Browsing by Author "Lei, Benfang"

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1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus
(2014-04) Fonner, Brittany A.; Tripet, Brian P.; Lui, Mengyao; Zhu, Hui; Lei, Benfang; Copie, Valerie
Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdBN1). Herein, we report the near complete 1H, 13C and 15N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.
Axial Ligand Replacement Mechanism in Heme Transfer from Streptococcal Heme-Binding Protein Shp to HtsA of the HtsABC Transporter
(2013-09) Ran, Yanchao; Malmirchegini, G. Reza; Clubb, Robert T.; Lei, Benfang
The heme-binding protein Shp of Group A Streptococcus rapidly transfers its heme to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. Heme axial residue-to-alanine replacement mutant proteins of Shp and HtsA (ShpM66A, ShpM153A, HtsAM79A, and HtsAH229A) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric ShpM66A at high pH and ShpM153A have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsAM79A efficiently acquires heme from ferric Shp but alters the reaction mechanism to two kinetic phases from a single phase in the wild-type protein reactions. In contrast, apoHtsAH229A cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShpM66A into pentacoordinate holoHtsAH229A involves an intermediate, whereas holoHtsAH229A is directly formed from pentacoordinate holoShpM153A. Conversely, apoHtsAM79A reacts with holoShpM66A and holoShpM153A in mechanisms with one and two kinetic phases, respectively. These results imply that the Met79 and His229 residues of HtsA displace the Met66 and Met153 residues of Shp, respectively. Structural docking analysis supports this mechanism of the specific axial residue displacement. Furthermore, the rates of the cleavage of the axial bond in Shp in the presence of a replacing HtsA axial residue are greater than that in the absence of a replacing HtsA axial residue. These findings reveal a novel heme transfer mechanism of the specific displacement of the Shp axial residues with the HtsA axial residues and the involvement of the HtsA axial residues in the displacement.
Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That Are Involved in Innate Immune Evasion
(2013-06-17) Liu, Guanghui; Liu, Mengyao; Xie, Gang; Lei, Benfang
Human 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). SsEM1 and SsE produced by serotype M28 GAS (SsEM28) have a 37% sequence difference. This study aims at determining whether SsEM28 is 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 in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsEM28 or SeE was converted into lyso-PAF. SsEM1 and SsEM28 had kcat values of 373 s−1 and 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 in kcat and Km in hydrolyzing triglycerides, acetyl esters, and PAF indicates that the SsE proteins are more potent hydrolases against PAF and have high affinity for PAF. SsEM28 possesses much lower esterase activities against triglycerides and other esters than SsEM1 but have similar potency with SsEM1 in PAF hydrolysis. Deletion of sseM28 in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsEM28 in 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.
Direct Heme Transfer Reactions in the Group A Streptococcus Heme Acquisition Pathway
(2012-05) Lu, C.; Xie, G.; Liu, Mengyao; Zhu, Hui; Lei, Benfang
The heme acquisition machinery in Group A Streptococcus (GAS) consists of the surface proteins Shr and Shp and ATP-binding cassette transporter HtsABC. Shp cannot directly acquire heme from methemoglobin (metHb) but directly transfers its heme to HtsA. It has not been previously determined whether Shr directly relays heme from metHb to Shp. Thus, the complete pathway for heme acquisition from metHb by the GAS heme acquisition machinery has remained unclear. In this study, the metHb-to-Shr and Shr-to-Shp heme transfer reactions were characterized by spectroscopy, kinetics and protein-protein interaction analyses. Heme is efficiently transferred from the β and α subunits of metHb to Shr with rates that are 7 and 60 times greater than those of the passive heme release from metHb, indicating that Shr directly acquires heme from metHb. The rapid heme transfer from Shr to Shp involves an initial heme donor/acceptor complex and a spectrally and kinetically detectable transfer intermediate, implying that heme is directly channeled from Shr to Shp. The present results show that Shr speeds up heme transfer from metHb to Shp, whereas Shp speeds up heme transfer from Shr to HtsA. Furthermore, the findings demonstrate that Shr can interact with metHb and Shp but not HtsA. Taken together with our published results on the Shp/HtsA reaction, these findings establish a model of the heme acquisition pathway in GAS in which Shr directly extracts heme from metHb and Shp relays it from Shr to HtsA.
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.
¹H, ¹³C, ¹⁵N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus
(2013-05) Fonner, Brittany A.; Tripet, Brian P.; Lui, Mengyao; Lei, Benfang; Copie, Valerie
Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdBN1). Herein, we report the near complete 1H, 13C and 15N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.
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, Mengyao
Natural 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.
IgG Endopeptidase SeMac does not Inhibit Opsonophagocytosis of Streptococcus equi Subspecies equi by Horse PolymorphonuclearLeukocytes
(2010-08) Liu, Mengyao; Lei, Benfang
The secreted Mac protein made by group A Streptococcus (GAS) inhibits opsonophagocytosis of GAS by human polymorphonuclear leukocytes (PMNs). This protein also has the endopeptidase activity against human immunoglobulin G (IgG), and the Cys94, His262 and Asp284 are critical for the enzymatic activity. The horse pathogen Streptococcus equi subspecies equi produces a homologue of Mac (SeMac). SeMac was characterized to determine whether SeMac has IgG endopeptidase activity and inhibits opsonophagocytosis of S. equi by horse PMNs. The gene was cloned and recombinant SeMac was overexpressed in Escherichia coli and purified to homogeneity. Mice with experimental S. equi infection and horses with strangles caused by S. equi seroconverted to SeMac, indicating that SeMac is produced in vivo during infection. SeMac has endopeptidase activity against human IgG. However, the protein just cleaves a small fraction, which may be IgG1 only, of horse IgG. Replacement of Cys102 with Ser or His272 with Ala abolishes the enzymatic activity of SeMac, and the Asp294Ala mutation greatly decreases the enzymatic activity. SeMac does not inhibit opsonophagocytosis of S. equi by horse PMNs but opsonophagocytosis of GAS by human PMNs. Thus, SeMac is a cysteine endopeptidase with a limited activity against horse IgG and must have other function.
Lipid Oxidation in Trout Muscle Is Strongly Inhibited by a Protein That Specifically Binds Hemin Released from Hemoglobin
(2013-05) Cai, He; Grunwald, Eric W.; Park, Sung Yong; Lei, Benfang; Richards, Mark P.
The recombinant streptococcal protein apoShp can be used as a probe for hemoglobin (Hb) reactivity in fish muscle due to its specific affinity for hemin that is released from Hb at post-mortem pH values. Hemin affinity measurements indicated that apoShp binds hemin released from Hb but not myoglobin (Mb). Hemin affinity of holoShp was higher at pH 5.7 compared to pH 8.0. This may be attributed to enhanced electrostatic interaction of His58 with the heme-7-propionate at lower pH. ApoShp readily acquired hemin that was released from trout IV metHb in the presence of washed cod muscle during 2 °C storage at pH 6.3. This was based on increases in redness in the washed cod matrix, which occurs when apoShp binds hemin that is released from metHb. ApoShp prevented Hb-mediated lipid oxidation in washed cod muscle during 2 °C storage. The prevention of Hb-mediated lipid oxidation by apoShp was likely due to bis-methionyl coordination of hemin that dissociated from metHb. This hexacoordination of hemin appears to prevent peroxide-mediated redox reactions, and there is no component in the matrix capable of dissociating hemin from Shp. ApoShp was also added to minced muscle from rainbow trout (Oncorhynchus mykiss) to examine the degree to which Hb contributes to lipid oxidation in trout muscle. Addition of apoShp inhibited approximately 90% of the lipid oxidation that occurred in minced trout muscle during 9 days of 2 °C storage on the basis of lipid peroxide, hexanal, and thiobarituric acid reactive substances (TBARS) values. These results strongly suggest that Hb is the primary promoter of lipid oxidation in trout muscle.
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, Benfang
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 (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.
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.
Non-Heme-Binding Domains and Segments of the Staphylococcus aureus IsdB Protein Critically Contribute to the Kinetics and Equilibriumof Heme Acquisition from Methemoglobin
(2014-06) Zhu, Hui; Li, Dengfeng; Liu, Mengyao; Copie, Valerie; Lei, Benfang
The hemoglobin receptor IsdB rapidly acquires heme from methemoglobin (metHb) in the heme acquisition pathway of Staphylococcus aureus. IsdB consists of N-terminal segment (NS), NEAT1 (N1), middle (MD), and heme binding NEAT2 (N2) domains, and C-terminal segment (CS). This study aims to elucidate the roles of these domains or segments in the metHb/IsdB reaction. Deletion of CS does not alter the kinetics and equilibrium of the reaction. Sequential deletions of NS and N1 in NS-N1-MD-N2 progressively reduce heme transfer rates and change the kinetic pattern from one to two phases, but have no effect on the equilibrium of the heme transfer reaction, whereas further deletion of MD reduces the percentage of transferred metHb heme. MD-N2 has higher affinity for heme than N2. MD in trans reduces rates of heme dissociation from holo-N2 and increases the percentage of metHb heme captured by N2 by 4.5 fold. NS-N1-MD and N2, but not NS-N1, MD, and N2, reconstitute the rapid metHb/IsdB reaction. NS-N1-MD-NIsdC, a fusion protein of NS-N1-MD and the NEAT domain of IsdC, slowly acquires heme from metHb by itself but together with N2 results in rapid heme loss from metHb. Thus, NS-N1 and MD domains specifically and critically contribute to the kinetics and equilibrium of the metHb/IsdB reaction, respectively. These findings support a mechanism of direct heme acquisition by IsdB in which MD enhances the affinity of N2 for heme to thermodynamically drive heme transfer from metHb to IsdB and in which NS is required for the rapid and single phase kinetics of the metHb/IsdB reaction.
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.
A periplasmic arsenite-binding protein involved in regulating arsenite oxidation
(2011-12) Liu, Guanghui; Liu, Mengyao; Kim, Eun-Hae; Maaty, Walid S.; Bothner, Brian; Lei, Benfang; Rensing, Christopher; Wang, Gejiao; McDermott, Timothy R.
Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical speciation, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal transduction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a periplasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An ΔaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a KD of 2.4 µM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.
Pneumococcal surface protein A contributes to secondary Streptococcus pneumoniae infection after influenza virus infection
(2009-08) King, Quinton O.; Lei, Benfang; Harmsen, Allen G.
We compared the growth of Streptococcus pneumoniae mutants with a disruption in the gene for either pneumococcal surface protein A (PspA−), neuraminidase A (NanA−), or hyaluronidase (Hyl−) to that of the parental strain D39 by means of a competitive growth model in mice with and those without prior influenza virus infection. The numbers of total bacteria recovered from mice with prior influenza virus infection were significantly greater than those recovered from mice without prior influenza virus infection. Although the Hyl− and NanA− mutants did not display attenuation in mice with or without prior influenza virus infection, the PspA− mutant exhibited attenuation both in mice with and in mice without prior influenza virus infection. This defect was severe in influenza virus–infected mice, for which growth of the PspA− mutant was 1800-fold lower than that of the parental strain D39. Furthermore, PspA immunization significantly reduced secondary bacterial lung burdens and concentrations of specific markers of lung damage in mice receiving serotypes 2, 3, and 4 pneumococci. Our findings indicate that PspA contributes to secondary S. pneumoniae infection after influenza virus infection and that PspA immunization mitigates early secondary pneumococcal lung infections.
Production, Characterization, and Flocculation Mechanism of Cation Independent, pH Tolerant, and Thermally Stable Bioflocculant from Enterobacter sp. ETH-2
(2014-12) Tang, Wei; Song, Liyan; Li, Dou; Qiao, Jing; Zhao, Tiantao; Zhao, Heping; Lei, Benfang
Synthetic high polymer flocculants, frequently utilized for flocculating efficiency and low cost, recently have been discovered as producing increased risk to human health and the environment. Development of a more efficient and environmentally sound alternative flocculant agent is investigated in this paper. Bioflocculants are produced by microorganisms and may exhibit a high rate of flocculation activity. The bioflocculant ETH-2, with high flocculating activity (2849 mg Kaolin particle/mg ETH-2), produced by strain Enterobacter sp. isolated from activated sludge, was systematically investigated with regard to its production, characterization, and flocculation mechanism. Analyses of microscopic observation, zeta potential and ETH-2 structure demonstrates the bridging mechanism, as opposed to charge neutralization, was responsible for flocculation of the ETH-2. ETH-2 retains high molecular weight (603 to 1820 kDa) and multi-functional groups (hydroxyl, amide and carboxyl) that contributed to flocculation. Polysaccharides mainly composed of mannose, glucose, and galactose, with a molar ratio of 1:2.9:9.8 were identified as the active constituents in bioflocculant. The structure of the long backbone with active sites of polysaccharides was determined as a primary basis for the high flocculation activity. Bioflocculant ETH-2 is cation independent, pH tolerant, and thermally stable, suggesting a potential fit for industrial application.
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.
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, Benfang
Hypervirulent 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.
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.
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, Benfang
The 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.