Peroxide Responsive Regulator PerR of group A Streptococcus Is Required for the Expression of Phage-Associated DNase Sda1 under Oxidative Stress

The peroxide regulator (PerR) is a ferric uptake repressor-like protein, which is involved in adaptation to oxidative stress and iron homeostasis in group A streptococcus. A perR mutant is attenuated in surviving in human blood, colonization of the pharynx, and resistance to phagocytic clearance, indicating that the PerR regulon affects both host environment adaptation and immune escape. Sda1 is a phage-associated DNase which promotes M1T1 group A streptococcus escaping from phagocytic cells by degrading DNA-based neutrophil extracellular traps. In the present study, we found that the expression of sda1 is up-regulated under oxidative conditions in the wild-type strain but not in the perR mutant. A gel mobility shift assay showed that the recombinant PerR protein binds the sda1 promoter. In addition, mutation of the conserved histidine residue in the metal binding site of PerR abolished sda1 expression under hydrogen peroxide treatment conditions, suggesting that PerR is directly responsible for the sda1 expression under oxidative stress. Our results reveal PerR-dependent sda1 expression under oxidative stress, which may aid innate immune escape of group A streptococcus.     

Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes

Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.     

Pathogenic mechanisms of invasive group A Streptococcus infections by influenza virus–group A Streptococcus superinfection

Group A Streptococcus (GAS) are pathogenic bacteria of the genus Streptococcus and cause severe invasive infections that comprise a wide range of diverse diseases, including acute respiratory distress syndrome, renal failure, toxic shock‐like syndrome, sepsis, cellulitis and necrotizing fasciitis. The essential virulence, infected host and external environmental factors required for invasive GAS infections have not yet been determined. Superinfection with influenza virus and GAS induced invasive GAS infections was demonstrated by our team in a mouse model, after which clinical cases of invasive GAS infections secondary to influenza virus infection were reported by other investigators in Japan, USA, Canada, UK China, and other countries. However, the pathogenic mechanisms underlying influenza virus‐GAS superinfection are not yet fully understood. The present review describes the current knowledge about invasive GAS infections by superinfection. Topics addressed include the bacteriological, virological and immunological mechanisms impacting invasion upon superinfection on top of underlying influenza virus infection by GAS and other bacteria (i.e., Streptococcus pneumoniae and Staphylococcus aureus). Future prospects are also discussed.

     

Stabilization and polysaccharide storage in group A Streptococcus pyogenes

Water-shock treatment of group A Streptococcus pyogenes released a mixture of nucleotide-like substances and small amounts of protein. The amount of protein was much less than found with osmotic shock of Gram-negative bacteria. In group A S. pyogenes the osmolytes released exhibited as much as a 6-fold change in respect to different growth phases. Osmolyte release was dependent on the stabilization agent used and independent of cellular metabolic activity. The released osmolytes were found to be required for optimal intracellular iodophilic polysaccharide (IPS) storage. Stabilization of washing solutions, and IPS storage medium with metabolically inert non-ionic organic compounds prevented osmolyte loss and enhanced IPS storage. Polyvinyl pyrrolidone and polyethyleneglycol (MW greater than 1000) exhibited the same protective effects as found with calf serum. Smaller non-ionic organic compounds provided similar protective action but the bacteria were more susceptible to osmotic stress.     

Immunoglobulin receptors of group A Streptococcus]

It was shown in the gel precipitation tests that absorption of human and rabbit IgG or Fc-fragments obtained from human IgG group A streptococcal cultures results in inhibition of the reactions of these preparations with immunoglobulin sera. The reactions of F(ab')2-fragments with the corresponding sera are not inhibited during their absorption by the same cultures. The results obtained support the presence in a number of group A streptococcal cultures of immunoglobulin receptors (Ig-receptors) capable of reacting with Fc-parts of human and rabbit IgG. Pepsin treatment destroys Ig-receptors. These receptors could not be found by the method used in hydrochloric acid extracts prepared from streptococci containing the receptors. The method can be applied for determination of Ig-receptors in streptococcal cultures.     

The role of non-type-specific protein antigens of the cell wall in Streptococcus group A in developing delayed hypersensitivity

The role of the type-nonspecific (TNS) cell-wall antigens of group A streptococci has been determined. The study has been made on guinea pigs sensitized with whole microbial cells or HCl extracts containing TNS antigens. To determine delayed hypersensitivity, the in vitro cytotoxic test on adhering lymph-node cells in the autologous system has been used. The study has shown that sensitization with group A streptococci of different types or with TNS antigens induces the development of delayed hypersensitivity to TNS antigens (or antigen), common for different types of group A streptococci, but specific for this group. HCl extracts containing TNS antigens can be recommended as the preparation for testing delayed hypersensitivity to antigens, specific for group A streptococci.     

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection.     

A locus of group A Streptococcus involved in invasive disease and DNA transfer

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengths-transposon-mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of approximately 10-8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.     

Phage sensitivity of Streptococcus group A strains isolated in glomerulonephritis

The study of phage-sensitive strains of group A streptococci of different serological types, isolated from glomerulonephritis patients in Czechoslovakia, has confirmed the data previously obtained in Moscow. As revealed in this study, all strains of type 12, containing M-antigen, are sensitive to group 1 phage from the phage collection used in the investigation. Some of the strains of type 12, containing M-antigen and isolated from healthy carriers, have also proved to be sensitive to group 1 phages. Thus, streptococcal strains of type 12, containing M-substance and found to be the main causative agents of glomerulonephritis, have shown similar phage sensitivity when isolated in different geographical zones. For this reason, group 1 phages can be used for the detection of some "nephritogenic" strains among the population.     

Isolation and characteristics of surface proteins from Streptococcus group A

Cell wall surface proteins of group A Streptococcus type 29 were extracted with 1 M hydroxylamine pH 6.0. The purification procedure included fractionation with ammonium sulfate and gel filtration on Sephadex G-150. SDS polyacrylamide gel electrophoresis revealed a number of proteins (approximately 20) with molecular mass of 70 kD; the difference in Mr between the proteins was 5-10 kD. Isoelectrofocusing demonstrated that the proteins are either acid (pI = 3.7) or weakly alkaline (pI = 7.7). Possible reasons for the heterogeneity of Streptococcus cell wall surface proteins are discussed.     

Glycoprotein inhibitors and iodophilic polysaccharide storage in group A Streptococcus pyogenes

Inhibitors of glycoprotein synthesis in eucaryotic cells also inhibited iodophilic polysaccharide (IPS) storage in group A streptococcus pyogenes. Addition of bacitracin or amphomycin, known inhibitors of polyisoprenol phosphate metabolism or lipid-linked oligosaccharide synthesis, indicated that a key intermediate must be synthesized before IPS storage could be detected. Based on inhibitor action and energy requirements the intermediate was most likely an undecaprenol pyrophosphoryl maltosaccharide. Biosynthesis of the maltosaccharide had an ATP requirement as shown by arsenate action but IPS synthesis via amylomaltase was energy independent if the lipid-linked saccharides were preformed. Maltosaccharide acceptor site blockade with 2-deoxy-d-glucose immediately inhibited IPS storage, which demonstrated the need of acceptor glucose residues for transglycosylation activity of amylomaltase. Tunicamycin failed to inhibit IPS synthesis although it was added in lethal concentrations.     

A role for Streptococcus pneumoniae in virus-associated pneumonia

Here we show, in a double-blind, randomized, placebo-controlled trial in 37,107 fully immunized infants in Soweto, South Africa, that a 9-valent pneumococcal conjugate vaccine, PncCV, prevents 31% (95% confidence interval = 15-43%) of pneumonias associated with any of seven respiratory viruses in children in hospital. These data suggest that the pneumococcus has a major role in the development of pneumonia associated with these viruses and that viruses contribute to the pathogenesis of bacterial pneumonia.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.