Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion

Liu, Guanghui; Liu, Mengyao; Xie, Gary; Lei, Benfang

doi:10.1128/iai.00398-13

Cited by 11 publications

(21 citation statements)

References 49 publications

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“…We next checked the SsE expression of the 11 SpeB AϪ variants as an indicator of whether these covRS mutants enhanced expression of virulence genes. SsE solely confers the enzymatic activity of hydrolyzing platelet-activating factor (PAF) in GAS culture supernatants, and the PAF hydrolysis activity increases when the covS gene is deleted or has a null mutation (14,25,36). The PAF hy- Aϩ colonies in the same test.…”

Section: Time Course Of In Vivo Accumulation Of Mgas2221 Spebmentioning

confidence: 81%

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

Liu

Feng

et al. 2014

Infect Immun

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A؊ % 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 SpeB A؊ variants in CXCR2 ؊/؊ mice. One randomly chosen SpeB A؊ 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 SpeB A؊ variants from a mouse all had nonsynonymous covRS mutations that resulted in the SpeB A؊ 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.

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Section: Time Course Of In Vivo Accumulation Of Mgas2221 Spebmentioning

confidence: 81%

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

Liu

Feng

et al. 2014

Infect Immun

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“…The two PCR fragments were fused together in subsequent overlay PCR via the 21-bp complementary sequences that are underlined in primers 2 and 3 using primers 1 and 4. The fused PCR product first was cloned into the donor vector pDONR221 in the BP Clonase reaction and then into pGRV-RFA (29) in the LR Clonase reaction using the Gateway cloning kit. pGRV-RFA was renamed p740-RFA.…”

Section: Methodsmentioning

confidence: 99%

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

Liu

Feng

et al. 2015

Infect Immun

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bInvasive M1T1 group A Streptococcus (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 for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in ⌬sda1 mutants with (5448 ⌬sda1 M؉ strain) and without (5448 ⌬sda1 M؊ strain) M protein production. The 5448 ⌬sda1 M؉ strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 ⌬sda1 M؊ strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections. The human pathogen group A Streptococcus (GAS) causes about 700 million cases of relatively mild, noninvasive pharyngitis and superficial skin infections annually. However, severe GAS infections, including severe invasive infections and acute rheumatic fever/rheumatic heart disease, can occur, causing approximately 517,000 deaths in the world each year (1). A globally disseminated M1T1 clone of serotype M1 GAS most frequently is associated with severe invasive infections in the United States (2-4). It is believed that the original M1T1 GAS clone evolved from the acquisitions of DNase Sda1-and superantigen SpeA-encoding prophages and an interserotype exchange of a 36-kb chromosomal region containing the toxin genes encoding NAD ϩ -glycohydrolase (NADase) and streptolysin O (SLO) with serotype M12 GAS (5-7). More recently, hypervirulent M1T1 GAS strains have been isolated, usually having mutations in the two-component regulatory system CovRS (also known as CsrRS) (8-12).CovRS is known to negatively regulate multiple virulence genes in GAS, including the capsule synthetase gene hasA, the interleukin-8 (IL-8)/CXC chemokine peptidase gene spyCEP, and the platelet-activating factor acetylhydrolase gene sse (13-18). Natural CovS mutations of invasive M1T1 isolates usually enhance the expression of these virulence genes and downregulate the production of the protease SpeB (SpeB AϪ , for the lack of the...

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“…SsE is required for the skin invasion by hypervirulent serotype M1 GAS isolates in a mouse model of necrotizing fasciitis (18). The basis for the role of SsE in skin invasion has been proposed to be mediated by SsE inhibition of neutrophil recruitment in GAS skin infections (19,24), and the SsE inhibition of the neutrophil response is partly mediated by SsE-catalyzed hydrolysis of PAF (19). In this study, we first demonstrated that the MGAS5005 derivative that produced an enzymatically inactive SsE S178A mutant protein had the same phenotype as the reported phenotype of an sse deletion mutant of MGAS5005, with a reduction in skin invasion and enhancement in neutrophil recruitment during subcutaneous infection of mice (18,19).…”

Section: Discussionmentioning

confidence: 99%

“…SsE in 40 mM Tris-HCl, pH 8.0, was incubated with 2B11 at various concentrations in wells of a 96-well plate at room temperature for 5 min, and the PAF acetylhydrolase activity of the reaction mixtures was then assayed using a 2-thio-PAF assay as previously described (24). Briefly, the SsE-2B11 solutions were mixed with 30 l of solution containing 0.88 mM 2-thio-PAF and 1.26 mM 5,5=-dithiobis-(2-nitrobenzoic acid) (DTNB).…”

Section: Methodsmentioning

confidence: 99%

“…SsE is a protective antigen and is required for the skin invasion and dissemination of hypervirulent M1T1 GAS in a mouse model of necrotizing fasciitis (23). SsE has potent PAF acetylhydrolase activity and critically contributes to the inhibition of neutrophil recruitment by GAS (19,24). If the enzymatic activity of SsE is critical for its functions, an inhibitory antibody of SsE is likely protective and can have the potential to be developed as a therapeutic agent for treatment of invasive GAS infection.…”

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

et al. 2015

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Group 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 (SsE S178A ), 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. G roup A Streptococcus (GAS) is a major human pathogen that commonly causes pharyngitis and superficial skin infections (1). This pathogen can also cause severe invasive infections, such as necrotizing fasciitis, streptococcal toxic shock syndrome, pneumonia, and bacteremia. Streptococcal necrotizing fasciitis results from GAS infection of the subcutaneous tissue, which progresses rapidly, causes necrosis of the fascia and subcutaneous tissue, and leads to systemic infection (2). Annually in the United States, there are more than 10 million cases of streptococcal pharyngitis and about 10,000 cases of invasive GAS infections (3.5 cases per 100,000 persons), with a fatality rate of 13.7%, and invasive infections are most frequently caused by serotype M1, M3, and M12 GAS strains (3).While antibiotic treatment is effective to treat pharyngitis patients, it is not effective for treating severe invasive GAS infections (4). Prompt surgical debridement, fluid and electrolyte management, and analgesia are mainstays of therapy for necrotizing fasciitis (5-7). Clindamycin, hyperbaric oxygen therapy, and intravenous immunoglobulin are used as adjunctive treatments of severe invasive GAS infections (4-7). New strategies to treat severe GAS inf...

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Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion

Cited by 11 publications

References 49 publications

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

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

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