scholarly journals The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Brady T. Russo ◽  
Yetunde A. Ayinuola ◽  
Damini Singh ◽  
Katelyn Carothers ◽  
Vincent A. Fischetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Membrane ◽  
Cell Surface ◽  
Streptococcus Pyogenes ◽  
Cytoplasmic Membrane ◽  
M Protein ◽  
Sortase A ◽  
Content Type ◽  
M Proteins ◽  
Group A

ABSTRACT Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface. IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

scholarly journals M-Protein Analysis of Streptococcus pyogenes Isolates Associated with Acute Rheumatic Fever in New Zealand

2015 ◽  
Vol 53 (11) ◽  
pp. 3618-3620 ◽  
Author(s):  
Deborah A. Williamson ◽  
Pierre R. Smeesters ◽  
Andrew C. Steer ◽  
John D. Steemson ◽  
Adrian C. H. Ng ◽  
...  
Keyword(s):  
New Zealand ◽  
Rheumatic Fever ◽  
Streptococcus Pyogenes ◽  
Acute Rheumatic Fever ◽  
Skin Infection ◽  
Protein Analysis ◽  
M Protein ◽  
Content Type ◽  
Typing System ◽  
Group A

We applied anemmcluster typing system to group AStreptococcusstrains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenicemmtypes but found a high proportion ofemmtypes previously associated with pyoderma, further suggesting a role for skin infection in ARF.

scholarly journals Surface Analyses and Immune Reactivities of Major Cell Wall-Associated Proteins of Group A Streptococcus

2005 ◽  
Vol 73 (5) ◽  
pp. 3137-3146 ◽  
Author(s):  
Jason N. Cole ◽  
Ruben D. Ramirez ◽  
Bart J. Currie ◽  
Stuart J. Cordwell ◽  
Steven P. Djordjevic ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Streptococcus Pyogenes ◽  
Proteomic Analysis ◽  
Group A Streptococcus ◽  
Gas Cell ◽  
Distinct Cell ◽  
Associated Proteins ◽  
Group A

ABSTRACT A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

scholarly journals Host Pathways of Hemostasis that Regulate Group A Streptococcus pyogenes Pathogenicity

2020 ◽  
Vol 21 (2) ◽  
pp. 193-201
Author(s):  
Victoria A. Ploplis ◽  
Francis J. Castellino
Keyword(s):  
Streptococcus Pyogenes ◽  
Inflammatory Responses ◽  
Group A Streptococcus ◽  
Protein A ◽  
M Protein ◽  
Severe Group ◽  
Group A ◽  
Flow Through ◽  
Hallmark Feature ◽  
Major Target

A hallmark feature of severe Group A Streptococcus pyogenes (GAS) infection is dysregulated hemostasis. Hemostasis is the primary pathway for regulating blood flow through events that contribute towards clot formation and its dissolution. However, a number of studies have identified components of hemostasis in regulating survival and dissemination of GAS. Several proteins have been identified on the surface of GAS and they serve to either facilitate invasion to host distal sites or regulate inflammatory responses to the pathogen. GAS M-protein, a surface-exposed virulence factor, appears to be a major target for interactions with host hemostasis proteins. These interactions mediate biochemical events both on the surface of GAS and in the solution when M-protein is released into the surrounding environment through shedding or regulated proteolytic processes that dictate the fate of this pathogen. A thorough understanding of the mechanisms associated with these interactions could lead to novel approaches for altering the course of GAS pathogenicity.

scholarly journals Antigenic domains of the streptococcal Pep M5 protein. Localization of epitopes crossreactive with type 6 M protein and identification of a hypervariable region of the M molecule.

1986 ◽  
Vol 163 (1) ◽  
pp. 129-138 ◽  
Author(s):  
B N Manjula ◽  
A S Acharya ◽  
T Fairwell ◽  
V A Fischetti
Keyword(s):  
Protein Localization ◽  
Hypervariable Region ◽  
Coiled Coil ◽  
M Protein ◽  
Terminal Domain ◽  
Coil Structure ◽  
M Proteins ◽  
Group A ◽  
Antigenic Domains ◽  
Antigenic Epitopes

Pep M5, the pepsin-derived N-terminal half of the group A streptococcal type 5 M protein exhibits immunologic crossreaction with type 6 M protein, localizing some of the M6-crossreactive epitope(s) within this segment of the M5 protein. Based on the amino acid sequence of the Pep M5 protein, two structurally distinct domains have been recognized within its coiled-coil structure. We have now found that peptides derived from both the structurally distinct domains of the Pep M5 protein contain antigenic epitopes. Furthermore, only the peptides from the C-terminal domain of the Pep M5 protein crossreacted with rabbit anti-M6 sera, whereas those from the N-terminal domain did not. Consistent with this, sequence analyses of the arginyl peptides of the Pep M6 protein, the pepsin-derived N-terminal half of the M6 protein, revealed extensive homology of some of these peptides with regions within the C-terminal domain of the Pep M5 molecule. While an arginyl peptide of the Pep M6 protein exhibits 84% homology with region 150-186 of the Pep M5 protein, the C-terminal hexadecapeptide of the Pep M6 protein is virtually identical with the corresponding region of the Pep M5 protein. These results are suggestive of conformational similarities in the region around the pepsin-susceptible site within the M5 and M6 proteins. In addition, one or more epitopes of the M5 protein that are crossreactive with the M6 protein may be placed close to the pepsin-susceptible site of the M5 protein. Previous studies have suggested the N-terminal half of the M proteins to be the variable part of the molecule among the different M protein serotypes. The present results suggest that the N-terminal quarter of the M protein may represent the hypervariable domain of the M molecule.

scholarly journals The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus

2015 ◽  
Vol 197 (23) ◽  
pp. 3720-3730 ◽  
Author(s):  
Jessica L. Danger ◽  
Nishanth Makthal ◽  
Muthiah Kumaraswami ◽  
Paul Sumby
Keyword(s):  
Cell Surface ◽  
Virulence Factors ◽  
Binding Proteins ◽  
Mrna Translation ◽  
Regulatory Rna ◽  
Collagen Binding ◽  
Content Type ◽  
Small Regulatory Rna ◽  
Group A ◽  
Fibronectin Binding

ABSTRACTThe group AStreptococcus(GAS;Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to theprtF1andprtF2mRNAs within their 5′ untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and theprtF1andprtF2mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection.IMPORTANCEMore than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens.

scholarly journals CovRS-Regulated Transcriptome Analysis of a Hypervirulent M23 Strain of Group A Streptococcus pyogenes Provides New Insights into Virulence Determinants

2015 ◽  
Vol 197 (19) ◽  
pp. 3191-3205 ◽  
Author(s):  
Yun-Juan Bao ◽  
Zhong Liang ◽  
Jeffrey A. Mayfield ◽  
Shaun W. Lee ◽  
Victoria A. Ploplis ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Broad Spectrum ◽  
Virulence Genes ◽  
Expression Profiles ◽  
Altered Expression ◽  
Content Type ◽  
Metabolic Genes ◽  
A Genome ◽  
Group A ◽  
Regulated Gene Expression

ABSTRACTThe two-componentcontrolofvirulence (Cov) regulator (R)-sensor (S) (CovRS) regulates the virulence ofStreptococcus pyogenes(group AStreptococcus[GAS]). Inactivation of CovS during infection switches the pathogenicity of GAS to a more invasive form by regulating transcription of diverse virulence genes via CovR. However, the manner in which CovRS controls virulence through expression of extended gene families has not been fully determined. In the current study, the CovS-regulated gene expression profiles of a hypervirulentemm23GAS strain (M23ND/CovS negative [M23ND/CovS−]) and a noninvasive isogenic strain (M23ND/CovS+), under different growth conditions, were investigated. RNA sequencing identified altered expression of ∼349 genes (18% of the chromosome). The data demonstrated that M23ND/CovS−achieved hypervirulence by allowing enhanced expression of genes responsible for antiphagocytosis (e.g.,hasABC), by abrogating expression of toxin genes (e.g.,speB), and by compromising gene products with dispensable functions (e.g.,sfb1). Among these genes, several (e.g.,parEandparC) were not previously reported to be regulated by CovRS. Furthermore, the study revealed that CovS also modulated the expression of a broad spectrum of metabolic genes that maximized nutrient utilization and energy metabolism during growth and dissemination, where the bacteria encounter large variations in available nutrients, thus restructuring metabolism of GAS for adaption to diverse growth environments. From constructing a genome-scale metabolic model, we identified 16 nonredundant metabolic gene modules that constitute unique nutrient sources. These genes were proposed to be essential for pathogen growth and are likely associated with GAS virulence. The genome-wide prediction of genes associated with virulence identifies new candidate genes that potentially contribute to GAS virulence.IMPORTANCEThe CovRS system modulates transcription of ∼18% of the genes in theStreptococcus pyogenesgenome. Mutations that inactivate CovR or CovS enhance the virulence of this bacterium. We determined complete transcriptomes of a naturally CovS-inactivated invasive deep tissue isolate of anemm23strain ofS. pyogenes(M23ND) and its complemented avirulent variant (CovS+). We identified diverse virulence genes whose altered expression revealed a genetic switching of a nonvirulent form of M23ND to a highly virulent strain. Furthermore, we also systematically uncovered for the first time the comparative levels of expression of a broad spectrum of metabolic genes, which reflected different metabolic needs of the bacterium as it invaded deeper tissue of the human host.

scholarly journals The Streptococcal Protease SpeB Antagonizes the Biofilms of the Human Pathogen Staphylococcus aureus USA300 through Cleavage of the Staphylococcal SdrC Protein

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
Katelyn E. Carothers ◽  
Zhong Liang ◽  
Jeffrey Mayfield ◽  
Deborah L. Donahue ◽  
Mijoon Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Proteolytic Activity ◽  
Streptococcus Pyogenes ◽  
Human Pathogen ◽  
Content Type ◽  
Functional Studies ◽  
Human Proteins ◽  
Group A ◽  
Biofilm Disruption

ABSTRACT Streptococcus pyogenes, or group A Streptococcus (GAS), is both a pathogen and an asymptomatic colonizer of human hosts and produces a large number of surface-expressed and secreted factors that contribute to a variety of infection outcomes. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host; however, despite its broad proteolytic activity, studies on how this factor is utilized in polymicrobial environments are lacking. Here, we utilized various forms of SpeB protease to evaluate its antimicrobial and antibiofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies niches similar to those of GAS. For our investigation, we used a skin-tropic GAS strain, AP53CovS+, and its isogenic ΔspeB mutant to compare the production and activity of native SpeB protease. We also generated active and inactive forms of recombinant purified SpeB for functional studies. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface-expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche. IMPORTANCE Streptococcus pyogenes (GAS) causes a range of diseases in humans, ranging from mild to severe, and produces many virulence factors in order to be a successful pathogen. One factor produced by many GAS strains is the protease SpeB, which has been studied for its ability to cleave and degrade human proteins, an important factor in GAS pathogenesis. An understudied aspect of SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy niches similar to that of GAS. The significance of the research reported herein is the demonstration that SpeB can degrade the biofilms of the human pathogen Staphylococcus aureus, which has important implications for how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.

scholarly journals A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

2004 ◽  
Vol 186 (17) ◽  
pp. 5865-5875 ◽  
Author(s):  
Timothy C. Barnett ◽  
Aman R. Patel ◽  
June R. Scott
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Group A Streptococcus ◽  
Surface Protein ◽  
Surface Proteins ◽  
Human Pathogen ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Group A ◽  
Covalently Linked

ABSTRACT The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

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 Serum Opacity Factor (SOF) of Streptococcus pyogenes Evokes Antibodies That Opsonize Homologous and Heterologous SOF-Positive Serotypes of Group A Streptococci

2003 ◽  
Vol 71 (9) ◽  
pp. 5097-5103 ◽  
Author(s):  
Harry S. Courtney ◽  
David L. Hasty ◽  
James B. Dale
Keyword(s):  
Streptococcus Pyogenes ◽  
Rabbit Antiserum ◽  
M Protein ◽  
Group A Streptococci ◽  
Streptococcal Infections ◽  
Vaccine Candidates ◽  
Human Antibodies ◽  
Serum Opacity Factor ◽  
Group A

ABSTRACT Serum opacity factor (SOF) is a protein expressed by Streptococcus pyogenes that opacifies mammalian serum. SOF is also a virulence factor of S. pyogenes, but it has not been previously shown to elicit a protective immune response. Herein, we report that SOF evokes bactericidal antibodies against S. pyogenes in humans, rabbits, and mice. Rabbit antiserum against purified recombinant SOF2 opsonized SOF-positive M type 2, 4, and 28 S. pyogenes in human blood but had no effect on SOF-negative M type 5 S. pyogenes. Furthermore, affinity-purified human antibodies against SOF2 also opsonized SOF-positive streptococci. A combination of antisera against M2 and SOF2 proteins was dramatically more effective in killing streptococci than either antiserum alone, indicating that antibodies against SOF2 enhance the opsonic efficiency of M protein antibodies. Mice tolerated an intravenous injection of 100 μg of SOF without overt signs of toxicity, and immunization with SOF protected mice against challenge infections with M type 2 S. pyogenes. These data indicate that SOF evokes opsonic antibodies that may protect against infections by SOF-positive serotypes of group A streptococci and suggest that different serotypes of SOF have common epitopes that may be useful vaccine candidates to protect against group A streptococcal infections.

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