Correlation of Histopathologic Findings with Clinical Outcome in Necrotizing Fasciitis

The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.Severe Streptococcus pyogenes infections, such as streptococcal toxic shock syndrome and necrotizing fasciitis, result in high mortality rates that range from 25% to 50% (7, 43). The severity and incidence of these invasive S. pyogenes diseases have increased since the 1980s despite what had been considered near eradication of S. pyogenes infections in the previous decades of the 20th century due to the development of antibiotics (13, 43). The reasons for this resurgence remain vague, despite numerous reports indicating the prevalence of particular strains associated with severe S. pyogenes infections (9, 13). Recent studies assessed factors that are present in various serotypes, but they failed to determine specific pathogen and host factors that are common denominators of invasive S. pyogenes disease (34, 55). Moreover, from the host perspective, Kotb et al. eloquently illustrated that certain human leukocyte class II alleles confer a predisposition to severe S. pyogenes disease (29). Undoubtedly, the complexity and versatility of this organism, particularly its remarkable ability to alter its broad spectrum of virulence factors in order to survive in different environments, are the foundation for its success.Although in vitro studies have permitted analysis of virulence determinants, the conditions are selective, as the systems cannot completely mimic ho...

Section: Generation Ofsupporting

confidence: 83%

First Streptococcus pyogenes Signature-Tagged Mutagenesis Screen Identifies Novel Virulence Determinants

Kizy

Neely

2009

“…All of these three factors are regulated by the two-component regulatory system CovRS (33,43,44). Hypervirulent GAS can almost completely inhibit neutrophil infiltration (4,8,(45)(46)(47) and have enhanced expression of the spyCEP and sse genes (33,43,44). Null deletion and mutations of the covS gene cause the severe inhibition of neutrophil infiltration, and SsE is the most critical factor among SsE, ScpA, and SpyCEP for the covS deletion/mutation-mediated enhancement in innate immune evasion and virulence (17).…”

Section: Discussionmentioning

confidence: 99%

“…Western blotting was performed as described previously (33). Proteins in 10 l of the obtained samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (BioTrace NT; Pall Corp.) with Towbin transfer buffer using a Trans-Blot SD semidry transfer cell (Bio-Rad) at 15 V for 45 To prepare anti-SsE M28 antisera, 2 female BALB/c mice (5 weeks old) obtained from Charles River Laboratories were immunized subcutaneously on day 1 and then boosted with 50 g of purified recombinant SsE M28 suspended in 50 l of aluminum hydroxide gel (Sigma) on day 14. Immune antiserum was collected 2 weeks after the boost.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion

Liu

Xie

et al. 2013

Human pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsE M1 ) reduces neutrophil recruitment by targeting platelet-activating factor (PAF M28 in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE M28 in GAS reduced neutrophil infiltration and increased skin invasion in subcutaneous infection of mice. These results suggest that the SsE proteins evolved to target PAF for enhancing innate immune evasion and skin invasion.

“…This model would predict that growth of S. pyogenes in tissues that the organism has not evolved to infect may not provide the expected cues for the regulatory program, which could lead to an unbalanced manipulation of the host's response. In turn, this could produce consequences like the sustained dampening of the inflammatory response that has been noted to be associated with poor outcomes for infection of the fascia and muscle (1,32); this model is also consistent with studies that have established that the rate of bacterial killing is determined by the concentration of neutrophils and not the ratio of neutrophils to bacteria (46). A delay in the recruitment of neutrophils to extravascular sites could allow a small number of bacteria to proliferate to high numbers, causing significant local tissue damage.…”

Section: Discussionmentioning

confidence: 99%

“…As a general rule, the late stages of all these diseases are characterized by robust inflammation (6,14). However, a distinct absence of inflammation is not an uncommon presentation for advanced necrotizing fasciitis (32), and a deficiency of polymorphonuclear leukocytes (PMNs) upon histopathological examination of infected tissues is associated with a significantly increased risk of mortality from this disease (1). Thus, S. pyogenes appears to have the capacity to both suppress and stimulate inflammation, and the balance between these activities may be influenced by strain differences and/or stage-and tissue-specific cues in a manner that can affect the outcome of infection.…”

mentioning

confidence: 99%

Streptolysin S Inhibits Neutrophil Recruitment during the Early Stages ofStreptococcus pyogenesInfection

Lin¹,

Loughman

Zinselmeyer³

et al. 2009

In contrast to infection of superficial tissues, Streptococcus pyogenes infection of deeper tissue can be associated with a significantly diminished inflammatory response, suggesting that this bacterium has the ability to both promote and suppress inflammation. To examine this, we analyzed the behavior of an S. pyogenes mutant deficient in expression of the cytolytic toxin streptolysin S (SLS ؊ ) and evaluated events that occur during the first few hours of infection by using several models including injection of zebrafish (adults, larvae, and embryos), a transepithelial polymorphonuclear leukocyte (PMN) migration assay, and two-photon microscopy of mice in vivo. In contrast to wild-type S. pyogenes, the SLS ؊ mutant was associated with the robust recruitment of neutrophils and significantly reduced lethal myositis in adult zebrafish. Similarly, the mutant was attenuated in embryos in its ability to cause lethality. Infection of larva muscle allowed an analysis of inflammation in real time, which revealed that the mutant had recruited PMNs to the infection site. Analysis of transepithelial migration in vitro suggested that SLS inhibited the host cells' production of signals chemotactic for neutrophils, which contrasted with the proinflammatory effect of an unrelated cytolytic toxin, streptolysin O. Using two-photon microscopy of mice in vivo, we showed that the extravasation of neutrophils during infection with SLS ؊ mutant bacteria was significantly accelerated compared to infection with wild-type S. pyogenes. Taken together, these data support a role for SLS in the inhibition of neutrophil recruitment during the early stages of S. pyogenes infection.