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

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.     

Attenuation of virulence in multiple serotypes (M1, M3, and M28) of Group A Streptococcus after the loss of secreted esterase

IntroductionGroup A Streptococcus (GAS) can produce streptococcal secreted esterase (Sse), which inhibits neutrophil recruitment to the site of infection and is crucial for GAS pathogenesis. As an effective esterase, Sse hydrolyzes the sn-2 ester bond of human platelet-activating factor, inactivating it and abolishing its ability to recruit neutrophils.ObjectivesThe purpose of this study was to investigate the effects of sse deletion on the virulence of multiple serotypes of GAS.MethodsIsogenic strains that lack the sse gene (Δsse) were derived from the parent strains MGAS5005 (serotype M1, CovRS mutant), MGAS2221 (serotype M1, wild-type CovRS), MGAS315 (serotype M3, CovRS mutant) and MGAS6180 (serotype M28, wild-type CovRS) and were used to study the differences in virulence and pathogenicity of GAS serotypes.ResultsIn a subcutaneous infection model, mice infected with MGAS5005^Δsse exhibited higher survival rates but decreased dissemination to the organs compared with mice infected with MGAS5005. When mice were infected with the four Δsse mutants, the MPO activity and IFN-γ, TNF-α, IL-2 and IL-6 levels increased, but the skin lesion sizes decreased. In an intraperitoneal infection model, the absence of Sse significantly reduced the virulence of GAS, leading to increased mouse survival rates and decreased GAS burdens in the organs in most of the challenge experiments. In addition, the numbers of the four Δsse mutants were greatly reduced 60 min after incubation with isolated rat neutrophils.ConclusionOur results suggest that Sse participates in the pathogenesis of multiple GAS serotypes (MGAS5005, MGAS2221, MGAS315 and MGAS6180), particularly the hypervirulent CovS mutant strains MGAS5005 and MGAS315. These strain differences were positively correlated with the virulence of the serotype.     

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

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 k_cat 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 k_cat and K_m 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.     

A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response

Circumvention of the host innate immune response is critical for bacterial pathogens to infect and cause disease. Here we demonstrate that the group A Streptococcus (GAS; Streptococcus pyogenes) protease SpyCEP (S. pyogenes cell envelope protease) cleaves granulocyte chemotactic protein 2 (GCP-2) and growth-related oncogene alpha (GROα), two potent chemokines made abundantly in human tonsils. Cleavage of GCP-2 and GROα by SpyCEP abrogated their abilities to prime neutrophils for activation, detrimentally altering the innate immune response. SpyCEP expression is negatively regulated by the signal transduction system CovR/S. Purified recombinant CovR bound the spyCEP gene promoter region in vitro, indicating direct regulation. Immunoreactive SpyCEP protein was present in the culture supernatants of covR/S mutant GAS strains but not in supernatants from wild-type strains. However, wild-type GAS strains do express SpyCEP, where it is localized to the cell wall. Strain MGAS2221, an organism representative of the highly virulent and globally disseminated M1T1 GAS clone, differed significantly from its isogenic spyCEP mutant derivative strain in a mouse soft tissue infection model. Interestingly, and in contrast to previous studies, the isogenic mutant strain generated lesions of larger size than those formed following infection with the parent strain. The data indicate that SpyCEP contributes to GAS virulence in a strain- and disease-dependent manner.     

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

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.     

Identification and immunogenicity of group A Streptococcus culture supernatant proteins

Extracellular proteins made by group A Streptococcus (GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed in Escherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.     

Ubiquitous occurrence of virR and scpA genes in group A streptococci

Until now a few serotypes of M-class I group A streptococci (GAS) have been shown to encode VirR, a positive regulatory factor for the coordinate expression of the M protein (emm) and C5a peptidase (scpA) genes. The polymerase chain reaction technique has been applied to the genomic template of 36 GAS serotypes to demonstrate the general presence of VirR (virR) genes and scpA in GAS of both M classes. A virR gene region conserved in size was demonstrated for every strain investigated. Differences between virR genes from GAS of the two M classes were mainly confined to the 3 end of the gene and a region upstream of the gene's promoter. Every M-class II strain and some M-class I isolates were shown to possess a scpA gene of 4.6 kb, the rest of the M-class I GAS harbors a 3.5-kb scpA gene. The additional segment of 1.1 kb in the large-size scpA genes was located within a region of direct repeats at the 3 end of the gene. Among the serotypes encoding a large-size scpA gene a minority exhibits additional sequence variation downstream of the region of direct repeats.     

Streptococcus pyogenes nuclease A (SpnA) mediated virulence does not exclusively depend on nuclease activity

BackgroundStreptococcus pyogenes, or Group A Streptococcus (GAS), is a human pathogen that causes a wide range of diseases, including pharyngitis, necrotizing fasciitis and toxic shock syndrome. The bacterium produces a large arsenal of virulence factors, including the cell wall-anchored Streptococcus pyogenes nuclease A (SpnA), which facilitates immune evasion by degrading the DNA backbone of neutrophil extracellular traps. SpnA consists of a C-terminal endo/exonuclease domain and a N-terminal domain of unknown function.MethodsRecombinant SpnA mutants were generated by alanine conversion of selected residues that were predicted to play a role in the enzymatic activity and tested for their ability to degrade DNA. A GAS spnA deletion mutant was complemented with a plasmid-borne catalytic site mutant and analyzed for virulence in a Galleria mellonella (wax moth) infection model.ResultsSeveral predicted residues were experimentally confirmed to play a role in SpnA enzymatic activity. These include Glu592, Arg696, His716, Asp767, Asn769, Asp810 and Asp842. Complementation of a GAS spnA deletion mutant with a spnA H716A mutant gene partially restored virulence in wax moth larvae, whereas complementation with the spnA wt gene completely restored activity. Furthermore, complementation with a secreted form of SpnA showed reduced virulence.ConclusionOur results show that abolishing the enzymatic activity of SpnA only partially reduces virulence suggesting that SpnA has an additional virulence function, which might be located on the N-terminal domain. Furthermore, cell wall-anchoring of SpnA results in higher virulence compared to secreted SpnA, probably due to a higher local density of the enzyme.     

Staphopains Modulate Staphylococcus aureus Biofilm Integrity

Staphylococcus aureus is a known cause of chronic biofilm infections that can reside on medical implants or host tissue. Recent studies have demonstrated an important role for proteinaceous material in the biofilm structure. The S. aureus genome encodes many secreted proteases, and there is growing evidence that these enzymes have self-cleavage properties that alter biofilm integrity. However, the specific contribution of each protease and mechanism of biofilm modulation is not clear. To address this issue, we utilized a sigma factor B (ΔsigB) mutant where protease activity results in a biofilm-negative phenotype, thereby creating a condition where the protease(s) responsible for the phenotype could be identified. Using a plasma-coated microtiter assay, biofilm formation was restored to the ΔsigB mutant through the addition of the cysteine protease inhibitor E-64 or by using Staphostatin inhibitors that specifically target the extracellular cysteine proteases SspB and ScpA (called Staphopains). Through construction of gene deletion mutants, we determined that an sspB scpA double mutant restored ΔsigB biofilm formation, and this recovery could be replicated in plasma-coated flow cell biofilms. Staphopain levels were also found to be decreased under biofilm-forming conditions, possibly allowing biofilm establishment. The treatment of S. aureus biofilms with purified SspB or ScpA enzyme inhibited their formation, and ScpA was also able to disperse an established biofilm. The antibiofilm properties of ScpA were conserved across S. aureus strain lineages. These findings suggest an underappreciated role of the SspB and ScpA cysteine proteases in modulating S. aureus biofilm architecture.     

Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients

Hasegawa T, Okamoto A, Kamimura T, Tatsuno I, Hashikawa S‐N, Yabutani M, Matsumoto M, Yamada K, Isaka M, Minami M, Ohta M. Detection of invasive protein profile of Streptococcus pyogenes M1 isolates from pharyngitis patients. APMIS 2010; 118: 167–78. Streptococcal toxic shock syndrome (STSS) is a re‐emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two‐component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two‐dimensional gel electrophoresis (2‐DE) and pulsed‐field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two‐component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2‐DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection.     

Molecular and Clinical Diagnosis of Group A Streptococcal Pharyngitis in Children

Group A Streptococcus (GAS) pharyngitis is a very common condition causing significant morbidity in children. Accurate diagnosis followed by appropriate antimicrobial therapy is recommended to prevent postinfectious sequelae. Diagnosis of GAS pharyngitis by a rapid antigen detection test (RADT) or culture in the absence of discriminating clinical findings remains challenging. Validation of new sensitive rapid diagnostic tests is therefore a priority. The performance of a loop-mediated isothermal amplification (LAMP) assay (illumigene assay) for the diagnosis of GAS pharyngitis was compared with that of a RADT and standard culture in 361 pediatric throat swab samples. Discrepant results were resolved using an alternate molecular assay. Test results were correlated with clinical presentations in patients positive by either method. The closest estimate of the true prevalence of GAS pharyngitis was 19.7% (71/361 samples). The illumigene assay alone detected 70/71 GAS-positive samples; RADT and culture detected 35/71 and 55/71 samples, respectively. RADT followed by culture confirmation of RADT-negative specimens detected 58/71 cases. The illumigene assay increased identification among children eligible for testing by American College of Physicians (ACP)/American Academy of Family Physicians (AAFP) criteria from 31 to 39 positive cases, five of which were false positives. Analysis of clinical data in GAS-positive patients indicated that a significantly greater proportion of children with McIsaac scores of ≥4 tested positive by the illumigene assay versus RADT and culture. Overall, the illumigene assay was much more sensitive and was similarly specific for GAS detection, compared to culture alone, RADT alone, or the ACP/AAFP RADT/culture algorithm. Combining high sensitivity with rapidly available results, the illumigene GAS assay is an appropriate alternative to culture for the laboratory diagnosis of GAS pharyngitis in patients for whom testing is clinically indicated.     

Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachia-induced keratitis

The filarial nematode Onchocerca volvulus is the causative organism of river blindness. Our previous studies demonstrated an essential role for endosymbiotic Wolbachia bacteria in corneal disease, which is characterized by neutrophil infiltration into the corneal stroma and the development of corneal haze. To determine the role of Toll-like receptors (TLRs) in neutrophil recruitment and activation, we injected a soluble extract of O. volvulus containing Wolbachia bacteria into the corneal stromata of C57BL/6, TLR2^−/−, TLR4^−/−, TLR2/4^−/−, and TLR9^−/− mice. We found an essential role for TLR2, but not TLR4 or TLR9, in neutrophil recruitment to the cornea and development of corneal haze. Furthermore, chimeric mouse bone marrow studies showed that resident bone marrow-derived cells in the cornea can initiate this response. TLR2 expression was also essential for CXC chemokine production by resident cells in the cornea, including corneal fibroblasts, and for neutrophil activation. Taken together, these findings indicate that Wolbachia activates TLR2 on resident bone marrow-derived cells in the corneal stroma to produce CXC chemokines, leading to neutrophil recruitment to the corneal stroma, and that TLR2 mediates O. volvulus/Wolbachia-induced neutrophil activation and development of corneal haze.     

You Say that You Want a Molecular Revolution? Changing from the Group A Streptococcus Antigen and Culture Paradigm to Molecular Testing

Group A Streptococcus (GAS) (Streptococcus pyogenes) is the most common bacterial cause of acute pharyngitis. Diagnosed cases of GAS pharyngitis should be treated with antibiotics to prevent primary and secondary complications. Commonly, testing for GAS pharyngitis can be done rapidly (in minutes) using rapid antigen detection tests (RADTs), which generally have relatively high specificity (>90%) but lower sensitivity (70 to 90%). The low sensitivity of GAS RADTs requires that culture be performed to maximize sensitivity, which delays the final result by 24 to 48 hours. New molecular GAS assays are on the market and offer relatively rapid (i.e., minutes to tens of minutes) and highly sensitive results so that culture is not required for negative results. We describe our experience with moving from RADT and culture testing for GAS to solely molecular testing, providing quicker results for improved patient care.     

Genome Sequence of an M3 Strain of Streptococcus pyogenes Reveals a Large-Scale Genomic Rearrangement in Invasive Strains and New Insights into Phage Evolution

Group Astreptococcus (GAS) is a gram-positive bacterial pathogen that causes various suppurative infections and nonsuppurative sequelae. Since the late 1980s, streptococcal toxic-shock like syndrome (STSS) and severe invasive GAS infections have been reported globally. Here we sequenced the genome of serotype M3 strain SSI-1, isolated from an STSS patient in Japan, and compared it with those of other GAS strains. The SSI-1 genome is composed of 1,884,275 bp, and 1.7 Mb of the sequence is highly conserved relative to strain SF370 (serotype M1) and MGAS8232 (serotype M18), and almost completely conserved relative to strain MGAS315 (serotype M3). However, a large genomic rearrangement has been shown to occur across the replication axis between the homologous rrn-comX1 regions and between two prophage-coding regions across the replication axis. Atotal of 1 Mb of chromosomal DNA is inverted across the replication axis. Interestingly, the recombinations between the prophage regions are within the phage genes, and the genes encoding superantigens and mitogenic factors are interchanged between two prophages. This genomic rearrangement occurs in 65% of clinical isolates (64/94) collected after 1990, whereas it is found in only 25% of clinical isolates (7/28) collected before 1985. These observations indicate that streptococcal phages represent important plasticity regions in the GAS chromosome where recombination between homologous phage genes can occur and result not only in new phage derivatives, but also in large chromosomal rearrangements.     

Inhibition of neutrophil infiltration by a malleable protein matrix of lactic acid bacteria-fermented whey proteins in vivo

Objective:  A novel nutraceutical ingredient, the Malleable Protein Matrix (MPM), has previously demonstrated a significant anti-inflammatory effect in a systemic inflammatory disease model, comparable to conventional drugs. The objective of this study was to investigate the potential anti-inflammatory effects of MPM on neutrophil infiltration in vivo, phagocytosis activity as well as cytokine and chemokine production. Methods:  Groups of ten C57BL\6J mice received water or MPM per os for a period of 2 weeks prior to the creation of a murine air pouch. The subsequent neutrophil recruitment and activities were characterized following lipopolysaccharide injection. Results:  In the water control group, the number of recruited cells was 1.8X10⁷ cells/pouch, which was reduced to 9X10⁶ cells/pouch with oral MPM consumption, representing an inhibition of 50% of infiltrating leukocytes. A considerable reduction in the cytokine and chemokine production, mostly TNFα, IL-1β and IL-6 production in the MPM-treated group, suggested an inhibition of the mediators responsible for leukocyte extravasation. On the other hand, MPM consumption had no effect on neutrophil phagocytosis activity. Conclusion:  MPM administration demonstrates a significant reduction of neutrophil infiltration associated with an inhibition of cytokine and chemokine production. The air pouch model shares similarities with in vivo characteristics of rheumatoid arthritis and neutrophilic diseases, both of which would benefit from this 50% inhibition of neutrophil infiltration induced by MPM. Received 14 May 2007; returned for revision 7 October 2007; received from final revision 25 April 2008; accepted by S. Stimpson 27 June 2008