple Targets for Naturally Occurring Antibodies That Mediate Monocyte-Dependent Parasite Killing
Authors: Singh Subhash , Soe Soe , Roussilhon Christian , Corradin Giampietro , Druilhe Pierre
Published: January 2005

Plasmodium falciparum
MSP6 is a merozoite surface antigen that shows organization and sequence homologies similar to those of MSP3. Within its C-terminus conserved region, it presents some epitopes that are cross-reactive with MSP3 and others that are not, both being targets of naturally occurring antibodies that block the
P. falciparum
erythrocytic cycle in cooperation with monocytes.
CaNAT1
, a Heterologous Dominant Selectable Marker for Transformation of
Candida albicans
and Other Pathogenic
Candida
Species
Authors: Shen Junqing , Guo Weihui , Köhler Julia R.
Published: January 2005

A dominant selectable marker for
Candida albicans
and other
Candida
species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of
C. albicans
genes. Neither growth nor filamentous development was affected in strains expressing this marker.
Interaction of Enteropathogenic
Escherichia coli
with Human Intestinal Mucosa: Role of Effector Proteins in Brush Border Remodeling and Formation of Attaching and Effacing Lesions
Authors: Shaw Robert K , Cleary Jennifer , Murphy Michael S. , Frankel Gad , Knutton Stuart
Published: January 2005

Enteropathogenic
Escherichia coli
(EPEC) strains deliver effector proteins Tir, EspB, Map, EspF, EspH, and EspG into host cells to induce brush border remodeling and produce attaching and effacing (A/E) lesions on small intestinal enterocytes. In this study, the role of individual EPEC effectors in brush border remodeling and A/E lesion formation was investigated with an in vitro human small intestinal organ culture model of EPEC infection and specific effector mutants.
tir
,
map
,
espB
, and
espH
mutants produced “footprint” phenotypes due to close bacterial adhesion but subsequent loss of bacteria; an
espB
mutant and other type III secretion system mutants induced a “noneffacing footprint” associated with intact brush border microvilli, whereas a
tir
mutant was able to efface microvilli resulting in an “effacing footprint”;
map
and
espH
mutants produced A/E lesions, but loss of bacteria resulted in a “pedestal footprint.” An
espF
mutant produced typical A/E lesions without associated microvillous elongation. An
espG
mutant was indistinguishable from the wild type. These observations indicate that Tir, Map, EspF, and EspH effectors play a role in brush border remodeling and production of mature A/E lesions.
Mutagenesis of a Novel Gene in the
prcA-prtP
Protease Locus Affects Expression of
Treponema denticola
Membrane Complexes
Authors: Bian Xue-lin , Wang Hong-tao , Ning Yu , Lee Si Young , Fenno J. Christopher
Published: January 2005

A novel gene was identified in the
Treponema denticola prcA-prtP
protease operon. Strains with mutations in either the
prcA-prtP
or the
msp
region showed altered expression of a product(s) of the other locus. Together, these results provide information on the assembly of outer membrane complexes involved in
T. denticola
interaction with host cells and tissue.
Characterization of an Extracellular Dipeptidase from
Streptococcus gordonii
FSS2
Authors: Goldstein J. M. , Kordula T. , Moon J. L. , Mayo J. A. , Travis J.
Published: January 2005

PepV, a dipeptidase found in culture fluids of
Streptococcus gordonii
FSS2, was purified and characterized, and its gene was cloned. PepV is a monomeric metalloenzyme of approximately 55 kDa that preferentially degrades hydrophobic dipeptides. The gene encodes a polypeptide of 467 amino acids, with a theoretical molecular mass of 51,114 Da and a calculated pI of 4.8. The
S. gordonii
PepV gene is homologous to the PepV gene family from
Lactobacillus
and
Lactococcus
spp.
A Type IV Pilin, PilA, Contributes to Adherence ofBurkholderia pseudomalleiand Virulence In Vivo
Authors: Essex-Lopresti Angela E. , Boddey Justin A. , Thomas Richard , Smith Martin P. , Hartley M. Gill , Atkins Timothy , Brown Nat F. , Tsang Chuk Hai , Peak Ian R. A. , Hill Jim , Beacham Ifor R. , Titball Richard W.
Published: January 2005

TheBurkholderia pseudomalleiK96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). ApilAdeletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili inB. pseudomalleivirulence.
Oral Mucosal Endotoxin Tolerance Induction in Chronic Periodontitis
Authors: Muthukuru Manoj , Jotwani Ravi , Cutler Christopher W.
Published: January 2005

The oral mucosa is exposed to a high density and diversity of gram-positive and gram-negative bacteria, but very little is known about how immune homeostasis is maintained in this environment, particularly in the inflammatory disease chronic periodontitis (CP). The cells of the innate immune response recognize bacterial structures via the Toll-like receptors (TLR). This activates intracellular signaling and transcription of proteins essential for the induction of an adaptive immune response; however, if unregulated, it can lead to destructive inflammatory responses. Using single-immunoenzyme labeling, we show that the human oral mucosa (gingiva) is infiltrated by large numbers of TLR2+and TLR4+cells and that their numbers increase significantly in CP, relative to health (P< 0.05, Student'sttest). We also show that the numbers of TLR2+but not TLR4+cells increase linearly with inflammation (r2= 0.33,P< 0.05). Double-immunofluorescence analysis confirms that TLR2 is coexpressed by monocytes (MC)/macrophages (mφ) in situ. Further analysis of gingival tissues by quantitative real-time PCR, however, indicates that despite a threefold increase in the expression of interleukin-1β (IL-1β) mRNA during CP, there is significant (30-fold) downregulation of TLR2 mRNA (P< 0.05, Student'sttest). Also showing similar trends are the levels of TLR4 (ninefold reduction), TLR5 (twofold reduction), and MD-2 (sevenfold reduction) mRNA in CP patients compared to healthy persons, while the level of CD14 was unchanged. In vitro studies with human MC indicate that MC respond to an initial stimulus of lipopolysaccharide (LPS) fromPorphyromonas gingivalis(PgLPS) orEscherichia coli(EcLPS) by upregulation of TLR2 and TLR4 mRNA and protein; moreover, IL-1β mRNA is induced and tumor necrosis factor alpha (TNF-α), IL-10, IL-6, and IL-8 proteins are secreted. However, restimulation of MC with either PgLPS or EcLPS downregulates TLR2 and TLR4 mRNA and protein and IL-1β mRNA and induces a ca. 10-fold reduction in TNF-α secretion, suggesting the induction of endotoxin tolerance by either LPS. Less susceptible to tolerance than TNF-α were IL-6, IL-10, and IL-8. These studies suggest that certain components of the innate oral mucosal immune response, most notably TLRs and inflammatory cytokines, may become tolerized during sustained exposure to bacterial structures such as LPS and that this may be one mechanism used in the oral mucosa to attempt to regulate local immune responses.
Bioluminescence Imaging of
Toxoplasma gondii
Infection in Living Mice Reveals Dramatic Differences between Strains
Authors: Saeij Jeroen P. J. , Boyle Jon P. , Grigg Michael E. , Arrizabalaga Gustavo , Boothroyd John C.
Published: January 2005

We examined the in vivo growth, dissemination, and reactivation of strains of the protozoan parasite
Toxoplasma gondii
using a bioluminescence-based imaging system. Two
T. gondii
strains, one with a highly virulent disease phenotype in mice (S23) and the other with a 1,000-fold-lower virulence phenotype (S22), were engineered to stably express the light-emitting protein luciferase. One clone of each wild-type strain was isolated, and the two clones (S23-luc7 and S22-luc2) were found to express similar levels of luciferase. Mice were infected intraperitoneally with S23-luc7 (50 or 5 parasites) or S22-luc2 (500, 50, or 5 parasites), and the progress of the infections was examined noninvasively following injection of the substrate for luciferase,
d
-luciferin. In mice infected with 50 S23-luc7 parasites, the parasites grew exponentially within the peritoneal cavity (as measured by light emitted from luciferase-expressing parasites) during days 1 to 10 p.i., and this proliferation continued until there was severe disease. In mice infected with 500 S22-luc2 parasites, the parasites proliferated in a fashion similar to the S23-luc7 proliferation during days 1 to 6, but this was followed by a precipitous drop in the signal to levels below the limit of detection. Using this technique, we were also able to observe the process of reactivation of
T. gondii
in chronically infected mice. After treatment with dexamethasone, we detected reactivation of toxoplasmosis in mice infected with S23-luc7 and S22-luc2. During reactivation, growth of S23-luc7 was initially detected primarily in the head and neck area, while in S22-luc2-infected mice the parasites were detected primarily in the abdomen. This method has great potential for identifying important differences in the dissemination and growth of different
T. gondii
strains, especially strains with dramatically different disease outcomes.
Class I Major Histocompatibility Complex Presentation of Antigens That Escape from the Parasitophorous Vacuole ofToxoplasma gondii
Authors: Gubbels Marc-Jan , Striepen Boris , Shastri Nilabh , Turkoz Mustafa , Robey Ellen A.
Published: January 2005

The intracellular parasiteToxoplasma gondii, the causative agent of toxoplasmosis, induces a protective CD8 T-cell response in its host; however, the mechanisms by whichT. gondiiproteins are presented by the class I major histocompatibility complex remain largely unexplored.T. gondiiresides within a specialized compartment, the parasitophorous vacuole, that sequesters the parasite and its secreted proteins from the host cell cytoplasm, suggesting that an alternative cross-priming pathway might be necessary for class I presentation ofT. gondiiantigens. Here we used a strain ofT. gondiiexpressing yellow fluorescent protein and a secreted version of the model antigen ovalbumin to investigate this question. We found that presentation of ovalbumin secreted by the parasite requires the peptide transporter TAP (transporter associated with antigen processing) and occurs primarily in actively infected cells rather than bystander cells. We also found that dendritic cells are a major target ofT. gondiiinfection in vivo and account for much of the antigen-presenting activity in the spleen. Finally, we obtained evidence that Cre protein secreted byT. gondiican mediate recombination in the nucleus of the host cell. Together, these results indicate thatToxoplasmaproteins can escape from the parasitophorous vacuole into the host cytoplasm and be presented by the endogenous class I pathway, leading to direct recognition of infected cells by CD8 T cells.
HtrA Stress Protein Is Involved in Intramacrophagic Replication of Adherent and Invasive
Escherichia coli
Strain LF82 Isolated from a Patient with Crohn's Disease
Authors: Bringer Marie-Agnès , Barnich Nicolas , Glasser Anne-Lise , Bardot Olivier , Darfeuille-Michaud Arlette
Published: January 2005

Adherent and invasive
Escherichia coli
(AIEC) bacteria isolated from Crohn's disease patients are able to greatly replicate within macrophages without escaping from the phagosome and without inducing macrophage death. In the present study, evidence is provided that in AIEC strain LF82 the
htrA
gene encoding the stress protein HtrA is essential for intracellular replication within J774-A1 macrophages. Deletion of the
htrA
gene in strain LF82 induced increased sensitivity of the isogenic mutant to oxidative stress caused by hydrogen peroxide and a reduced rate of growth in an acid and nutrient-poor medium partly reproducing the microenvironment of the phagosome. In vitro experiments using an LF82
htrA
gene promoter fusion with the
lacZ
gene revealed a 38-fold activation of the promoter in AIEC LF82 intramacrophagic bacteria. The CpxRA two-component signaling pathway was not involved in this activation. In addition, the activation of the LF82
htrA
gene promoter was not observed in the nonpathogenic
E. coli
K-12 intramacrophagic bacteria, indicating that the AIEC LF82 genetic background is crucial for induction of
htrA
gene transcription during phagocytosis.
Contribution of the ATP-Dependent Protease ClpCP to the Autolysis and Virulence of
Streptococcus pneumoniae
Authors: Ibrahim Yasser Musa , Kerr Alison R. , Silva Nuno A. , Mitchell Tim J.
Published: January 2005

The ATP-dependent caseinolytic proteases (Clp) are fundamental for stress tolerance and virulence in many pathogenic bacteria. The role of ClpC in the autolysis and virulence of
Streptococcus pneumoniae
is controversial. In this study, we tested the role of ClpC in a number of
S. pneumoniae
strains and found that the contribution of ClpC to autolysis is strain dependent. ClpC is required for the release of autolysin A and pneumolysin in serotype 2
S. pneumoniae
strain D39. In vivo, ClpC is required for the growth of the pneumococcus in the lungs and blood in a murine model of disease, but it does not affect the overall outcome of pneumococcal disease. We also report the requirement of ClpP for the growth at elevated temperature and virulence of serotype 4 strain TIGR4 and confirm its contribution to the thermotolerance, oxidative stress resistance, and virulence of D39.
Evaluation of the Role of the Bvg Intermediate Phase in
Bordetella pertussis
during Experimental Respiratory Infection
Authors: Vergara-Irigaray Nuria , Chávarri-Martínez Alberto , Rodríguez-Cuesta Juan , Miller Jeff F. , Cotter Peggy A. , Martínez de Tejada Guillermo
Published: January 2005

The BvgAS system of
Bordetella pertussis
was traditionally considered to mediate a transition between two phenotypic phases (Bvg
+
and Bvg
−
) in response to environmental signals. We characterized a third state, the intermediate (Bvg
i
) phase, which can be induced by introducing a 1-bp substitution into
bvgS
(the
bvgS
-I1 mutation) or by growing
B. pertussis
under conditions intermediate between those leading to the Bvg
+
and Bvg
−
phases. Like
B. bronchiseptica
,
B. pertussis
displays in its Bvg
i
phase a characteristic colony morphology and hemolytic activity and expresses a Bvg
i
-phase-specific polypeptide called BipA, whose synthesis is regulated by
bvgAS
at the transcriptional level. Based on our results, we hypothesize that the Bvg
i
phase of
B. pertussis
may be involved in facilitating transmission between hosts. Thus, a
B. pertussis
mutant carrying the
bvgS
-I1 mutation (GMT1i) persisted at wild-type levels only in the upper murine respiratory tract. Interestingly, a
bipA
deletion derivative of GMT1i displayed a reduced ability to colonize the nasal cavity of mice compared with GMT1i. However, in experimental mixed infections GMT1i expressing the Bvg
i
phase could establish an initial colonization in the nose and trachea of mice as efficiently as GMT1, but the wild-type strain outcompeted GMT1i at a later time point at all sites of the respiratory tract, suggesting that the Bvg
i
phase does not serve as a phenotypic phase specialized in colonization. Finally, even though
B. pertussis
expresses in vitro the Bvg
i
phase at the human nasal temperature, anti-BipA antibodies were undetectable in a large collection of sera from pertussis patients.
Helicobacter pyloriInduces Transendothelial Migration of Activated Memory T Cells
Authors: Enarsson Karin , Brisslert Mikael , Backert Steffen , Quiding-Järbrink Marianne
Published: January 2005

Helicobacter pyloriinfection is associated with pronounced infiltration of granulocytes and lymphocytes into the gastric mucosa, resulting in active chronic gastritis that may develop into duodenal ulcer disease or gastric adenocarcinoma. Infiltrating T cells play a major role in the pathology of these diseases, but the signals involved in recruitment of T cells from blood toH. pylori-infected tissues are not well understood. We therefore examinedH. pylori-induced T-cell transendothelial migration (TEM). The Transwell system, employing a monolayer of human umbilical vein endothelial cells, was used as a model to study TEM.H. pyloriinduced a significant T-cell migration, compared to spontaneous migration. CD4+and CD8+T cells migrated to the same extent in response toH. pylori, whereas there was significantly larger transmigration of memory T cells compared to naive T cells. BothH. pyloriculture filtrate and urease induced migration, and the presence of theH. pylori cagpathogenicity island increased TEM. T-cell TEM was mediated by LFA-1-ICAM-1 interactions in accordance with an increased ICAM-1 expression on the endothelial cells after contact withH. pylori. Migrating T cells had increased expression of activation marker CD69 and chemokine receptors CXCR3, CCR4, and CCR9. Furthermore, T cells migrating in response toH. pylorisecreted Th1 but not Th2 cytokines upon stimulation. In conclusion, our data indicate that liveH. pyloriand its secreted products contribute to T-cell recruitment to the gastric mucosa and that the responding T cells have an activated memory Th1 phenotype.
Enhanced Killing ofCandida albicansby Human Macrophages Adherent to Type 1 Collagen Matrices via Induction of Phagolysosomal Fusion
Authors: Newman Simon L. , Bhugra Bindu , Holly Angela , Morris Randal E.
Published: January 2005

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candidaactivity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes inC. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candidaactivity and thatC. albicanspathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.
Rhizopus oryzae
Adheres to, Is Phagocytosed by, and Damages Endothelial Cells In Vitro
Authors: Ibrahim Ashraf S. , Spellberg Brad , Avanessian Valentina , Fu Yue , Edwards John E.
Published: January 2005

Rhizopus oryzae
is the most common cause of zygomycosis, a life-threatening infection that usually occurs in immunocompromised patients. A characteristic hallmark of zygomycosis is angioinvasion by the fungus, resulting in thrombosis and subsequent tissue necrosis. Interactions between
R. oryzae
and vascular endothelial cells are therefore likely of central importance to the organism's pathogenetic strategy. We studied the ability of
R. oryzae
to adhere to and damage human umbilical vein endothelial cells (HUVECs) in vitro. We report that
R. oryzae
spores and germ tubes adhere to HUVECs, whereas only spores adhere to subendothelial matrix proteins. Additionally,
R. oryzae
damages endothelial cells. This endothelial cell damage requires direct contact and subsequent phagocytosis of the fungus. Surprisingly,
R. oryzae
viability was not required for damage, but phagocytosis was required for dead
R. oryzae
to cause damage. These results elucidate the nature of
R. oryzae
-endothelial cell interactions, which are likely central to the angioinvasion and tissue necrosis seen during zygomycotic infections. The fact that dead
R. oryzae
damage human endothelial cells may, in part, explain the lack of efficacy of fungicidal agents during clinical disease.
Closely Related Mycobacterial Strains Demonstrate Contrasting Levels of Efficacy as Antitumor Vaccines and Are Processed for Major Histocompatibility Complex Class I Presentation by Multiple Routes in Dendritic Cells
Authors: Cheadle Eleanor J. , O'Donnell Dearbhaile , Selby Peter J. , Jackson Andrew M.
Published: January 2005

Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growingMycobacterium smegmatisand the slow-growingM. bovis M. bovisBCG, were engineered to express a model tumor antigen, the Kb-restricted dominant cytotoxic T-lymphocyte epitope OVA257-264. RecombinantM. bovisBCG but not recombinantM. smegmatisconferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinantM. smegmatisinfection led to a greater degree of dendritic cell maturation than recombinantM. bovisBCG infection. Antigen from recombinantM. smegmatiswas processed and presented as OVA257-264on Kbmolecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinantM. bovisBCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growingM. bovisBCG but not that vectored by fast-growing, readily destroyedM. smegmatisis processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.
A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized
Bacillus anthracis
Spore Challenge
Authors: Mohamed Nehal , Clagett Michelle , Li Juan , Jones Steven , Pincus Steven , D'Alia Giovanni , Nardone Linda , Babin Michael , Spitalny George , Casey Leslie
Published: January 2005

We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge (∼60 to 450 times the 50% lethal dose of
Bacillus anthracis
Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of
B. anthracis
spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax.
Chemotaxis Plays Multiple Roles during
Helicobacter pylori
Animal Infection
Authors: Terry Karianne , Williams Susan M. , Connolly Lynn , Ottemann Karen M.
Published: January 2005

Helicobacter pylori
is a human gastric pathogen associated with gastric and duodenal ulcers as well as specific gastric cancers.
H. pylori
infects approximately 50% of the world's population, and infections can persist throughout the lifetime of the host. Motility and chemotaxis have been shown to be important in the infection process of
H. pylori
. We sought to address the specific roles of chemotaxis in infection of a mouse model system. We found that mutants lacking
cheW
,
cheA
, or
cheY
are all nonchemotactic and infect FVB/N mice with an attenuated phenotype after 2 weeks of infection. If infections proceeded for 6 months, however, this attenuation disappeared. Histological and culture analysis revealed that nonchemotactic mutants were found only in the corpus of the stomach, while the wild type occupied both the corpus and the antrum. Further analysis showed that nonchemotactic
H. pylori
isolates had an increased 50% infectious dose and were greatly outcompeted when coinfected with the wild type. If nonchemotactic mutants were allowed to establish an infection, subsequent infection with the wild type partially displaced the nonchemotactic mutants, indicating a role for chemotaxis in maintenance of infection. The data presented here support four roles for chemotaxis in
H. pylori
mouse infections: (i) establishing infection, (ii) achieving high-level infection, (iii) maintaining an infection when there are competing
H. pylori
present, and (iv) colonizing all regions of the stomach.