scholarly journals The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes toIn VivoSelection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

2015 ◽  
Vol 83 (11) ◽  
pp. 4293-4303 ◽  
Author(s):  
Guanghui Liu ◽  
Wenchao Feng ◽  
Dengfeng Li ◽  
Mengyao Liu ◽  
Daniel C. Nelson ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Regulatory System ◽  
Innate Immune ◽  
M Protein ◽  
Content Type ◽  
Group A ◽  
Innate Immune Evasion ◽  
Selection Of

ABSTRACTInvasive M1T1 group AStreptococcus(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 forin vivoselection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion ofsda1or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion ofsda1in strain 5448 resulted in Δsda1mutants with (5448 Δsda1M+strain) and without (5448 Δsda1M−strain) M protein production. The 5448 Δsda1M+strain accumulated CovRS mutationsin vivoand resisted killing in the bloodstream, whereas the 5448 Δsda1M−strain lostin vivoselection of CovRS mutations and was sensitive to killing. The deletion ofemmand a spontaneous Mga mutation in MGAS2221 reduced and preventedin vivoselection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical forin vivoselection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to thein vivoselection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

scholarly journals Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

2014 ◽  
Vol 82 (4) ◽  
pp. 1579-1590 ◽  
Author(s):  
Jinquan Li ◽  
Guanghui Liu ◽  
Wenchao Feng ◽  
Yang Zhou ◽  
Mengyao Liu ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Regulatory System ◽  
Lavage Fluid ◽  
Content Type ◽  
Enhanced Expression ◽  
Group A ◽  
Skin Invasion ◽  
Selection For ◽  
Innate Immune Evasion ◽  
Selection Of

ABSTRACTPathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group AStreptococcus(GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeBA−]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeBA−variants (SpeBA−%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeBA−% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2−/−mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2−/−mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeBA−variants in CXCR2−/−mice. One randomly chosen SpeBA−mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeBA−variants from a mouse all had nonsynonymouscovRSmutations that resulted in the SpeBA−phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneouscovRSmutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants.

scholarly journals Regulation of Inhibition of Neutrophil Infiltration by the Two-Component Regulatory System CovRS in Subcutaneous Murine Infection with Group A Streptococcus

2013 ◽  
Vol 81 (3) ◽  
pp. 974-983 ◽  
Author(s):  
Jinquan Li ◽  
Hui Zhu ◽  
Wenchao Feng ◽  
Mengyao Liu ◽  
Yingli Song ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Group A Streptococcus ◽  
Synergistic Effects ◽  
Regulatory System ◽  
Neutrophil Infiltration ◽  
Neutrophil Recruitment ◽  
Innate Immune ◽  
Two Component ◽  
Group A ◽  
Innate Immune Evasion

ABSTRACTHypervirulent invasive group A streptococcus (GAS) isolates inhibit neutrophil infiltration more than pharyngitis isolates do, and the molecular basis of this difference is not well understood. This study was designed to first determine whether natural null mutation of the two-component regulatory system CovRS is responsible for the enhancement of the inhibition of neutrophil recruitment seen in hypervirulent GAS. Next, we examined the role of CovRS-regulated interleukin-8/CXC chemokine peptidase (SpyCEP), C5a peptidase (ScpA), and platelet-activating factor acetylhydrolase (SsE) in the enhanced innate immune evasion. Invasive isolate MGAS5005 induces less neutrophil infiltration and produced a greater lesion area than pharyngitis isolate MGAS2221 in subcutaneous infections of mice. It is known that MGAS5005, but not MGAS2221, has a natural 1-bp deletion in thecovSgene. Replacement ofcovSΔ1bpin MGAS5005 with wild-typecovSresulted in the MGAS2221 phenotype. Deletion ofcovSfrom MGAS2221 resulted in the MGAS5005 phenotype. Tests of single, double, and triple deletion mutants of the MGAS5005sse,spyCEP, andscpAgenes found that SsE plays a more important role than SpyCEP and ScpA in the inhibition of neutrophil recruitment and that SsE, SpyCEP, and ScpA do not have synergistic effects on innate immune evasion by MGAS5005. Deletion ofsse, but notspyCEPorscpA, of MGAS2221 enhances neutrophil recruitment. Thus,covSnull mutations can cause substantial inhibition of neutrophil recruitment by enhancing the expression of the chemoattractant-degrading virulence factors, and SsE, but not SpyCEP or ScpA, is required for CovRS-regulated GAS inhibition of neutrophil infiltration.

scholarly journals Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype

2014 ◽  
Vol 82 (5) ◽  
pp. 1744-1754 ◽  
Author(s):  
Tram N. Cao ◽  
Zhuyun Liu ◽  
Tran H. Cao ◽  
Kathryn J. Pflughoeft ◽  
Jeanette Treviño ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Mrna Levels ◽  
Bacterial Strains ◽  
Content Type ◽  
Small Regulatory Rna ◽  
Group A

ABSTRACTDespite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogenStreptococcus pyogenes(the group AStreptococcus[GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in thefasCgene of thefasBCAXregulatory system and an inactivating polymorphism in therivRregulator-encoding gene. ThefasCandrivRmutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of thefasCmutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of therivRmutation in M3 GAS restored the regulation ofgrabmRNA abundance but did not alter capsule mRNA levels. While important, thefasCandrivRmutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

scholarly journals The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Luis A. Vega ◽  
Kayla M. Valdes ◽  
Ganesh S. Sundar ◽  
Ashton T. Belew ◽  
Emrul Islam ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Immune Evasion ◽  
Immune Cell ◽  
Group A Streptococcus ◽  
Transcriptional Regulator ◽  
Innate Immune ◽  
Content Type ◽  
Human Host

ABSTRACTAs an exclusively human pathogen,Streptococcus pyogenes(the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene,cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators ofStreptococcus mutans(MetR),Streptococcus iniae(CpsY), andStreptococcus agalactiae(MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survivalin vivo. Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest thein vitrophenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE,speB,spd,nga[spn],prtS[SpyCEP], andsse) and cell surface-associated factors of GAS (emm1,mur1.2,sibA[cdhA], andM5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

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

2013 ◽  
Vol 81 (9) ◽  
pp. 3128-3138 ◽  
Author(s):  
Guanghui Liu ◽  
Mengyao Liu ◽  
Gang Xie ◽  
Benfang Lei
Keyword(s):  
Immune Evasion ◽  
Group A Streptococcus ◽  
Platelet Activating Factor ◽  
Neutrophil Recruitment ◽  
Innate Immune ◽  
Sequence Difference ◽  
Content Type ◽  
Paf Acetylhydrolase ◽  
Skin Invasion ◽  
Innate Immune Evasion

ABSTRACTHuman pathogen group A streptococcus (GAS) has developed mechanisms to subvert innate immunity. We recently reported that the secreted esterase produced by serotype M1 GAS (SsEM1) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsEM1and SsE produced by serotype M28 GAS (SsEM28) have a 37% sequence difference. This study aims at determining whether SsEM28is also a PAF acetylhydrolase and participates in innate immune evasion. We also examined whether SsE evolved to target PAF by characterizing the PAF acetylhydrolase (PAF-AH) activity and substrate specificity of SsEM1, SsEM28, SeE, the SsE homologue inStreptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsEM28or SeE was converted into lyso-PAF. SsEM1and SsEM28hadkcatvalues of 373 s−1and 467 s−1, respectively, that were ≥30-fold greater than that of hpPAF-AH (12 s−1). The comparison of SsEM1, SsEM28, and hpPAF-AH inkcatandKmin hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsEM28possesses much lower esterase activities against triglycerides and other esters than SsEM1but have similar potency with SsEM1in PAF hydrolysis. Deletion ofsseM28in acovSdeletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas intransexpression of SsEM28in 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.

scholarly journals Hypervirulent Group AStreptococcusof Genotypeemm3 Invades the Vascular System in Pulmonary Infection of Mice

2018 ◽  
Vol 86 (6) ◽  
Author(s):  
Benfang Lei ◽  
Dylan Minor ◽  
Wenchao Feng ◽  
Mengyao Liu
Keyword(s):  
Pulmonary Infection ◽  
Vascular System ◽  
Regulatory System ◽  
Virulence Gene ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Gram Staining ◽  
Systemic Dissemination ◽  
Group A ◽  
Innate Immune Evasion

ABSTRACTNatural mutations of the two-component regulatory system CovRS are frequently associated with invasive group AStreptococcus(GAS) isolates and lead to the enhancement of virulence gene expression, innate immune evasion, systemic dissemination, and virulence. How CovRS mutations enhance systemic dissemination is not well understood. A hypervirulent GAS isolate of theemm3 genotype, MGAS315, was characterized using a mouse model of pulmonary infection to understand systemic dissemination. This strain has a G1370T mutation in the sensor kinasecovSgene of CovRS. Intratracheal inoculation of MGAS315 led to the lung infection that displayed extensive Gram staining at the alveolar ducts, alveoli, and peribronchovascular and perivascular interstitium. The correction of thecovSmutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, thecovSmutation allowed MGAS315 to disrupt and degrade the smooth muscle and endothelial layers of the blood vessels, directly contributing to systemic dissemination. It is concluded that hypervirulentemm3 GAScovSmutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination.

scholarly journals Strain-Specific Regulatory Role of Eukaryote-Like Serine/Threonine Phosphatase in Pneumococcal Adherence

2012 ◽  
Vol 80 (4) ◽  
pp. 1361-1372 ◽  
Author(s):  
Shivangi Agarwal ◽  
Shivani Agarwal ◽  
Preeti Pancholi ◽  
Vijay Pancholi
Keyword(s):  
High Efficiency ◽  
Regulatory System ◽  
Gene Encoding ◽  
Content Type ◽  
Knockout Mutants ◽  
Catalytically Active

ABSTRACTStreptococcus pneumoniaeexploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase ofS. pneumoniae(StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerlessphpPknockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknownin vitroandin vivoevidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpPmutants. In particular, StkP (threonine)-phosphorylated RR06 bound to thecbpApromoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.

scholarly journals Serine/Threonine Protein Kinase Stk Is Required for Virulence, Stress Response, and Penicillin Tolerance in Streptococcus pyogenes

2011 ◽  
Vol 79 (10) ◽  
pp. 4201-4209 ◽  
Author(s):  
Julia Bugrysheva ◽  
Barbara J. Froehlich ◽  
Jeffrey A. Freiberg ◽  
June R. Scott
Keyword(s):  
Streptococcus Pyogenes ◽  
Fatty Acid Biosynthesis ◽  
Group A Streptococcus ◽  
Acid Biosynthesis ◽  
Regulation Of Expression ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Group A

ABSTRACTGenes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogenStreptococcus pyogenes(group A streptococcus [GAS]). We report that in GAS,stkis required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that thestkdeletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.

scholarly journals Natural Variation in the Promoter of the Gene Encoding the Mga Regulator Alters Host-Pathogen Interactions in Group A Streptococcus Carrier Strains

2013 ◽  
Vol 81 (11) ◽  
pp. 4128-4138 ◽  
Author(s):  
Anthony R. Flores ◽  
Randall J. Olsen ◽  
Andrea Wunsche ◽  
Muthiah Kumaraswami ◽  
Samuel A. Shelburne ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Group A Streptococcus ◽  
Ex Vivo ◽  
Asymptomatic Carrier ◽  
Carrier State ◽  
Wild Type ◽  
Gene Encoding ◽  
Content Type ◽  
Group A

ABSTRACTHumans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group AStreptococcus(GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator. Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in themgapromoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange, we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier strain. Compared to strains with the wild-typemgapromoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewermgaand Mga-regulated gene transcripts. Consistent with decreasedmgatranscripts, strains containing the variantmgapromoter were also significantly less virulent inin vivoandex vivomodels of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to themgapromoter and that the 12-bp deletion in themgapromoter reduces CodY-mediatedmgatranscription. We conclude that the naturally occurring 12-bp deletion in themgapromoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new insight into the molecular basis of the carrier state of an important human pathogen.

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