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Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of betahemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671-1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www .genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.Group A streptococcus (GAS), specifically Streptococcus pyogenes, is a common cause of pharyngitis, impetigo, and many other human respiratory tract and soft tissue infections. Recently, there has been a dramatic increase in reports of severe invasive GAS infections, including necrotizing fasciitis and toxic shock syndrome (44). Although GAS produces a wide array of virulence factors, those responsible for the rapid bacterial spread and tissue injury seen with invasive GAS infections are unknown.A hallmark feature of GAS in the clinical laboratory is the zone of beta-hemolysis observed surrounding colonies grown on the surface of blood agar media. The factor responsible for the beta-hemolytic phenotype is streptolysin S (SLS), the oxygen-stable cytolytic toxin of GAS. Despite detailed investigations over several decades, the exact chemical nature of SLS has remained a great mystery of GAS biology. SLS can exist in intracellular, cell-surface-bound, and extracellular forms (16), but attempts at purification are complicated by instability of the cytolytic activity in the absence of high-molecular-weight carriers, such as yeast RNA core or albumin (47).We have adopted a molecular genetic approach towards the identification of SLS and the study of...

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22-to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.The serine protease of Staphylococcus aureus strain V8 (Ssp, also known as V8 protease) was one of the first secreted enzymes of S. aureus to be purified and characterized in detail (16). It is a member of the glutamyl endopeptidase family of enzymes (24) and has been widely used in this capacity as a specific tool for determining protein structure. However, its contributions to the growth and survival of S. aureus have not been elucidated. S. aureus is a major cause of infectious morbidity and mortality in both the community and hospital settings (30), and although capable of expressing several different toxins, it is not generally equated with other pathogens that cause illness primarily through the elaboration of specific toxins (17). Rather, the hallmarks of S. aureus disease are its rapid multiplication and induction of inflammation at the site of infection and its ability to disseminate and initiate metastatic infection (50, 51). This is facilitated by an accessory gene regulator locus, agr, which at high cell density is responsible for inducing the expression of secreted toxins and exoenzymes, while simultaneously promoting the reduced expression of cell surface adhesins and colonization factors (18,38,41,48). Therefore, agr-null mutants demonstrate enhanced expression of colonization factors and a pleiotropic defect in expression of secreted virulence factors.Due to the inability to express secreted virulence factors, agr-null strai...

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Clinical antimicrobial susceptibility breakpoints are used to predict the clinical outcome of antimicrobial treatment. In contrast, microbiologic breakpoints are used to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. Currently, the National Committee for Clinical Laboratory Standards (NCCLS) guidelines utilize clinical breakpoints to characterize the activity of the fluoroquinolones against Streptococcus pneumoniae. To determine whether levofloxacin breakpoints can identify isolates that harbor recognized resistance mechanisms, we examined 115 S. pneumoniae isolates with a levofloxacin MIC of >2 μg/mL for first-step parC mutations. A total of 48 (59%) of 82 isolates with a levofloxacin MIC of 2 μg/mL, a level considered susceptible by NCCLS criteria, had a first-step mutation in parC. Whether surveillance programs that use levofloxacin data can effectively detect emerging resistance and whether fluoroquinolones can effectively treat infections caused by such isolates should be evaluated.

We report on amino acid substitutions in the quinolone resistance-determining region of type II topisomerases and the prevalence of reserpine-inhibited efflux for 70 clinical isolates of S. pneumoniae for which the ciprofloxacin MIC is >4 g/ml and 28 isolates for which the ciprofloxacin MIC is <2 g/ml.

. S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.

Streptococcus iniae causes meningoencephalitis and death in commercial fish species and has recently been identified as an emerging human pathogen producing fulminant soft tissue infection. As identified by pulsedfield gel electrophoresis (PFGE), strains causing disease in either fish or humans belong to a single clone, whereas isolates from nondiseased fish are genetically diverse. In this study, we used in vivo and in vitro models to examine the pathogenicity of disease-associated isolates. Strains with the clonal (disease-associated) PFGE profile were found to cause significant weight loss and bacteremia in a mouse model of subcutaneous infection. As little as 10 2 CFU of a disease-associated strain was sufficient to establish bacteremia, with higher inocula (10 7 ) resulting in increased mortality. In contrast, non-disease-associated (commensal) strains failed to cause bacteremia and weight loss, even at inocula of 10 8 CFU. In addition, disease-associated strains were more resistant to phagocytic clearance in a human whole blood killing assay compared to commensal strains, which were almost entirely eradicated. Disease-associated strains were also cytotoxic to human endothelial cells as measured by lactate dehydrogenase release from host cells. However, both disease-associated and commensal strains adhered to and invaded cultured human epithelial and endothelial cells equally well. While cellular invasion may still contribute to the pathogenesis of invasive S. iniae disease, resistance to phagocytic clearance and direct cytotoxicity appear to be discriminating virulence attributes of the disease-associated clone.