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 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 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 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 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 A Neutralizing Monoclonal IgG1 Antibody of Platelet-Activating Factor Acetylhydrolase SsE Protects Mice against Lethal Subcutaneous Group A Streptococcus Infection

2015 ◽  
Vol 83 (7) ◽  
pp. 2796-2805 ◽  
Author(s):  
Mengyao Liu ◽  
Wenchao Feng ◽  
Hui Zhu ◽  
Benfang Lei
Keyword(s):  
Enzymatic Activity ◽  
Soft Tissues ◽  
Group A Streptococcus ◽  
Platelet Activating Factor ◽  
Passive Immunization ◽  
Neutrophil Infiltration ◽  
Content Type ◽  
Paf Acetylhydrolase ◽  
Group A ◽  
Skin Invasion

Group AStreptococcus(GAS) can cause life-threatening invasive infections, including necrotizing fasciitis. There are no effective treatments for severe invasive GAS infections. The platelet-activating factor (PAF) acetylhydrolase SsE produced by GAS is required for invasive GAS to evade innate immune responses and to invade soft tissues. This study determined whether the enzymatic activity of SsE is critical for its function in GAS skin invasion and inhibition of neutrophil recruitment and whether SsE is a viable target for immunotherapy for severe invasive GAS infections. An isogenic derivative of M1T1 strain MGAS5005 producing SsE with an S178A substitution (SsES178A), an enzymatically inactive SsE mutant protein, was generated. This strain induced higher levels of neutrophil infiltration and caused smaller lesions than MGAS5005 in subcutaneous infections of mice. This phenotype is similar to that of MGAS5005ssedeletion mutants, indicating that the enzymatic activity of SsE is critical for its function. An anti-SsE IgG1 monoclonal antibody (MAb), 2B11, neutralized the PAF acetylhydrolase activity of SsE. Passive immunization with 2B11 increased neutrophil infiltration, reduced skin invasion, and protected mice against MGAS5005 infection. However, 2B11 did not protect mice when it was administered after MGAS5005 inoculation. MGAS5005 induced vascular effusion at infection sites at early hours after GAS inoculation, suggesting that 2B11 did not always have access to infection sites. Thus, the enzymatic activity of SsE mediates its function, and SsE has the potential to be included in a vaccine but is not a therapeutic target. An effective MAb-based immunotherapy for severe invasive GAS infections may need to target virulence factors that are critical for systemic survival of GAS.

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 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 Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia

2020 ◽  
Vol 65 (1) ◽  
pp. e01284-20
Author(s):  
Punyawee Dulyayangkul ◽  
Karina Calvopiña ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Regulatory System ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Antimicrobial Drug Resistance ◽  
Regulatory Systems ◽  
Selection Of

ABSTRACTFluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr. Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

scholarly journals RivR Is a Negative Regulator of Virulence Factor Expression in Group A Streptococcus

2012 ◽  
Vol 81 (1) ◽  
pp. 364-372 ◽  
Author(s):  
Jeanette Treviño ◽  
Zhuyun Liu ◽  
Tram N. Cao ◽  
Esmeralda Ramirez-Peña ◽  
Paul Sumby
Keyword(s):  
Mutant Strain ◽  
Virulence Factor ◽  
Regulatory Networks ◽  
Group A Streptococcus ◽  
Human Keratinocytes ◽  
Negative Regulator ◽  
Upstream Region ◽  
Content Type ◽  
Factor Expression ◽  
Group A

The bacterial pathogen group AStreptococcus(GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α2-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given thatgrabandhasABCare also negatively regulated by the two-component system CovR/S (controlofvirulence), we tested whether RivR functions through CovR/S. A comparison ofrivandcovsingle and double mutant strains showed that RivR requires CovR activity forgrabandhasABCregulation. Analysis of the upstream region ofrivRidentified a novel promoter the deletion of which reducedrivRmRNA abundance by 70%. ArivRmutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. TherivRmutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.

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