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 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 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 A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Ulrike Resch ◽  
James Anthony Tsatsaronis ◽  
Anaïs Le Rhun ◽  
Gerald Stübiger ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Lipid Composition ◽  
Secretory Pathway ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Surface Proteins ◽  
Streptococcal Toxic Shock Syndrome ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Two Component ◽  
Group A

ABSTRACT Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

scholarly journals Complement-mediated Opsonization of Invasive Group A Streptococcus pyogenes Strain AP53 Is Regulated by the Bacterial Two-component Cluster of Virulence Responder/Sensor (CovRS) System

2013 ◽  
Vol 288 (38) ◽  
pp. 27494-27504 ◽  
Author(s):  
Garima Agrahari ◽  
Zhong Liang ◽  
Jeffrey A. Mayfield ◽  
Rashna D. Balsara ◽  
Victoria A. Ploplis ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Death Rate ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Factor H ◽  
Human Neutrophils ◽  
Two Component ◽  
Group A ◽  
Allelic Alteration ◽  
High Level

Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR+S−. However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR+S− cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS− to wild-type covS (covS+), a dramatic loss of FH and C4BP binding to the AP53/covR+S+ cells was observed. This resulted in elevated C3b deposition on AP53/covR+S+ cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR+S+. We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively.

scholarly journals Metal-Mediated Modulation of Streptococcal Cysteine Protease Activity and Its Biological Implications

2014 ◽  
Vol 82 (7) ◽  
pp. 2992-3001 ◽  
Author(s):  
Karthickeyan Chella Krishnan ◽  
Santhosh Mukundan ◽  
Julio A. Landero Figueroa ◽  
Joseph A. Caruso ◽  
Malak Kotb
Keyword(s):  
Cysteine Protease ◽  
Metal Binding ◽  
Group A Streptococcus ◽  
Iron Chelation ◽  
Regulatory System ◽  
Bacterial Virulence ◽  
Bacterial Survival ◽  
Metal Binding Sites ◽  
Two Component ◽  
Group A

ABSTRACTStreptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues47Cys and195His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections.

scholarly journals The SaeR/S Gene Regulatory System Is Essential for Innate Immune Evasion byStaphylococcus aureus

2009 ◽  
Vol 199 (11) ◽  
pp. 1698-1706 ◽  
Author(s):  
Jovanka M. Voyich ◽  
Cuong Vuong ◽  
Mark DeWald ◽  
Tyler K. Nygaard ◽  
Stanislava Kocianova ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Regulatory System ◽  
Innate Immune ◽  
S Gene ◽  
Gene Regulatory System ◽  
Gene Regulatory ◽  
Innate Immune Evasion

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.

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