Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.

Synthesis of the Streptococcus pneumoniae type 3 capsule requires the pathway glucose-6-phosphate (Glc-6-P) 3 Glc-1-P 3 UDP-Glc 3 UDP-glucuronic acid (UDP-GlcUA) 3 (GlcUA-Glc) n . The UDP-Glc dehydrogenase and synthase necessary for the latter two steps, and essential for capsule production, are encoded by genes (cps3D and cps3S, respectively) located in the type 3 capsule locus. The phosphoglucomutase (PGM) and Glc-1-P uridylyltransferase activities necessary for the first two steps are derived largely through the actions of cellular enzymes. Homologues of these enzymes, encoded by cps3M and cps3U in the type 3 locus, are not required for capsule production. Here, we show that cps3M and cps3U also are not required for mouse virulence. In contrast, nonencapsulated isolates containing defined mutations in cps3D and cps3S were avirulent, as were reduced-capsule isolates containing mutations in pgm. Insertion mutants that lacked PGM activity were avirulent in both immunologically normal (BALB/cByJ) and immunodeficient (CBA/N) mice. In contrast, a mutant (JY1060) with reduced PGM activity was avirulent in the former but had only modestly reduced virulence in the latter. The high virulence in CBA/N mice was not due to the lack of antibodies to phosphocholine but reflected a growth environment distinct from that found in BALB/cByJ mice. The reduced PGM activity of JY1060 resulted in enhanced binding of complement and antibodies to surface antigens. However, decomplementation of BALB/cByJ mice did not enhance the virulence of this mutant. Suppressor mutations, only some of which resulted in increased capsule production, increased the virulence of JY1060 in BALB/cByJ mice. The results suggest that PGM plays a critical role in pneumococcal virulence by affecting multiple cellular pathways.

Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (HlyA1) are toxins produced by uropathogenic Escherichia coli (UPEC). We previously showed that these toxins contribute to the inflammation and tissue damage seen in a mouse model of ascending urinary tract infection. CNF1 constitutively activates small Rho GTPases by deamidation of a conserved glutamine residue, and HlyA1 forms pores in eukaryotic cell membranes. In this study, we used cDNA microarrays of bladder tissue isolated from mice infected intraurethrally with wild-type CP9, CP9cnf1, or CP9⌬hlyA to further evaluate the role that each toxin plays in the host response to UPEC. Regardless of the strain used, we found that UPEC itself elicited a significant change in host gene expression 24 h after inoculation. The largest numbers of upregulated genes were in the cytokine and chemokine signaling and Toll-like receptor signaling pathways. CNF1 exerted a strong positive influence on expression of genes involved in innate immunity and signal transduction and a negative impact on metabolism-and transport-associated genes. HlyA1 evoked an increase in expression of genes that encode innate immunity factors and a decrease in expression of genes involved in cytoskeletal and metabolic processes. Multiplex cytokine and myeloperoxidase assays corroborated our finding that a strong proinflammatory response was elicited by all strains tested. Bladders challenged intraurethrally with purified CNF1 displayed pathology similar to but significantly less intense than the pathology that we observed in CP9-challenged mice. Our data demonstrate substantial roles for CNF1 and HlyA1 in initiation of a strong proinflammatory response to UPEC in the bladder. U rinary tract infections (UTIs) are the most common bacterial infection in women and affect over 50% of women at some time throughout their lifetime (1). The clinical manifestations of UTIs vary from mild to severe, and common symptoms include dysuria, hematuria, pyuria, urinary frequency and urgency, suprapubic pain, and fever. The annual cost in the United States for diagnosis and treatment of UTIs is over $3 billion (2). Uncomplicated UTIs occur when commensal bacteria from the gastrointestinal (GI) tract transit to the periurethral area or vaginal introitus. The bacteria are then introduced into the urethra and ascend into the bladder to cause cystitis and, in more severe cases, into the kidneys to cause pyelonephritis (3-5). The etiological agents of 85% of uncomplicated UTIs are uropathogenic Escherichia coli (UPEC), a subgroup of extraintestinal pathogenic E. coli (1). UPEC expresses virulence factors that specifically facilitate colonization of and replication within the urinary tract.Cytotoxic necrotizing factor 1 (CNF1) is an ϳ115-kDa toxin that is expressed by ϳ40% of UPEC isolates and up to 30% of diarrheal E. coli isolates (6). CNF1 constitutively activates small Rho-family GTPases via deamidation of glutamine 63 of RhoA and glutamine 61 in Rac1 and Cdc42 (7,8). Constitutive activation of these small GTPases by C...

SummaryRegulation of chain length is essential to the proper functioning of prokaryotic and eukaryotic polysaccharides. Modulation of polymer size by substrate concentration is an attractive but unexplored control mechanism that has been suggested for many polysaccharides. The Streptococcus pneumoniae capsular polysaccharide is essential for virulence, and regulation of its size is critical for survival in different host environments. GlcUA) is catalysed by the type 3 synthase, a processive b-glycosyltransferase, and requires a UDP-Glc dehydrogenase for conversion of UDP-Glc to UDP-GlcUA. Strains containing mutant UDP-Glc dehydrogenases exhibited reduced levels of UDPGlcUA, along with reductions in total capsule amount and polymer chain length. In both the parent and mutant strains, UDP-Glc levels far exceeded UDPGlcUA levels, which were very low to undetectable in the absence of blocking synthase activity. The in vivo observations were consistent with in vitro conditions that effect chain termination and ejection of the polysaccharide from the synthase when one substrate is limiting. These data are the first to demonstrate modulation of polysaccharide chain length by substrate concentration and to enable a model for the underlying mechanism. Further, they may have implications for the control of chain length in both prokaryotic and eukaryotic polymers synthesized by similar mechanisms. Synthesis of the type 3 capsule [-4)-b-D-Glc-(1-3)-b-D-GlcUA-(1-] from UDPglucose (UDP-Glc) and UDP-glucuronic acid (UDP-

Bacillus cereus G9241 was isolated from a welder with a pulmonary anthrax-like illness. The organism contains two megaplasmids, pBCXO1 and pBC218. These plasmids are analogous to the Bacillus anthracis Ames plasmids pXO1 and pXO2 that encode anthrax toxins and capsule, respectively. Here we evaluated the virulence of B. cereus G9241 as well as the contributions of pBCXO1 and pBC218 to virulence. B. cereus G9241 was avirulent in New Zealand rabbits after subcutaneous inoculation and attenuated 100-fold compared to the published 50% lethal dose (LD 50 ) values for B. anthracis Ames after aerosol inoculation. A/J and C57BL/6J mice were comparably susceptible to B. cereus G9241 by both subcutaneous and intranasal routes of infection. However, the LD 50 s for B. cereus G9241 in both mouse strains were markedly higher than those reported for B. anthracis Ames and more like those of the toxigenic but nonencapsulated B. anthracis Sterne. Furthermore, B. cereus G9241 spores could germinate and disseminate after intranasal inoculation into A/J mice, as indicated by the presence of vegetative cells in the spleen and blood of animals 48 h after infection. Lastly, B. cereus G9241 derivatives cured of one or both megaplasmids were highly attenuated in A/J mice. We conclude that the presence of the toxin-and capsule-encoding plasmids pBCXO1 and pBC218 in B. cereus G9241 alone is insufficient to render the strain as virulent as B. anthracis Ames. However, like B. anthracis, full virulence of B. cereus G9241 for mice requires the presence of both plasmids.

Pneumonia caused by Staphylococcus aureus is a growing concern in the health care community. We hypothesized that characterization of the early innate immune response to bacteria in the lungs would provide insight into the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcus aureus pneumonia was utilized to define the early events in the innate immune response and to assess the changes in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs of mice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinoculation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway. Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 h postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or S. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionation followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the airway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation were observed. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with S. aureus, with the increase dominated by inflammatory and coagulation proteins. The data presented here provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host airway during the earliest stages of S. aureus pneumonia.Staphylococcus aureus is a leading cause of hospital-acquired and health care-associated pneumonia and may be increasing in importance as a cause of severe community-acquired pneumonia. In the inpatient setting, it is the most common grampositive bacterium implicated in cases of ventilator-associated and hospital-acquired pneumonia (1, 9, 31). In addition, S. aureus is a frequent cause of health care-associated pneumonia occurring in residents of long-term-care facilities, individuals recently discharged from acute-care hospitals, and patients receiving outpatient treatment at hospitals and dialysis centers (1, 27, 30). A steady increase in the isolation of methicillinresistant strains of S. aureus from patients with hospital-acquired pneumonia and, more recently, community-acquired pneumonia underscores the importance of identifying host and bacterial factors that facilitate the progression of staphylococcal pneumonia.Mice have been used extensively to study pneumonia caused by a variety of bacteria (2,6,26,35,36,45,55,63,64). Murine models of airborne infection with S. aureus have been useful in characterizing host responses during the first 4 to 8 h of lung infection but do not mimic the natural route of infection and result in self-limited disease, even in immunocompromised animals (28,53,56). In these studies, proinflammatory cytokines and chemokines were released and neutrophils ...

AtxA is a critical transcriptional regulator of plasmid-encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax-like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT-PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence.

To ascertain whether epithelial cells of oral cavity origin may be infected with human immunodeficiency virus (HIV-1), a study to determine susceptibility to infection of salivary gland epithelial cell lines (HSY and HSG) was undertaken. Because of the potential for oral-genital transmission, an endometrial cell line, HEC-1, was also studied. Epithelial cell monolayers were infected with cell-free HTLVIIIB or a primary HIV-1 isolate. Several lines of evidence indicated that inoculation of these cell lines with HIV-1 led to productive infection: 1) p24 antigen was present in supernatants, with levels peaking on days 3-4; 2) provirus was found in cells by polymerase chain reaction; 3) virions present in supernatants were infectious as confirmed by coculture with the T-lymphoblastoid line CEM-NKr. Following a period of virus production, HIV-1 entered a latency phase over 10 weeks. All epithelial cell lines were positive for galactosylceramide (GalC) and CXCR4. HSY was weakly positive for surface CD4, and also expressed mRNA for CD4 and CCR5, as did HEC-1. Blocking studies indicated that anti-GalC, but not anti-CD4, significantly reduced productive infection, and that regulated on activation normal T cell expressed and secreted (RANTES) but not stromal cell-derived factor (SDF-1) could partially block infection of the M-tropic primary isolate. These results suggest that epithelial cells in the oral cavity and the genital tract might be targets of HIV-1 and potentially serve as a mediator of systemic infection.