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 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 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 Differential Recognition of Surface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs

2002 ◽  
Vol 184 (8) ◽  
pp. 2181-2191 ◽  
Author(s):  
Timothy C. Barnett ◽  
June R. Scott
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Surface Proteins ◽  
Reading Frame ◽  
Gram Positive Bacteria ◽  
Differential Recognition ◽  
Surface Localization ◽  
Group A ◽  
Protein F

ABSTRACT The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-type srtA gene was provided in trans. The surface localization of T6, however, was unaffected by the srtA mutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented by srtB in trans. An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains. We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.

scholarly journals The Relevance of IL-1-Signaling in the Protection against Gram-Positive Bacteria

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 132
Author(s):  
Angelina Midiri ◽  
Giuseppe Mancuso ◽  
Concetta Beninati ◽  
Elisabetta Gerace ◽  
Carmelo Biondo
Keyword(s):  
Host Defense ◽  
Group A Streptococcus ◽  
Bacterial Species ◽  
Interleukin 1 ◽  
Group B Streptococcus ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Group A ◽  
Group B ◽  
Deficient Mice

Previous studies performed using a model of group B streptococcus (GBS)-induced peritoneal inflammation indicate that the interleukin-1 receptor (IL-1R) family plays an important role in the innate host defense against this encapsulated Gram-positive bacteria. Since the role of IL-1-dependent signaling in peritoneal infections induced by other Gram-positive bacteria is unknown, in the present study we sought to investigate the contribution of IL-1R signaling in host defenses against Streptococcus pyogenes (group A streptococcus or GAS) or Staphylococcus aureus, two frequent and global human Gram-positive extracellular pathogens. We analyzed here the outcome of GAS or S. aureus infection in IL-1R-deficient mice. After inoculated intraperitoneal (i.p.) inoculation with group A Streptococcus or S. aureus, all the wild-type (WT) control mice survived the challenge, while, respectively, 63% or 50% of IL-1-defective mice died. Lethality was due to the ability of both bacterial species to replicate and disseminate to the target organs of IL-1R-deficient mice. Moreover, the experimental results indicate that IL-1 signaling promotes the production of leukocyte attractant chemokines CXCL-1 and CXCL-2 and recruitment of neutrophils to bacterial infection sites. Accordingly, the reduced neutrophil recruitment in IL-1R-deficient mice was linked with decreased production of neutrophil chemokines. Collectively, our findings indicate that IL-1 signaling, as previously showed in host defense against GBS, plays a fundamental role also in controlling the progression and outcome of GAS or S. aureus disease.

scholarly journals ExPortal and the LiaFSR Regulatory System Coordinate the Response to Cell Membrane Stress in Streptococcus pyogenes

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Yibin Lin ◽  
Misu A. Sanson ◽  
Luis Alberto Vega ◽  
Brittany Shah ◽  
Shrijana Regmi ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Antimicrobial Peptides ◽  
Cell Envelope ◽  
Regulatory System ◽  
Immunogold Electron Microscopy ◽  
Membrane Stress ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Envelope Stress ◽  
Group A

ABSTRACT LiaFSR is a gene regulatory system important for response to cell membrane stress in Gram-positive bacteria but is minimally studied in the important human pathogen group A Streptococcus (GAS). Using immunofluorescence and immunogold electron microscopy, we discovered that LiaF (a membrane-bound repressor protein) and LiaS (a sensor kinase) reside within the GAS membrane microdomain (ExPortal). Cell envelope stress induced by antimicrobials resulted in ExPortal disruption and activation of the LiaFSR system. The only human antimicrobial peptide whose presence resulted in ExPortal disruption and LiaFSR activation was the alpha-defensin human neutrophil peptide 1 (hNP-1). Elimination of membrane cardiolipin through targeted gene deletion resulted in loss of LiaS colocalization with the GAS ExPortal and activation of LiaFSR, whereas LiaF membrane localization was unaffected. Isogenic mutants lacking either LiaF or LiaS revealed a critical role of LiaF in ExPortal integrity. Thus, LiaF and LiaS colocalize with the GAS ExPortal by distinct mechanisms, further supporting codependence. These are the first data identifying a multicomponent signal system within the ExPortal, thereby providing new insight into bacterial intramembrane signaling in GAS that may serve as a paradigm for Gram-positive bacteria. IMPORTANCE Bacterial two-component systems sense and induce transcriptional changes in response to environmental stressors, including antimicrobials and human antimicrobial peptides. Since the stresses imposed by the host’s defensive responses may act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression. The mechanism by which bacteria recognize these stresses and subsequently induce transcriptional responses remains not well understood. In this study, we showed that LiaFSR senses cell envelope stress through colocalization of LiaF and LiaS with the group A Streptococcus (GAS) ExPortal and is activated in direct response to ExPortal disruption by antimicrobials or human antimicrobial peptides. Our studies shed new light on the sensing of cell envelope stress in Gram-positive bacteria and may contribute to the development of therapies targeting these processes.

scholarly journals CovS Inactivates CovR and Is Required for Growth under Conditions of General Stress in Streptococcus pyogenes

2004 ◽  
Vol 186 (12) ◽  
pp. 3928-3937 ◽  
Author(s):  
Tracy L. Dalton ◽  
June R. Scott
Keyword(s):  
Gene Expression ◽  
Streptococcus Pyogenes ◽  
Sigma Factor ◽  
Regulatory System ◽  
Stress Conditions ◽  
Mild Stress ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
General Stress ◽  
Two Component

ABSTRACT The gram-positive human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes diseases ranging from mild and often self-limiting infections of the skin or throat to invasive and life-threatening illnesses. To cause such diverse types of disease, the GAS must be able to sense adverse environments and regulate its gene expression accordingly. The CovR/S two-component signal transduction regulatory system in GAS represses about 15% of the GAS genome, including many genes involved in virulence, in response to the environment. We report that CovR is still able to repress transcription from several promoters in the absence of the putative histidine kinase sensor for this system, CovS. We also show that a phosphorylation site mutant (D53A) of CovR is unable to repress gene expression. In addition, we report that a strain with a nonpolar mutation in CovS does not grow at a low pH, elevated temperature, or high osmolarity. The stress-related phenotypes of the CovS mutant were complemented by expression of covS from a plasmid. Selection for growth of a CovS mutant under stress conditions resulted in isolation of second-site mutations that inactivated covR, indicating that CovR and CovS act in the same pathway. Also, at 40°C in the wild-type strain, CovR appeared to be less active on the promoter tested, which is consistent with the hypothesis that it was partially inactivated by CovS. We suggest that under mild stress conditions, CovS inactivates CovR, either directly or indirectly, and that this inactivation relieves repression of many GAS genes, including the genes needed for growth of GAS under stress conditions and some genes that are necessary for virulence. Growth of many gram-positive bacteria under multiple-stress conditions requires alteration of promoter recognition produced by RNA polymerase association with the general stress response sigma factor, σB. We provide evidence that for GAS, which lacks a sigB ortholog, growth under stress conditions requires the CovR/S two-component regulatory system instead. This two-component system in GAS thus appears to perform a function for which other gram-positive bacteria utilize an alternative sigma factor.

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