The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse ) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δ sse MGAS5005 ) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K M and k cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δ sse MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.

The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS 1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covS wt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covS wt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.

Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse Aϩ ] and normal SpeB activity [SpeB Aϩ ]). Sse Aϩ SpeB Aϩ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse Aϩ SpeB Aϩ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse Aϩ SpeB Aϩ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.KEYWORDS CovRS, PAF acetylhydrolase, RocA, SpeB, Streptococcus pyogenes, group A Streptococcus, in vivo expression, mutation, virulence, virulence regulation T he human pathogen group A Streptococcus (GAS) commonly causes pharyngitis and superficial skin infections. GAS also causes severe invasive infections, such as necrotizing fasciitis and sepsis. The most recently available data indicate that from 2005 to 2012 severe invasive infections in the United States were most frequently associated with GAS of the M protein serotypes M1, M12, M28, M89, and M3 (1), and the currently circulating M1 GAS belongs to the pandemic M1T1 clone (2). GAS produces an

bInvasive M1T1 group A Streptococcus (GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in ⌬sda1 mutants with (5448 ⌬sda1 M؉ strain) and without (5448 ⌬sda1 M؊ strain) M protein production. The 5448 ⌬sda1 M؉ strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 ⌬sda1 M؊ strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections. The human pathogen group A Streptococcus (GAS) causes about 700 million cases of relatively mild, noninvasive pharyngitis and superficial skin infections annually. However, severe GAS infections, including severe invasive infections and acute rheumatic fever/rheumatic heart disease, can occur, causing approximately 517,000 deaths in the world each year (1). A globally disseminated M1T1 clone of serotype M1 GAS most frequently is associated with severe invasive infections in the United States (2-4). It is believed that the original M1T1 GAS clone evolved from the acquisitions of DNase Sda1-and superantigen SpeA-encoding prophages and an interserotype exchange of a 36-kb chromosomal region containing the toxin genes encoding NAD ϩ -glycohydrolase (NADase) and streptolysin O (SLO) with serotype M12 GAS (5-7). More recently, hypervirulent M1T1 GAS strains have been isolated, usually having mutations in the two-component regulatory system CovRS (also known as CsrRS) (8-12).CovRS is known to negatively regulate multiple virulence genes in GAS, including the capsule synthetase gene hasA, the interleukin-8 (IL-8)/CXC chemokine peptidase gene spyCEP, and the platelet-activating factor acetylhydrolase gene sse (13-18). Natural CovS mutations of invasive M1T1 isolates usually enhance the expression of these virulence genes and downregulate the production of the protease SpeB (SpeB AϪ , for the lack of the...

(2013) The sagA/pel locus does not regulate the expression of the M protein of the M1T1 lineage of group A Streptococcus, Virulence, 4:8,[698][699][700][701][702][703][704][705][706]