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ABSTRACTStreptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus(GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the Δsclmutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.

ABSTRACTBacillus anthracis, the causative agent of anthrax, relies on multiple virulence factors to subvert the host immune defense. Using Caenorhabditis elegansas an infection model, we screened approximately 5,000 transposon mutants of B. anthracisSterne for decreased virulence. One of the attenuated mutants resulted in loss of expression of yceGand yceH, the last two genes in a six-gene cluster of tellurite resistance genes. We generated an analogous insertional mutant to confirm the phenotype and characterize the role of yceGHin resistance to host defenses. Loss of yceGHrendered the mutants more sensitive to tellurite toxicity as well as to host defenses such as reactive oxygen species and the cathelicidin family of antimicrobial peptides. Additionally, we see decreased survival in mammalian models of infection, including human whole blood and in mice. We identify a novel role for the yceGHgenes in B. anthracisSterne virulence and suggest that C. elegans is a useful infection model to study anthrax pathogenesis.

ABSTRACTStreptococcus iniaerepresents a major health and economic problem in fish species worldwide. Random Tn917mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniaemutants. The Tn917insertion in one mutant disrupted an S. iniaehomologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with ΔPGM mutant cells reaching sizes ∼3-fold larger than those of the wild type (WT). The ΔPGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the ΔPGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgmgene on a plasmid vector restored antimicrobial peptide resistance and virulence to the ΔPGM mutant. In analysis of the aborted infectious process, we found that ΔPGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the ΔPGM mutant and later challenged with a lethal dose of WT S. iniaesurvived. We conclude that the pgmgene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniaeΔPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). 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.

ABSTRACTStreptococcus suisserotype 2 is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease, including meningitis. The pathogenesis of S. suisinfections is poorly understood. Bacteria circulate in the bloodstream in the nonimmune host until they come in contact with brain microvascular endothelial cells (BMEC) forming the blood-brain barrier. The bacterial polysaccharide capsule confers antiphagocytic properties. It is known that group B streptococci (GBS) invade and damage BMEC, which may be a primary step in the pathogenesis of neonatal meningitis. Interactions betweenS. suisand human endothelial cells were studied to determine if they differ from those between GBS and endothelial cells. Invasion assays performed with BMEC and human umbilical vein endothelial cells demonstrated that unlike GBS, S. suisserotype 2 could not invade either type of cell. Adherence assays showed that S. suisadhered only to BMEC, whereas GBS adhered to both types of cell. These interactions were not affected by the presence of a capsule, since acapsular mutants from both bacterial species adhered similarly compared to the wild-type strains. Lactate dehydrogenase release measurements indicated that some S. suisstrains were highly cytotoxic for BMEC, even more than GBS, whereas others were not toxic at all. Cell damage was related to suilysin (S. suishemolysin) production, since only suilysin-producing strains were cytotoxic and cytotoxicity could be inhibited by cholesterol and antisuilysin antibodies. It is possible that hemolysin-positive S. suisstrains use adherence and suilysin-induced BMEC injury, as opposed to direct cellular invasion, to proceed from the circulation to the central nervous system.

ABSTRACTStaphylococcus aureusexpresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrDfrom S. aureussubsp. aureusstrain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureusproteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactispromoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureussubsp. aureusNCTC8325-4 ΔsdrDbacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureussurvival and the ability to escape the innate immune system in blood.