Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the alpha-subunit of Mac-1, a leukocyte beta2 integrin required for innate immunity to invading microbes. The GAS Mac-1-like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcgammaRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.

Recently, we observed that Staphylococcus aureus strains newly isolated from patients had twofold-higher aconitase activity than a strain passaged extensively in vitro, leading us to hypothesize that aconitase specific activity decreases over time during in vitro passage. To test this hypothesis, a strain recovered from a patient with toxic shock syndrome was serially passaged for 6 weeks, and the aconitase activity was measured. Aconitase specific activity decreased 38% (P < 0.001) by the sixth week in culture. During serial passage, S. aureus existed as a heterogeneous population with two colony types that had pronounced (wild type) or negligible zones of beta-hemolytic activity. The cell density-sensing accessory gene regulatory (agr) system regulates beta-hemolytic activity. Surprisingly, the percentage of colonies with a wild-type beta-hemolytic phenotype correlated strongly with aconitase specific activity ( ‫؍‬ 0.96), suggesting a common cause of the decreased aconitase specific activity and the variation in percentage of beta-hemolytic colonies. The loss of the beta-hemolytic phenotype also coincided with the occurrence of mutations in the agrC coding region or the intergenic region between agrC and agrA in the derivative strains. Our results demonstrate that in vitro growth is sufficient to result in mutations within the agr operon. Additionally, our results demonstrate that S. aureus undergoes significant phenotypic and genotypic changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.The ability of Staphylococcus aureus to evade the host immune response and cause disease is due to an extensive repertoire of known and putative virulence factors, including four hemolysins, two lipases, several proteases, exotoxins, and enterotoxins. The production of many virulence factors is regulated by the accessory gene regulatory (agr) operon (22,27) and several other global regulatory loci. The agr locus consists of two divergently transcribed mRNAs designated RNA II and RNA III (19). RNA II encodes a two-component regulatory system (AgrA and AgrC) that senses the level of cyclic thiolactone peptides generated from agrB and agrD, also encoded by RNA II (15). RNA III is an RNA effector molecule that reciprocally regulates the transcription of cell-associated adherence factors and secreted proteins (25). RNA III also regulates the translation of alpha-toxin mRNA (25). Transcription of the agr operon is autoregulated by agrA in a cell densitydependent manner and by at least two other global regulatory proteins: the staphylococcal accessory regulator (SarA) (6) and ArlR, the regulatory moiety of the ArlS-ArlR two-component regulatory system (10). Recently, we determined that aconitase affects the synthesis of several S. aureus virulence factors and the expression of the global gene regulators RNA III and sarA (G. A. Somerville, unpublished data). Aconitase (citrate [isocitrate] hydrolyase, EC 4.2.1.3) is a citric acid cycle enzyme ...

Recent genomic studies have revealed extensive variation in natural populations of many pathogenic bacteria. However, the evolutionary processes which contribute to much of this variation remain unclear. A previous whole-genome DNA microarray study identified variation at a large chromosomal region (RD13) of Staphylococcus aureus which encodes a family of proteins with homology to staphylococcal and streptococcal superantigens, designated staphylococcal exotoxin-like (SET) proteins. In the present study, RD13 was found in all 63 S. aureus isolates of divergent clonal, geographic, and disease origins but contained a high level of variation in gene content in different strains. A central variable region which contained from 6 to 10 different set genes, depending on the strain, was identified, and DNA sequence analysis suggests that horizontal gene transfer and recombination have contributed to the diversification of RD13. Phylogenetic analysis based on the RD13 DNA sequence of 18 strains suggested that loss of various set genes has occurred independently several times, in separate lineages of pathogenic S. aureus, providing a model to explain the molecular variation of RD13 in extant strains. In spite of multiple episodes of set deletion, analysis of the ratio of silent substitutions in set genes to amino acid replacements in their products suggests that purifying selection (selective constraint) is acting to maintain SET function. Further, concurrent transcription in vitro of six of the seven set genes in strain COL was detected, indicating that the expression of set genes has been maintained in contemporary strains, and Western immunoblot analysis indicated that multiple SET proteins are expressed during the course of human infections. Overall, we have shown that the chromosomal region RD13 has diversified extensively through episodes of gene deletion and recombination. The coexpression of many set genes and the production of multiple SET proteins during human infection suggests an important role in host-pathogen interactions.

SummaryHeparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp-2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N-terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin-mediated uptake of MS11-OpaA. This inhibition could be mimicked by an RGD-containing hexapeptide and by ␣5␤1 integrin-specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp-2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side-chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co-receptors facilitating fibronectinmediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking the Opa-proteoglycan complex with host cell integrin receptors.

). To study mac variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 ؎ 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.

We isolated lipopolysaccharides (LPSs) from phase variants of Coxiella burnetii Nine Mile and compared the isolated LPS and C. burnetii cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The LPSs were found to be the predominant component which varied structurally and antigenically between virulent phase I and avirulent phase II. A comparison of techniques historically used to extract the phase I antigenic component revealed that the aqueous phase of phenol-water, trichloroacetic acid, and dimethyl sulfoxide extractions of phase I C. burnetii cells all contained phase I LPS, although the efficiency and specificity of extraction varied. Our studies provide additional evidence that phase variation in C. burnetii is analogous to the smooth-to-rough LPS variation of gram-negative enteric bacteria, with phase I LPS being equivalent to smooth LPS and phase II being equivalent to rough LPS. In addition, we identified a variant with a third LPS chemotype with appears to have a structural complexity intermediate to phase I and II LPSs. All three C. burnetii LPSs contain a 2-keto-3-deoxyoctulosonic acid-like substance, heptose, and gel Limulus amoebocyte lysates in subnanogram amounts. The C. burnetii LPSs were nontoxic to chicken embryos at doses of over 80 ,ug per embryo, in contrast to Salmonella typhimurium smooth-and rough-type LPSs, which were toxic in nanogram amounts. Coxiella burnetii, the etiologic agent of Q fever, is an obligately intracellular bacterium that multiplies within the

SummaryNeisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified 35 SO 4 -labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc-proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.

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