Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3).

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.

Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr.

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.

Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes.

Bacteria encounter oxidative stress by exposure to reactive oxygen species (ROS) present in the aerobic environment and during immune responses. In Streptococcus pyogenes, Dpr has been identified as a stress protein conferring resistance to hydrogen peroxide and multiple other stresses. The expression of Dpr is under perR (peroxide stress response regulator) control. However, the exact molecular mechanism of PerR regulation of Dpr is not clear. In this study, a perR deletion mutant was constructed by double cross-over mutagenesis. The profile of Dpr expression, performed by Western blot assay, revealed growth-phase dependency under normal culture conditions. Dpr expression decreased under iron-restricted conditions, whereas iron, zinc, nickel, and hydrogen peroxide induced its expression. The perR mutant does not induce Dpr as well when exposed to environmental signals. PerR binds the promoter region of dpr. Increased iron and hydrogen peroxide concentrations decreased PerR binding to the promoter region of dpr, suggesting that regulation of Dpr by environmental signals is mediated by PerR directly.

Crystal structures of Streptococcus pyogenes Dpr reveal a dodecameric iron-binding protein with a ferroxidase site.

DNA-binding protein from starved cells (Dps)-like proteins are key factors involved in oxidative stress protection in bacteria. They bind and oxidize iron, thus preventing the formation of harmful reactive oxygen species that can damage biomolecules, particularly DNA. Dps-like proteins are composed of 12 identical subunits assembled in a spherical structure with a hollow central cavity. The iron oxidation occurs at 12 intersubunit sites located at dimer interfaces. Streptococcus pyogenes Dps-like peroxide resistance protein (Dpr) has been previously found to protect the catalase-lacking S. pyogenes bacterium from oxidative stress. We have determined the crystal structure of S. pyogenes Dpr, the second Dpr structure from a streptococcal bacterium, in iron-free and iron-bound forms at 2.0- and 1.93-A resolution, respectively. The iron binds to well-conserved sites at dimer interfaces and is coordinated directly to Asp77 and Glu81 from one monomer, His50 from a twofold symmetry-related monomer, a glycerol molecule, and a water molecule. Upon iron binding, Asp77 and Glu81 change conformation. Site-directed mutagenesis of active-site residues His50, His62, Asp66, Asp77, and Glu81 to Ala revealed a dramatic decrease in iron incorporation. A short helix at the N-terminal was found in a different position compared with other Dps-like proteins. Two types of pores were identified in the dodecamer. Although the N-terminal pore was found to be similar to that of other Dps-like proteins, the C-terminal pore was found to be blocked by bulky Tyr residues instead of small residues present in other Dps-like proteins.

Rapid identification of Babesia canis and Babesia gibsoni (Asian genotype) in canine blood samples using a customized portable real-time PCR analyzer and TaqMan-based assay.

Canine babesiosis is a serious infectious disease in subtropical and tropical regions. Typically, clinical detection of canine babesiosis is performed by blood smear observation or the traditional polymerase chain reaction (PCR). Herein, we developed a new TaqMan-based real-time PCR assay combined with a customized portable real-time PCR platform for a rapid and accurate detection of canine babesiosis. Two new primer/probe pairs (B18S and BITS1) were designed based on 18S ribosomal RNA and an internal transcribed spacer 1 (ITS1) sequence to differentiate Babesia canis and B. gibsoni (Asian genotype) DNAs from canine blood samples. Additionally, a corresponding customized compact real-time PCR platform with low 6-carboxyfluorescein fluorescence detection (≤5 nM), including a fast and accurate thermal cycling ability with a user-friendly interface for thermal control and data analysis, was designed for the limited space use. Both assays (B18S and BITS1) demonstrated a sensitivity of 100 copies/reaction based on the 95 % confidence interval evaluation method. The self-developed customized portable real-time PCR analyzer presented high repeatability and reproducibility with the TaqMan-based assay. Moreover, 501 clinical specimens were collected for evaluating the performance of the proposed PCR. The positive and negative predictive values were 90 % (18 of 20) and 100 % (226 of 226), respectively, for samples suspected with B. canis infection and 98 % (55 of 56) and 100 % (199 of 199), respectively, for samples suspected with B. gibsoni infection.

An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses.

Streptococcus pyogenes does not produce catalase, but it can grow in aerobic environments and survive in the presence of peroxide. One of the stress proteins of this organism, peroxide resistance protein (Dpr), has been studied to examine its role in resistance to hydrogen peroxide, but the protective mechanism of Dpr is not clear. The aim of this study was to characterize the dpr gene and its role in dealing with different stresses. A dpr deletion mutant was constructed by double-crossover mutagenesis. The dpr mutant was more sensitive to H(2)O(2), and complementation could partially restore the defect in the mutant. Pretreatment with the iron chelator deferoxamine mesylate rescued the survival activity of the mutant under oxidative stress conditions. The dpr mutant also showed a low survival rate in the long-term stationary phase, when it was treated with extreme acids, and under alkaline pH conditions compared to the wild-type strain. The growth of the dpr mutant was slower than that of the wild-type strain in iron-limiting conditions. The dpr mutant showed high sensitivity to iron and zinc but not to manganese, copper, nickel, and calcium. Recombinant Dpr protein was purified and showed iron-binding activity, whereas no DNA-binding activity was found. These data indicate that an iron-binding protein, Dpr, provides protection from hydrogen peroxide stress by preventing the Fenton reaction, and Dpr was identified as a novel stress protein that protects against several stresses in group A streptococci.

The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes.

CovR/S is an important two component regulatory system, which regulates about 15% of the gene expression in Streptococcus pyogenes. The covR/S locus was identified as an operon generating an RNA transcript around 2.5-kb in size. In this study, we found the covR/S operon produced three RNA transcripts (around 2.5-, 1.0-, and 0.8-kb in size). Using RNA transcriptional terminator sequence prediction and transcriptional terminator analysis, we identified two atypical rho-independent terminator sequences downstream of the covR gene and showed these terminator sequences terminate RNA transcription efficiently. These results indicate that covR/S operon generates covR/S transcript and monocistronic covR transcripts.

Streptococcus pyogenes induces epithelial inflammatory responses through NF-kappaB/MAPK signaling pathways.

Innate immunity involves a cascade of inflammatory events, resulting in the secretion of chemokines and cytokines to recruit mediator cells in adaptive immunity. To study epithelial inflammatory responses initiated by Streptococcus pyogenes infection, we investigated chemotaxis ability in the supernatant of infected human respiratory epithelial HEp-2 cells. Our results showed that these supernatants showed significantly increased ability to attract monocytes, implying the release of inflammatory chemoattractants into the medium. Expression of interleukin (IL)-8 and IL-6 in HEp-2 cells was significantly increased at both the mRNA and protein levels after infection with S. pyogenes. Electrophoretic mobility shift and reporter-gene assays demonstrated that the transcription factors NF-kappaB and AP-1, regulated by mitogen-activated protein (MAP) kinase, were activated after streptococcal infection. The increases in mRNAs for IL-8 and IL-6 were abrogated by addition of NF-kappaB and MAP kinase inhibitors, suggesting that the upregulation of IL-8 and IL-6 is mediated through NF-kappaB and MAP kinase signaling pathways. Taken together, our results indicate that S. pyogenes infection of epithelial cells induces the secretion of pro-inflammatory chemokines/cytokines through activation of NF-kappaB and MAP kinase signaling pathways. These early innate responses initiated by S. pyogenes-infected respiratory epithelial cells may recruit immune cells to the airway and induce inflammation.

Distinct structural features of the peroxide response regulator from group A Streptococcus drive DNA binding.

Group A streptococcus (GAS, Streptococcus pyogenes) is a strict human pathogen that causes severe, invasive diseases. GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species (ROS) through novel mechanisms. The peroxide response regulator (PerR), a member of ferric uptake regulator (Fur) family, plays a key role for GAS to cope with oxidative stress by regulating the expression of multiple genes. Our previous studies have found that expression of an iron-binding protein, Dpr, is under the direct control of PerR. To elucidate the molecular interactions of PerR with its cognate promoter, we have carried out structural studies on PerR and PerR-DNA complex. By combining crystallography and small-angle X-ray scattering (SAXS), we confirmed that the determined PerR crystal structure reflects its conformation in solution. Through mutagenesis and biochemical analysis, we have identified DNA-binding residues suggesting that PerR binds to the dpr promoter at the per box through a winged-helix motif. Furthermore, we have performed SAXS analysis and resolved the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Overall, we provide structural insights into molecular recognition of DNA by PerR and define the hollow structural arrangement of PerR-30bpDNA complex, which displays a unique topology distinct from currently proposed DNA-binding models for Fur family regulators.

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.