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

Abstract

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...

Zebrafish as a Model Host for Candida albicans Infection

“…For example, Neely and colleagues found that Streptococcus pyogenes (a human pathogen) and Streptococcus iniae (a natural patho- on May 10, 2018 by guest http://iai.asm.org/ gen of fish) infect adult zebrafish at similar efficiencies, and the comparable results support the conclusion that the Streptococcus-zebrafish model can be used to study human pathogenic bacteria (31). By employing large-scale, signature-tagged mutagenesis, 1,200 different mutant S. pyogenes strains were screened with the zebrafish infection model to identify virulence genes of the bacterial pathogen, which is important in S. pyogenes pathogenesis (18). The zebrafish model has also been used in studies of other bacteria, including studies of chronic tuberculosis caused by Mycobacterium marinum infection, realtime analysis of Salmonella enterica serovar Typhimurium infection, and analysis of Listeria monocytogenes-phagocyte interactions (22,25,37,41).…”

Molecular Epidemiology of sil Locus in Clinical Streptococcus pyogenes Strains

“…Furthermore, a systematic sequencing of the silCR gene in emm14 strains is necessary to determine whether the described mutation is an exception or the rule among the emm14 strains (13,19). Nonetheless, a similar mutation was reported for an additional emm14 strain isolated in the United States (15).…”

“…This in turn activates the transcription of silCR (autoregulation) and silD and silE and represses that of silC (14). The silC gene product is linked to GAS virulence in the mouse model of human NF (13,15). SilCR is involved in the downregulation of the expression of the gene encoding the CXC chemokine protease ScpC, which impairs the recruitment and the activation of neutrophils to the soft tissue infection site (16,17).…”

Signature tagged mutagenesis in the functional genetic analysis of gastrointestinal pathogens

“…2,3,[5][6][7][8][9][10]15 In this review we will focus on STM screens associated with gastrointestinal (GI) infections ( Table 1). GI illness is a global public health concern and in the developed world it is usually self-limiting but has a considerable economic burden.…”

“…STM was initially developed to identify virulence genes in Salmonella enterica serovar typhimurium but has subsequently been used in many screens in other bacterial species as well as in the yeast Saccharomyces cerevisiae, the fungus Cryptococcus neoformans and the parasite Toxoplasma gondii. [1][2][3][4][5][6][7][8][9][10] For any bacterial pathogen there are several critical parameters that must be followed in order to ensure an efficient STM screen in vivo. First, the transposon chosen should insert randomly into the chromosome, a property which varies depending on the transposon.…”