Browsing by Author "Li, Jinquan"

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Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate InnateImmune Evasion
(2012-04) Liu, Mengyao; Zhu, Hui; Li, Jinquan; Garcia, C. C.; Feng, Wenchao; Kirpotina, Liliya N.; Hilmer, Jonathan K.; Tavares, L. P.; Layton, A. W.; Quinn, Mark T.; Bothner, Brian; Teixeira, M. M.; Lei, Benfang
The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (ΔsseMGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and kcat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ΔsseMGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.
Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice
(2014-04) Li, Jinquan; Liu, Guanghui; Feng, Wenchao; Zhou, Yang; Liu, Mengyao; Wiley, James A.; Lei, Benfang
Pathogen 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 A Streptococcus (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 nonsynonymous covRS mutations 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 spontaneous covRS mutations 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.
Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of in vitro and in vivo Protease SpeB Expression and Virulence
(2017-01) Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Li, Jinquan; Ishaq, Suzanne L.; Yeoman, Carl J.; Lei, Benfang
Group A Streptococcus (GAS) acquires mutations of virulence regulator CovRS in human and mouse infections that upregulate virulence genes and downregulate protease SpeB. To identify in vivo mutants with novel phenotype, GAS isolates from mouse infection were screened by enzymatic assays for SpeB and platelet-activating factor acetylhydrolase Sse, identifying a new type of variants that had enhanced Sse expression and normal SpeB production (Sse(A+)SpeB(A+)). Sse(A+)SpeB(A+) variants have transcripts levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse(A+)SpeB(A+) isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse(A+)SpeB(A+) isolates also had nonsense mutations or small indels of rocA RocA and CovS mutants have similar enhancement in expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA, but not CovS, do not downregulate speB transcription at stationary growth phase and in subcutaneous infection of mice. RocA and CovS mutations have greater enhancement in expression of hasA than spyCEP in mouse skin infection in comparison with wild type GAS. RocA mutants rank between wild type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infection of mice and exhibit distinct gene expression pattern and virulence from CovS mutants. The findings provide novel information for the understanding of GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.
Regulation of Inhibition of Neutrophil Infiltration by the Two-Component Regulatory System CovRS in Subcutaneous Murine Infection with GroupA Streptococcus
(2013-06) Li, Jinquan; Zhu, Hui; Feng, Wenchao; Liu, Mengyao; Song, Y.; Zhang, Xiaolan; Zhou, Yang; Bei, W.; Lei, Benfang
Hypervirulent 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 the covS gene. Replacement of covSΔ1bp in MGAS5005 with wild-type covS resulted in the MGAS2221 phenotype. Deletion of covS from MGAS2221 resulted in the MGAS5005 phenotype. Tests of single, double, and triple deletion mutants of the MGAS5005 sse, spyCEP, and scpA genes 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 of sse, but not spyCEP or scpA, of MGAS2221 enhances neutrophil recruitment. Thus, covS null 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.
The sagA / pel locus does not regulate the expression of the M protein of the M1T1 lineage of group A Streptococcus
(2013-11) Zhou, Yang; Hanks, Tracey S.; Feng, Wenchao; Li, Jinquan; Liu, Guanghui; Liu, Mengyao; Lei, Benfang
Altered expression of Group A Streptococcus (GAS) virulence factors, including the M protein, can result as a consequence of spontaneous genetic changes that occur during laboratory and animal passage. Occurrence of such secondary mutations during targeted gene deletion could confound the interpretation of effects attributable to the function of the gene being investigated. Contradicting reports on whether the sagA/pel locus regulates the M protein-encoding emm might be due to inconsistent occurrence of mutations unrelated with sagA. This study examined the possibility that altered emm expression observed in association with sagA/pel deletion mutants is artifactual. sagA deletion mutants (MGAS2221ΔsagA) of M1T1 isolate MGAS2221 obtained using liquid broth for GAS growth during the deletion process had diminished emm transcription and no detectable M protein production. In contrast, a ΔsagA mutant of another closely genetically related M1T1 isolate had normal emm expression. The sagB gene does not regulate emm; however, one of three MGAS2221ΔsagB mutants had diminished emm expression. The emm regulator mga was downregulated in these M protein expression-negative strains. These results argue that sagA deletion does not directly cause the downregulation of emm expression. Indeed, two MGAS2221ΔsagA mutants obtained using agar plates for GAS growth during the deletion process both had normal emm expression. We conclude that the sagA/pel locus does not regulate emm expression in the M1T1 lineage and provide a protocol for targeted gene deletion that we find less prone to the generation of mutants exhibiting downregulation in emm expression.