ABSTRACT
To understand protective immune responses against the onset of group A Streptococcus respiratory infection, we investigated whether MyD88 KO mice were susceptible to acute infection through transmission. After commingling with mice that had intranasal group A Streptococcus (GAS) inoculation, MyD88-/- recipient mice had increased GAS loads in the nasal cavity and throat that reached a mean throat colonization of 6.3 × 106 CFU/swab and mean GAS load of 5.2 × 108 CFU in the nasal cavity on day 7. Beyond day 7, MyD88-/- recipient mice became moribund, with mean 1.6 × 107 CFU/swab and 2.5 × 109 CFU GAS in the throat and nasal cavity, respectively. Systemic GAS infection occurred a couple of days after the upper respiratory infection. GAS infects the lip, the gingival sulcus of the incisor teeth, and the lamina propria of the turbinate but not the nasal cavity and nasopharyngeal tract epithelia, and C57BL/6J recipient mice had no or low levels of GAS in the nasal cavity and throat. Direct nasal GAS inoculation of MyD88-/- mice caused GAS infection, mainly in the lamina propria of the turbinate. In contrast, C57BL/6J mice with GAS inoculation had GAS bacteria in the nasal cavity but not in the lamina propria of the turbinates. Thus, MyD88-/- mice are highly susceptible to acute and lethal GAS infection through transmission, and MyD88 signaling is critical for protection of the respiratory tract lamina propria but not nasal and nasopharyngeal epithelia against GAS infection.
Subject(s)
Epithelium/microbiology , Host-Pathogen Interactions , Myeloid Differentiation Factor 88/deficiency , Respiratory Mucosa/microbiology , Respiratory Tract Infections/etiology , Streptococcal Infections/etiology , Streptococcal Infections/transmission , Streptococcus pyogenes/physiology , Animals , Biopsy , Disease Susceptibility , Epithelium/pathology , Genetic Predisposition to Disease , Immunohistochemistry , Mice , Mice, Knockout , Neutrophil Infiltration , Organ Specificity , Respiratory Mucosa/pathology , Respiratory Tract Infections/pathology , Streptococcal Infections/pathologyABSTRACT
Group 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.
Subject(s)
Host-Pathogen Interactions/immunology , Lung/enzymology , NADPH Oxidases/immunology , Neutrophils/enzymology , Streptococcal Infections/enzymology , Streptococcus pyogenes/pathogenicity , Animals , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Female , Lung/immunology , Lung/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/genetics , Neutrophil Infiltration , Neutrophils/immunology , Neutrophils/microbiology , Organ Specificity , Phagocytes/enzymology , Phagocytes/immunology , Skin/immunology , Skin/microbiology , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcus pyogenes/immunology , Streptolysins/deficiency , Streptolysins/genetics , Streptolysins/immunologyABSTRACT
Natural mutations of the two-component regulatory system CovRS are frequently associated with invasive group A Streptococcus (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 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.
Subject(s)
Genotype , Lung Diseases/microbiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Streptococcus pyogenes/pathogenicity , Animals , Bacteremia , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Cytokines/metabolism , Female , Immunity, Innate , Lung Diseases/blood , Lung Diseases/complications , Mice , Mice, Inbred C57BL , Mutation, Missense , Streptococcal Infections/blood , Streptococcal Infections/complications , VirulenceABSTRACT
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.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Bacterial Proteins/metabolism , Genotype , Immunologic Factors/metabolism , Neutrophil Infiltration/drug effects , Streptococcus pyogenes/pathogenicity , Streptolysins/metabolism , Virulence Factors/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , Antigens, Bacterial/genetics , Bacterial Load , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Carrier Proteins/genetics , Disease Models, Animal , Gene Deletion , Liver/microbiology , Mice, Inbred C57BL , Skin/microbiology , Spleen/microbiology , Streptococcal Infections/microbiology , Streptococcal Infections/physiopathology , Streptococcus pyogenes/classification , Streptolysins/geneticsABSTRACT
Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked 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 infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.
Subject(s)
Bacterial Proteins/genetics , Codon, Nonsense/genetics , Exotoxins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Streptococcal Infections/microbiology , Streptococcus pyogenes/genetics , Trans-Activators/genetics , Virulence/genetics , Animals , Down-Regulation/genetics , Female , Gene Expression Regulation, Bacterial/genetics , Histidine Kinase , Male , Mice , Mice, Inbred C57BL , Neutrophil Infiltration/genetics , Skin/microbiology , Transcription, Genetic/geneticsABSTRACT
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.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Enzyme Inhibitors/administration & dosage , Immunization, Passive , Streptococcal Infections/pathology , Streptococcus pyogenes/enzymology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , Female , Gene Deletion , Immunoglobulin G/administration & dosage , Mice, Inbred BALB C , Streptococcal Infections/microbiology , Streptococcus pyogenes/physiology , Survival Analysis , Treatment OutcomeABSTRACT
Invasive 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 Δsda1 mutants with (5448 Δsda1(M+) strain) and without (5448 Δsda1(M-) strain) M protein production. The 5448 Δsda1(M+) strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 Δsda1(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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/immunology , Deoxyribonuclease I/immunology , Immunity, Innate , Intracellular Signaling Peptides and Proteins/genetics , Repressor Proteins/genetics , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcus pyogenes/immunology , Animals , Deoxyribonuclease I/genetics , Histidine Kinase , Humans , Immune Evasion , Intracellular Signaling Peptides and Proteins/immunology , Male , Mice , Mice, Inbred C57BL , Regulon , Repressor Proteins/immunology , Streptococcus pyogenes/enzymology , Streptococcus pyogenes/geneticsABSTRACT
The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdB(N1)) spanning residues 125-272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdB(N1) is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdB(N1)) is disordered in solution. Analysis of Hb binding demonstrates that IsdB(N1) can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdB(N1) loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdB(N1) in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain.
Subject(s)
Cation Transport Proteins/chemistry , Hemoglobins/metabolism , Protein Structure, Tertiary , Amino Acid Sequence , Antigens, Bacterial/chemistry , Cation Transport Proteins/metabolism , Heme/metabolism , Hemoglobins/chemistry , Methemoglobin/chemistry , Nuclear Magnetic Resonance, Biomolecular , Phenylalanine/chemistry , Receptors, Cell Surface/chemistry , Staphylococcus aureus/metabolismABSTRACT
Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH(N2N3), Ala(326)-Asp(660)) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH(N2N3) extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.
Subject(s)
Antigens, Bacterial/metabolism , Cation Transport Proteins/metabolism , Heme/metabolism , Hemoglobins/metabolism , Receptors, Cell Surface/metabolism , Staphylococcus aureus/metabolism , Cloning, Molecular , Humans , Nuclear Magnetic Resonance, Biomolecular , ProteolysisABSTRACT
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 [SpeB(A-)]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeB(A-) variants (SpeB(A-)%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeB(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.
Subject(s)
Bacterial Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Mutation , Neutrophils/immunology , Protein Kinases/genetics , Streptococcal Infections/immunology , Streptococcus pyogenes/genetics , Analysis of Variance , Animals , Bacterial Proteins/metabolism , Bronchoalveolar Lavage Fluid/cytology , Cysteine Endopeptidases/metabolism , Disease Models, Animal , Exotoxins/metabolism , Female , Genetic Variation , Immune Evasion , Immunity, Innate/physiology , Liver/microbiology , Macrophages/cytology , Mice , Mice, Mutant Strains , Neutrophils/cytology , Skin/microbiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/immunology , Virulence/geneticsABSTRACT
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 (Δsse(MGAS5005)) 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. K(M) and k(cat) 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 Δsse(MGAS5005) 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.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/immunology , Immunity, Innate , Neutrophil Infiltration/immunology , Neutrophils/metabolism , Platelet Activating Factor/metabolism , Skin Diseases, Bacterial/immunology , Skin/immunology , Streptococcus/enzymology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Female , Gene Deletion , Humans , Male , Mice , Mice, Knockout , Neutrophils/immunology , Platelet Activating Factor/immunology , Skin/metabolism , Skin/microbiology , Skin/pathology , Skin Diseases, Bacterial/genetics , Skin Diseases, Bacterial/metabolism , Skin Diseases, Bacterial/pathology , Streptococcus/genetics , Streptococcus/immunology , Streptococcus/pathogenicityABSTRACT
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 (Shp(M66A), Shp(M153A), HtsA(M79A), and HtsA(H229A)) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric Shp(M66A) at high pH and Shp(M153A) have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsA(M79A) 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, apoHtsA(H229A) cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShp(M66A) into pentacoordinate holoHtsA(H229A) involves an intermediate, whereas holoHtsA(H229A) is directly formed from pentacoordinate holoShp(M153A). Conversely, apoHtsA(M79A) reacts with holoShp(M66A) and holoShp(M153A) 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.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Carrier Proteins/metabolism , Heme/metabolism , Hemeproteins/metabolism , Circular Dichroism , Heme-Binding Proteins , Hydrogen-Ion Concentration , Imidazoles/metabolism , Kinetics , Ligands , Streptococcus pyogenes/metabolismABSTRACT
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 (SsE(M1)) reduces neutrophil recruitment by targeting platelet-activating factor (PAF). SsE(M1) and SsE produced by serotype M28 GAS (SsE(M28)) have a 37% sequence difference. This study aims at determining whether SsE(M28) 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 SsE(M1), SsE(M28), SeE, the SsE homologue in Streptococcus equi, and human plasma PAF-AH (hpPAF-AH). PAF incubated with SsE(M28) or SeE was converted into lyso-PAF. SsE(M1) and SsE(M28) 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 SsE(M1), SsE(M28), 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. SsE(M28) possesses much lower esterase activities against triglycerides and other esters than SsE(M1) but have similar potency with SsE(M1) in PAF hydrolysis. Deletion of sse(M28) in a covS deletion mutant of GAS increased neutrophil recruitment and reduced skin infection, whereas in trans expression of SsE(M28) 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.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/immunology , Immune Evasion/immunology , Immunity, Innate/immunology , Streptococcus/immunology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Esterases/genetics , Esterases/immunology , Esterases/metabolism , Female , Humans , Hydrolysis , Immune Evasion/genetics , Immunity, Innate/genetics , Mice , Mice, Inbred BALB C , Neutrophil Infiltration/genetics , Neutrophil Infiltration/immunology , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/microbiology , Platelet Activating Factor/genetics , Platelet Activating Factor/immunology , Platelet Activating Factor/metabolism , Sequence Deletion/genetics , Sequence Deletion/immunology , Staphylococcal Skin Infections/genetics , Staphylococcal Skin Infections/immunology , Staphylococcal Skin Infections/metabolism , Staphylococcal Skin Infections/microbiology , Streptococcus/genetics , Streptococcus/metabolism , Substrate Specificity/genetics , Substrate Specificity/immunology , Triglycerides/genetics , Triglycerides/immunology , Triglycerides/metabolismABSTRACT
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 covS(Δ1bp) 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.
Subject(s)
Bacterial Proteins/classification , Bacterial Proteins/metabolism , Neutrophils/physiology , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcus pyogenes/metabolism , Animals , Bacterial Proteins/genetics , Female , Gene Deletion , Gene Expression Regulation, Bacterial/physiology , Immunity, Innate , Mice , Mice, Inbred BALB C , Streptococcus pyogenes/geneticsABSTRACT
Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the DeltamtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.
Subject(s)
Fasciitis, Necrotizing/genetics , Polymorphism, Single Nucleotide , Virulence/genetics , Animals , Fasciitis, Necrotizing/immunology , Fasciitis, Necrotizing/prevention & control , Genetic Variation , Humans , Macaca fascicularis/microbiology , Male , Mice , Neutrophils/physiology , Serotyping , Shock, Septic/microbiology , Streptococcus pyogenes/genetics , Up-RegulationABSTRACT
The M protein, a major virulence factor of Group A Streptococcus (GAS), is regulated by the multigene regulator Mga. An unexplained phenomena frequently occurring with in vitro genetic manipulation or culturing of M1T1 GAS strains is the loss of M protein production. This study was aimed at elucidating the basis for the loss of M protein production. The majority of M protein-negative (M-) variants had one C deletion at a tract of 8 cytidines starting at base 1,571 of the M1 mga gene, which is designated as c.1571C[8]. The C deletion led to a c.1571C[7] mga variant that has an open reading frame shift and encodes a Mga-M protein fusion protein. Transformation with a plasmid containing wild-type mga restored the production of the M protein in the c.1571C[7] mga variant. Isolates producing M protein (M+) were recovered following growth of the c.1571C[7] M protein-negative variant subcutaneously in mice. The majority of the recovered isolates with reestablished M protein production had reverted back from c.1571C[7] to c.1571C[8] tract and some M+ isolates lost another C in the c.1571C[7] tract, leading to a c.1571C[6] variant that encodes a functional Mga with 13 extra amino acid residues at the C-terminus compared with wild-type Mga. The nonfunctional c.1571C[7] and functional c.1571C[6] variants are present in M1, M12, M14, and M23 strains in NCBI genome databases, and a G-to-A nonsense mutation at base 1,657 of M12 c.1574C[7] mga leads to a functional c.1574C[7]/1657A mga variant and is common in clinical M12 isolates. The numbers of the C repeats in this polycytidine tract and the polymorphism at base 1,657 lead to polymorphism in the size of Mga among clinical isolates. These findings demonstrate the slipped-strand mispairing within the c.1574C[8] tract of mga as a reversible switch controlling M protein production phase variation in multiple GAS common M types.
ABSTRACT
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 K(D) 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.
Subject(s)
Agrobacterium tumefaciens/genetics , Arsenites/metabolism , Bacterial Proteins/metabolism , Oxidoreductases/metabolism , Periplasmic Binding Proteins/metabolism , Agrobacterium tumefaciens/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Oxidation-Reduction , Oxidoreductases/genetics , Sequence Deletion , Signal TransductionABSTRACT
Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and ß7/ß8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.
Subject(s)
Antigens, Bacterial/metabolism , Carrier Proteins/metabolism , Cell Wall/metabolism , Heme/metabolism , Staphylococcus aureus/metabolism , Antigens, Bacterial/chemistry , Biological Transport , Carrier Proteins/chemistry , Cell Wall/chemistry , Heme/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Staphylococcus aureus/chemistryABSTRACT
The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV-vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV-vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV-vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a K(d) of 40.9 muM but not with H229A HtsA, and cyanide binds to M79A and H229A with K(d) of 0.5 and 19.1 microM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.
Subject(s)
Carrier Proteins/chemistry , Heme/metabolism , Hemeproteins/chemistry , Streptococcus pyogenes/chemistry , Bacterial Proteins , Carbon Monoxide/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Heme-Binding Proteins , Hemeproteins/genetics , Hemeproteins/metabolism , Ligands , Mutation, Missense , Spectrum AnalysisABSTRACT
The development of new advances in understanding the role of neutrophils in inflammation requires effective procedures for isolating and purifying neutrophils. Methods for isolating human neutrophils are fairly standard, and some are covered in other chapters of this volume and previous editions. However, procedures for isolating neutrophils from nonhuman species used to model human diseases vary from those used in isolating human neutrophils and are not as well developed. Since neutrophils are highly reactive and sensitive to small perturbations, the methods of isolation are important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animal models (bovine, equine, ovine), small animal models (murine and rabbit), and nonhuman primates (cynomolgus macaques) and describe optimized details for obtaining the highest cell purity, yield, and viability.