Isogenic Mutant Strains Research Articles

Molecular characterization of Streptococcus mutans strains containing the cnm gene encoding a collagen-binding adhesin

Objective Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm. Design A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed. Results The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A. Conclusions Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.

Helicobacter pylori induces NF‐κB independent of CagA

We refer to the publication by Lamb et al (2009). This report describes the Helicobacter pylori virulence factor cytotoxin‐associated gene A (CagA), encoded in the pathogenicity island (PAI), as essential for the rapid activation of the transcription factor nuclear factor κB (NF‐κB) in epithelial cells. The authors reported fast NF‐κB activation in AGS (human gastric carcinoma) cells infected with H. pylori G27 wild type, but not with the isogenic CagA‐deficient strain, determined by IκBα phosphorylation and degradation, RelA phosphorylation, NF‐κB DNA‐binding, and interleukin 8 (IL‐8) mRNA expression. However, the profound CagA dependency of NF‐κB activation, representing the centrepiece of the study by Lamb et al (2009), is in clear contradiction to previously …

Response: strain‐specific activation of NF‐κB by Helicobacter pylori CagA

We appreciate the comments from Schweitzer et al in this issue about our discovery that Helicobacter pylori cytotoxin‐associated gene A (CagA) activates nuclear factor κB (NF‐κB), as they reveal the lack of consensus on the role that CagA plays during H. pylori infection. We are confident in our data for several reasons. First, we demonstrated in our studies that three independent H. pylori cagA ‐positive strains, but not their respective isogenic cagA mutants, activate NF‐κB. Second, because cagA is monocistronic and transcribed in a different direction than other genes comprising the cag pathogenicity island ( cag PAI; Censini et al , 1996), it is unlikely that our H. pylori isogenic cagA mutant strains are exerting “polar effects” on other genes within the cag PAI. Third, these …

Streptococcus mutans autolysin AtlA is a fibronectin‐binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis

Streptococcus mutans, a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104-90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.

Surface Protein EF3314 Contributes to Virulence Properties of Enterococcus faecalis

Identification of putative new virulence factors as additional targets for therapeutic approaches alternative to antibiotic treatment of multi-resistant enterococcal infections. The EF3314 gene, coding for a putative surface-exposed antigen, was identified by the analysis of the Enterococcus faecalis V583 genome for LPXTG-motif cell wall anchor surface protein genes. A non-polar EF3314 gene deletion mutant in the E. faecalis 12030 human clinical isolate was obtained. The wild type and the isogenic mutant strain were investigated for biofilm formation, adherence to Hela cells, survival in human macrophages and a Caenorhabditis elegans infection model. The aminoterminal portion of the EF3314 protein was overexpressed in E. coli to obtain mouse polyclonal antibodies for use in Western blotting and immunolocalization experiments. The EF3314 gene has an unusually high GC content (46.88% vs. an average of 37.5% in the E. faecalis chromosome) and encodes a protein of 1744 amino acids that presents a series of 14 imperfect repeats of 90 amino acids covering almost the entire length of the protein. Its global organization is similar to the alpha-like protein family of group B streptococci, enterococcal surface protein Esp and biofilm associated protein Bap from S. aureus. The EF3314 gene was always present and specific for E. faecalis strains of human, food and animal origin. Differences in size depended on variable numbers of repeats in the repetitive region. EF3314 is a newly described, surface exposed protein that contributes to the virulence properties of E. faecalis.

T Cell Memory Response to Pneumococcal Protein Antigens in an Area of High Pneumococcal Carriage and Disease

Streptococcus pneumoniae is a leading cause of vaccine-preventable disease worldwide. Pneumococcal protein antigens are currently under study as components of potential vaccines that offer protection against multiple serotypes. We have therefore characterized T cell pneumococcal immunity acquired through asymptomatic carriage. Peripheral blood mononuclear cells from 40 healthy Gambian adults were stimulated with supernatants derived from S. pneumoniae strain (D39), 2 isogenic mutant strains lacking either pneumolysin or choline binding protein A, and recombinant pneumolysin. Immune responses were measured by cellular proliferation and by interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot and bioplex cytokine assays. Nasopharyngeal swabs were cultured to determine carriage rates. S. pneumoniae nasopharyngeal carriage was detected in 60% of individuals. Both effector and resting (or central) CD4(+) T cell memory were frequently present to a range of pneumococcal antigens. However, the level of the effector memory response did not relate to current nasopharyngeal carriage. Pneumolysin was not immunodominant in these T cell responses but induced a distinct proinflammatory profile (high IFN-gamma, IL-12[p40], and L-17 levels and low IL-10 and IL-13 levels). In this population, T cell-mediated immunological memory potentially capable of pathogen clearance and immune surveillance is common but is not associated with the absolute interruption of pneumococcal carriage. How this naturally acquired immune memory influences pneumococcal vaccine efficacy remains to be determined.

Contribution of cell surface protein antigen c of Streptococcus mutans to platelet aggregation

Streptococcus mutans is considered to be one of the pathogens that cause infective endocarditis. The purpose of the present study was to examine the properties of S. mutans with regard to platelet aggregation by focusing on its high molecular protein antigen c (PAc). The platelet aggregation properties of six clinical strains and one isogenic mutant strain of S. mutans were analysed using an aggregometer and confocal microscopy, as well as with an inhibition assay of platelet aggregation using anti-PAc serum. S. mutans strains with PAc expression induced platelet aggregation, while a PAc-deficient mutant and two clinical isolates with no PAc expression did not. When platelets were pretreated with higher amounts of anti-PAc serum, the platelet aggregation rate was reduced in a dose-dependent manner, indicating that PAc binds directly to platelets. S. mutans PAc is involved in human platelet aggregation and may be one of the virulence factors in the pathogenesis of infective endocarditis.

Quorum sensing is necessary for the virulence of Pseudomonas aeruginosa during urinary tract infection

Quorum sensing is a signaling pathway used by bacteria to monitor their population density by detecting small diffusible molecules. To understand the role of quorum sensing in pathogenesis of urinary tract infections, wild type Pseudomonas aeruginosa, having both functional las and rhl quorum sensing systems, and its isogenic single and double mutants were used in a mouse model of ascending urinary tract infection. Mice were evaluated on the basis of bacteriological and histopathological examinations, detection of acyl homoserine lactone, and the pathologic index factors of renal and bladder tissue. Single as well as double mutant strains were able to reach renal tissue, but they were not able to multiply and cause tissue damage. Reduced rates of infection, mild histopathologic lesions, and decreased production of pathologic index factors like myeloperoxidase, malondialdehyde, and reactive nitrogen intermediates were found in these groups of mice as compared to mice infected with a standard strain (PAO1) possessing both functional sensing systems. Loss of any one of the quorum-sensing components significantly reduced the in vivo virulence of P. aeruginosa in this mouse model of ascending urinary tract infection. Our results suggests that quorum-sensing signals may act as virulence factors and are essential for P. aeruginosa multiplication and virulence during the course of urinary tract infection.

The Escherichia coli Common Pilus and the Bundle-Forming Pilus Act in Concert during the Formation of Localized Adherence by Enteropathogenic E. coli

Although the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described E. coli common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent E. coli HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the ecpA gene was also deleted. Furthermore, a DeltaespA DeltaecpA double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the DeltaespA and DeltaecpA single mutants, even in the presence of BFP. A Delta bfp DeltaespA DeltaecpA triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.

Cell Surface O-Glycans LimitStaphylococcus aureusAdherence to Corneal Epithelial Cells

The mucin-rich environment of the intact corneal epithelium is thought to contribute to the prevention of Staphylococcus aureus infection. This study examined whether O-glycans, which constitute the majority of the mucin mass of epithelial cell glycocalyces, prevented bacterial adhesion and growth. Abrogation of mucin O glycosylation using the chemical primer benzyl-alpha-GalNAc resulted in increased adherence of parental strain RN6390 to apical human corneal-limbal epithelial (HCLE) cells and to biotinylated cell surface protein in static and liquid phase adhesion assays, consistent with a role of mucin O-glycans in preventing bacterial adhesion. Comparable results were found with ALC135, an isogenic mutant strain defective in the accessory gene regulators agr and sar, indicating that the agr- and/or sar-regulated virulence factors did not play a major role in mediating adhesion to the corneal cell surface after mucin O-glycan truncation. In exoglycosidase digestion studies, treatment with sialidase from Arthrobacter ureafaciens--which hydrolyzed mucin-associated O-acetyl sialic acid--but not from Clostridium perfringens resulted in an increase in RN6390 and ALC135 adhesion. Abrogation of mucin O glycosylation in HCLE cell cultures did not affect bacterial growth. Overall, these data indicate that mucin O-glycans contribute to the prevention of bacterial adherence to the apical surface of corneal epithelial cells and suggest that alteration of cell surface glycosylation from disease or trauma, including that stemming from contact lens wear, could contribute to a higher risk of infection.

Regulation of the Helicobacter pylori Cellular Receptor Decay-accelerating Factor

Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulceration and distal gastric cancer, and adherence of H. pylori to gastric epithelial cells is critical for induction of inflammation. One H. pylori constituent that increases disease risk is the cag pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Decay-accelerating factor (DAF) is a cellular receptor for H. pylori and a mediator of the inflammatory response to this pathogen. H. pylori induces DAF expression in human gastric epithelial cells; therefore, we sought to define the mechanism by which H. pylori up-regulates DAF and to extend these findings into a murine model of H. pylori-induced injury. Co-culture of MKN28 gastric epithelial cells with the wild-type H. pylori cag(+) strain J166 induced transcriptional expression of DAF, which was attenuated by disruption of a structural component of the cag secretion system (cagE). H. pylori-induced expression of DAF was dependent upon activation of the p38 mitogen-activated protein kinase pathway but not NF-kappaB. Hypergastrinemic INS-GAS mice infected with wild-type H. pylori demonstrated significantly increased DAF expression in gastric epithelium versus uninfected controls or mice infected with an H. pylori cagE(-) isogenic mutant strain. These results indicate that H. pylori cag(+) strains induce up-regulation of a cognate cellular receptor in vitro and in vivo in a cag-dependent manner, representing the first evidence of regulation of an H. pylori host receptor by the cag pathogenicity island.

Molecular characterization of group A Streptococcus maltodextrin catabolism and its role in pharyngitis

We previously demonstrated that the cell-surface lipoprotein MalE contributes to GAS maltose/maltodextrin utilization, but MalE inactivation does not completely abrogate GAS catabolism of maltose or maltotriose. Using a genome-wide approach, we identified the GAS phosphotransferase system (PTS) responsible for non-MalE maltose/maltotriose transport. This PTS is encoded by an open reading frame (M5005_spy1692) previously annotated as ptsG based on homology with the glucose PTS in Bacillus subtilis. Genetic inactivation of M5005_spy1692 significantly reduced transport rates of radiolabelled maltose and maltotriose, but not glucose, leading us to propose its reannotation as malT for maltose transporter. The DeltamalT, DeltamalE and DeltamalE:malT strains were significantly attenuated in their growth in human saliva and in their ability to catabolize alpha-glucans digested by purified human salivary alpha-amylase. Compared with wild-type, the three isogenic mutant strains were significantly impaired in their ability to colonize the mouse oropharynx. Finally, we discovered that the transcript levels of maltodextrin utilization genes are regulated by competitive binding of the maltose repressor MalR and catabolite control protein A. These data provide novel insights into regulation of the GAS maltodextrin genes and their role in GAS host-pathogen interaction, thereby increasing the understanding of links between nutrient acquisition and virulence in common human pathogens.

Both leukotoxin and poly- N-acetylglucosamine surface polysaccharide protect Aggregatibacter actinomycetemcomitans cells from macrophage killing

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly- N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately twofold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans.

Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M. tuberculosis isolates to identify the role of embB306 mutations in any type of drug resistance. Replacing wild-type embB306 ATG from EMB-susceptible clinical M. tuberculosis strain 210 with embB306 ATA, ATC, CTG, or GTG increased the EMB MIC from 2 microg/ml to 7, 7, 8.5, and 14 microg/ml, respectively. Replacing embB306 ATC or GTG from two high-level EMB-resistant clinical strains with wild-type ATG lowered EMB MICs from 20 microg/ml or 28 microg/ml, respectively, to 3 microg/ml. All parental and isogenic mutant strains had identical isoniazid (INH) and rifampin (RIF) MICs. However, embB306 CTG mutants had growth advantages compared to strain 210 at sub-MICs of INH or RIF in monocultures and at sub-MICs of INH in competition assays. CTG mutants were also more resistant to the additive or synergistic activities of INH, RIF, or EMB used in different combinations. These results demonstrate that embB306 mutations cause an increase in the EMB MIC, a variable degree of EMB resistance, and are necessary but not sufficient for high-level EMB resistance. The unusual growth property of embB306 mutants in other antibiotics suggests that they may be amplified during treatment in humans and that a single mutation may affect antibiotic susceptibility against multiple first-line antibiotics.

Group A Streptococcal Cysteine Protease Degrades C3 (C3b) and Contributes to Evasion of Innate Immunity

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic DeltaspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the DeltaspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the DeltaspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.

A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus

Although central to pathogenesis, the molecular mechanisms used by microbes to regulate virulence factor production in specific environments during host-pathogen interaction are poorly defined. Several recent ex vivo and in vivo studies have found that the level of group A Streptococcus (GAS) virulence factor gene transcripts is temporally related to altered expression of genes encoding carbohydrate utilization proteins. These findings stimulated us to analyze the role in pathogenesis of catabolite control protein A (CcpA), a GAS ortholog of a key global regulator of carbohydrate metabolism in Bacillus subtilis. Inasmuch as the genomewide effects of CcpA in a human pathogen are unknown, we analyzed the transcriptome of a DeltaccpA isogenic mutant strain grown in nutrient-rich medium. CcpA influences the transcript levels of many carbohydrate utilization genes and several well characterized GAS virulence factors, including the potent cytolysin streptolysin S. Compared with the wild-type parental strain, the DeltaccpA isogenic mutant strain was significantly less virulent in a mouse model of invasive infection. Moreover, the isogenic mutant strain was significantly impaired in ability to colonize the mouse oropharynx. When grown in human saliva, a nutrient-limited environment, CcpA influenced production of several key virulence factors not influenced during growth in nutrient-rich medium. Purified recombinant CcpA bound to the promoter region of the gene encoding streptolysin S. Our discovery that GAS virulence and complex carbohydrate utilization are directly linked through CcpA provides enhanced understanding of a mechanism used by a Gram-positive pathogen to modulate virulence factor production in specific environments.

Efficient point mutagenesis in mycobacteria using single‐stranded DNA recombineering: characterization of antimycobacterial drug targets

Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10,000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage lambda Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.

Biosynthesis and IroC‐dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate-type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography-mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe-enterobactin or Fe-salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.

Degradation of Complement 3 by Streptococcal Pyrogenic Exotoxin B Inhibits Complement Activation and Neutrophil Opsonophagocytosis

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcus (GAS) infection. The inhibition of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we examined the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using an enzyme-linked immunosorbent assay, we found that SPE B-treated serum impaired the activation of the classical, the lectin, and the alternative complement pathways. In contrast, C192S, a SPE B mutant lacking protease activity, had no effect on complement activation. Further study showed that cleavage of serum C3 by SPE B, but not C192S, blocked zymosan-induced production of reactive oxygen species in neutrophils as a result of decreased deposition of C3 fragments on the zymosan surface. Reconstitution of C3 into SPE B-treated serum unblocked zymosan-mediated neutrophil activation dose dependently. SPE B-treated, but not C192S-treated, serum also impaired opsonization of C3 fragments on the surface of GAS strain A20. Moreover, the amount of C3 fragments on the A20 cell surface, a SPE B-producing strain, was less than that on its isogenic mutant strain, SW507, after opsonization with normal serum. A20 opsonized with SPE B-treated serum was more resistant to neutrophil killing than A20 opsonized with normal serum, and SPE B-mediated resistance was C3 dependent. These results suggest a novel SPE B mechanism, one which degrades serum C3 and enables GAS to resist complement damage and opsonophagocytosis.

A Chemokine-Degrading Extracellular Protease Made by Group AStreptococcusAlters 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 (GROalpha), two potent chemokines made abundantly in human tonsils. Cleavage of GCP-2 and GROalpha 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.