Microbiology & Cell Biology
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Item Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group AStreptococcusIs Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin(2019-09) Lei, Benfang; Minor, Dylan; Feng, Wenchao; Jerome, Maria; Quinn, Mark T.; Jutila, Mark A.; Liu, MengyaoGroup A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.Item Hypervirulent Group A Streptococcus of Genotype emm3 Invades the Vascular System in Pulmonary Infection of Mice(2018-06) Lei, Benfang; Minor, Dylan; Feng, Wenchao; Liu, MengyaoNatural mutations of the two-component regulatory system CovRS are frequently associated with invasive Group A Streptococcus (GAS) isolates and lead to the enhancement in 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 the emm3 genotype, MGAS315, was characterized using a mouse model of pulmonary infection to understand systemic dissemination. This strain has a G1370T mutation in the sensor kinase covS gene 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 the covS mutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, the covS mutation 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 hypervirulent emm3 GAS covS mutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination.Item Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice(2017-09) Feng, Wenchao; Minor, Dylan; Liu, Mengyao; Lei, BenfangHypervirulent group A streptococcus (GAS) can inhibit neutrophil recruitment and cause systemic infection in a mouse model of skin infection. The purpose of this study was to determine whether platelet-activating factor acetylhydrolase Sse and streptolysin S (SLS) have synergistic contributions to inhibition of neutrophil recruitment and systemic infection in subcutaneous infection of mice by MGAS315, a hypervirulent genotype emm3 GAS strain. Deletion of sse and sagA in MGAS315 synergistically reduced the skin lesion size and GAS burden in the liver and spleen. However, the mutants were persistent at skin sites and had similar growth factors in nonimmune blood. Thus, the low numbers of Δsse ΔsagA mutants in the liver and spleen were likely due to their reduction in the systemic dissemination. Few intact and necrotic neutrophils were detected at MGAS315 infection sites. In contrast, many neutrophils and necrotic cells were present at the edge of Δsse mutant infection sites on day 1 and at the edge of and inside Δsse mutant infection sites on day 2. ΔsagA mutant infection sites had massive numbers of and few intact neutrophils at the edge and center of the infection sites, respectively, on day 1 and were full of intact neutrophils or necrotic cells on day 2. Δsse ΔsagA mutant infection sites had massive numbers of intact neutrophils throughout the whole infection site. These sse and sagA deletion-caused changes in the histological pattern at skin infection sites could be complemented. Thus, the sse and sagA deletions synergistically enhance neutrophil recruitment. These findings indicate that both Sse and SLS are required but that neither is sufficient for inhibition of neutrophil recruitment and systemic infection by hypervirulent GAS.Item 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, BenfangGroup 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.Item 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, BenfangHypervirulent 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.Item 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, BenfangAltered 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.Item 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, BenfangPathogen 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.Item Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues(2015-06-05) Stetzner, Zachary W.; Li, Dengfeng; Feng, Wenchao; Liu, Mengyao; Liu, Guanghui; Wiley, James A.; Lei, BenfangThe M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covSwt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covSwt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.Item A Neutralizing Monoclonal IgG1 Antibody of Platelet-Activating Factor Acetylhydrolase SsE Protects Mice against Lethal Subcutaneous Group A StreptococcusInfection(2015-04) Lie, Mengyao; Feng, Wenchao; Zhu, Hui; Lei, BenfangGroup A Streptococcus (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 MGAS5005 sse deletion 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.Item Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues(2015-06) Stetzner, Zachary W.; Li, Dengfeng; Feng, Wenchao; Liu, Mengyao; Liu, Guanghui; Wiley, James A.; Lei, BenfangThe M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covSwt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covSwt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.