Angicin, a novel bacteriocin of Streptococcus anginosus.

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which provide a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from - 70 to 90 °C and pH values from 2 to 10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings.

Bicarbonate and unsaturated fatty acids enhance capsular polysaccharide synthesis gene expression in oral streptococci, Streptococcus anginosus.

We recently reported on the capsular polysaccharide (CP) synthesis (cps) genes of the oral streptococci, Streptococcus anginosus. In this study, we investigate the effects of carbon dioxide (CO2), bicarbonate (HCO3-) and unsaturated fatty acids (UFAs) on CP synthesis of S. anginosus. We found that CP production increased when bacteria were exposed to high concentrations of CO2. This increase was similarly observed in the presence of sodium bicarbonate (NaHCO3) under atmospheric condition. Since ectopic expression of carbonic anhydrase, which converts CO2 to HCO3-, eliminated the requirement for CO2 in CP production and growth of S. anginosus lacking this enzyme, it seemed that HCO3- is an essential factor for CP production. Furthermore, UFAs also stimulated the CP production. Promoter-reporter assay and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that stimulation of CP production occurs at the transcription level. The results of the promoter assays suggest that the expression and stimulation of cps genes by HCO3- or UFAs require the cpsA gene, which is located in the first position of the cps operon. With respect to the relationship between HCO3-and UFAs, HCO3- may stimulate UFA synthesis pathway at transcription level. Therefore, it is possible that UFAs are definitive signals for the CP production in S. anginosus.

The fibronectin-binding protein homologue Fbp62 of Streptococcus anginosus is a potent virulence factor.

Streptococcus anginosus appears to be able to adhere to cultured epithelial cells or fibronectin and this may be associated with bacterial pathogenicity. In the present study, the molecular characteristics and virulence of the fibronectin-binding protein (FBP), Fbp62, of S. anginosus were investigated in animal models to determine the role of the molecule in bacterial infection. fbp62 encodes a 549 amino acid residue with an apparent molecular mass of 62.8 kDa that lacks a membrane anchor motif and a leader peptide, suggesting that fbp62 codes for an atypical FBP. It has been observed that the S. anginosus Fbp62 is very similar to the FbpA of Streptococcus gordonii, PavA of Streptococcus pneumoniae, SmFnB of Streptococcus mutans and Fbp54 of Streptococcus pyogenes. Recombinant Fbp62 prepared from pGEX-4T-2 was found to bind to fibronectin in a dose-dependent manner and competitively inhibit the binding of S. anginosus to fibronectin. Furthermore, anti-Fbp62 antiserum abrogated the binding of S. anginosus to fibronectin. Adhesion of the isogenic mutant, Δfbp62, constructed from S. anginosus NCTC 10713 (wild-type, WT) by homologous recombination to HEp-2 cells and DOK cells was significantly weaker than that of S. anginosus WT. In addition, Δfbp62's lethality and ability to form abscesses were weaker in a mouse model of infection than in the WT strain. Taken together, these results suggest that Fbp62 is an important pathogenic factor of S. anginosus.

Regulation of the ß-hemolysin gene cluster of Streptococcus anginosus by CcpA.

Streptococcus anginosus is increasingly recognized as an opportunistic pathogen. However, our knowledge about virulence determinants in this species is scarce. One exception is the streptolysin-S (SLS) homologue responsible for the ß-hemolytic phenotype of the S. anginosus type strain. In S. anginosus the expression of the hemolysin is reduced in the presence of high glucose concentrations. To investigate the genetic mechanism of the hemolysin repression we created an isogenic ccpA deletion strain. In contrast to the wild type strain, this mutant exhibits hemolytic activity in presence of up to 25 mM glucose supplementation, a phenotype that could be reverted by ccpA complementation. To further demonstrate that CcpA directly regulates the hemolysin expression, we performed an in silico analysis of the promoter of the SLS gene cluster and we verified the binding of CcpA to the promoter by electrophoretic mobility shift assays. This allowed us to define the CcpA binding site in the SLS promoter region of S. anginosus. In conclusion, we report for the first time the characterization of a potential virulence regulator in S. anginosus.

AbiEi Binds Cooperatively to the Type IV abiE Toxin-Antitoxin Operator Via a Positively-Charged Surface and Causes DNA Bending and Negative Autoregulation.

Bacteria resist phage infection using multiple strategies, including CRISPR-Cas and abortive infection (Abi) systems. Abi systems provide population-level protection from phage predation, via "altruistic" cell suicide. It has recently been shown that some Abi systems function via a toxin-antitoxin mechanism, such as the widespread AbiE family. The Streptococcus agalactiae AbiE system consists of a bicistronic operon encoding the AbiEi antitoxin and AbiEii toxin, which function as a Type IV toxin-antitoxin system. Here we examine the AbiEi antitoxin, which belongs to a large family of transcriptional regulators with a conserved N-terminal winged helix-turn-helix domain. This winged helix-turn-helix is essential for transcriptional repression of the abiE operon. The function of the AbiEi C-terminal domain is poorly characterized, but it contributes to transcriptional repression and is sufficient for toxin neutralization. We demonstrate that a conserved charged surface on one face of the C-terminal domain assists sequence-specific DNA binding and negative autoregulation, without influencing antitoxicity. Furthermore, AbiEi binds cooperatively to two inverted repeats within the abiE promoter and bends the DNA by 72°. These findings demonstrate that the mechanism of DNA binding by the widespread family of AbiEi antitoxins and transcriptional regulators can contribute to negative autoregulation.

Streptolysin S of Streptococcus anginosus exhibits broad-range hemolytic activity.

Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent ß-hemolytic phenotype. A gene cluster (sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for ß-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions.

Antibiotic susceptibilities, streptococcal pyrogenic exotoxin gene profiles among clinical isolates of group C or G Streptococcus dysgalactiae subsp. equisimilis & of group G S. anginosus group at a tertiary care centre.

BACKGROUND & OBJECTIVES: Group C and group G streptococci (together GCGS) are often regarded as commensal bacteria and their role in streptococcal disease burden is under-recognized. While reports of recovery of GCGS from normally sterile body sites are increasing, their resistance to macrolides, fluoroquinolone further warrants all invasive ß haemolytic streptococci to be identified to the species level and accurately tested for antimicrobial susceptibility. This study was aimed to determine the prevalence, clinical profile, antimicrobial susceptibility and streptococcal pyrogenic exotoxin gene profile (speA, speB, speC, speF, smeZ, speI, speM, speG, speH and ssa) of GCGS obtained over a period of two years at a tertiary care centre from north India. METHODS: The clinical samples were processed as per standard microbiological techniques. ß-haemolytic streptococci (BHS) were characterized and grouped. Antimicrobial susceptibility of GCGS was performed using disk diffusion method. All GCGS were characterized for the presence of streptococcal pyrogenic exotoxins (spe) and spe genes were amplified by PCR method. RESULTS: GCGS (23 GGS, 2GCS) comprised 16 per cent of ß haemolytic streptococci (25/142 ßHS, 16%) isolated over the study period. Of the 25 GCGS, 22 (88%) were recovered from pus, two (8%) from respiratory tract, whereas one isolate was recovered from blood of a fatal case of septicaemia. Of the total 23 GGS isolates, 18 (78%) were identified as Streptococcus dysgalactiae subsp equisimilis (SDSE, large-colony phenotype), five (21%) were Streptococcus anginosus group (SAG, small-colony phenotype). The two GCS were identified as SDSE. All GCGS isolates were susceptible to penicillin, vancomycin, and linezolid. Tetracycline resistance was noted in 50 per cent of SDSE isolates. The rates of macrolide and fluoroquinolone resistance in SDSE were low. Twelve of the 20 SDSE isolates were positive for one or more spe genes, with five of the SDSE isolates simultaneously carrying speA+ speB+ smeZ+ speF or speB+ smeZ+speF, speI+speM+speG+speH or, speI+spe M+speH or speA+ speB+ speC+ smeZ+ speF. One notable finding was the presence of spe B in four of the five isolates of the Streptococcus anginosus group. No isolate was positive for ssa. INTERPRETATION & CONCLUSIONS: Our study showed no association between GCGS isolates harbouring streptococcal pyrogenic exotoxins and disease severity. This might be attributed to the small sample size of spe-positive isolates.

Novel twin streptolysin S-like peptides encoded in the sag operon homologue of beta-hemolytic Streptococcus anginosus.

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

Presumed hydrogen sulfide-mediated neurotoxicity after streptococcus anginosus group meningitis.

Hydrogen sulfide is an environmental toxicant and gaseous neurotransmitter. It is produced enterically by sulfur-reducing bacteria and invasive pathogens including Streptococcus anginosus group, Salmonella and Citrobacter. We describe putative focal hydrogen sulfide neurotoxicity after Streptococcus constellatus meningitis, treated with adjunctive sodium nitrite and hyperbaric oxygen therapy.

Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.

BACKGROUND: Hydrogen sulfide is one of the predominant volatile sulfur compounds (VSCs) produced by oral bacteria. This study developed and evaluated a system for detecting hydrogen sulfide production by oral bacteria. METHODS: L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (METase) and beta carbon-sulfur (beta C-S) lyase were used to degrade homocysteine and cysteine, respectively, to produce hydrogen sulfide. Enzymatic reactions resulting in hydrogen sulfide production were assayed by reaction with bismuth trichloride, which forms a black precipitate when mixed with hydrogen sulfide. The enzymatic activities of various oral bacteria that result in hydrogen sulfide production and the capacity of bacteria from periodontal sites to form hydrogen sulfide in reaction mixtures containing L-cysteine or DL-homocysteine were assayed. RESULTS: With L-cysteine as the substrate, Streptococcus anginosus FW73 produced the most hydrogen sulfide, whereas Porphyromonas gingivalis American Type Culture Collection (ATCC) 33277 and W83 and Fusobacterium nucleatum ATCC 10953 produced approximately 35% of the amount produced by the P. gingivalis strains. Finally, the hydrogen sulfide found in subgingival plaque was analyzed. Using bismuth trichloride, the hydrogen sulfide produced by oral bacteria was visually detectable as a black precipitate. CONCLUSIONS: Hydrogen sulfide production by oral bacteria was easily analyzed using bismuth trichloride. However, further innovation is required for practical use.

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction.

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.

Homocysteine biosynthesis pathways of Streptococcus anginosus.

A gene (cgs) encoding cystathionine gamma-synthase was cloned from Streptococcus anginosus, and its protein was purified and characterized. The cgs gene and the immediately downstream lcd gene were shown to be cotranscribed as an operon. High-performance liquid chromatography analyses showed that the S. anginosus Cgs not only has cystathionine gamma-synthase activity, but also expresses O-acetylhomoserine sulfhydrylase activity. These results suggest that S. anginosus has the capacity to utilize both the transsulfuration and direct sulfhydrylation pathways for homocysteine biosynthesis.

Isolation of a putative laminin binding protein from Streptococcus anginosus.

Viridans streptococci, including Streptococcus anginosus, are a common cause of infective endocarditis in humans. Adherence mechanisms involved in colonization of non-diseased native valves (present in 40% of native valve endocarditis) are unknown. We have previously shown that an endocarditis isolate of S. anginosus adheres to exposed basement membrane of human and porcine valve tissue in a laminin dependent manner. We now describe the partial purification of an 80 kDa putative laminin binding protein (PLBP) by biochemical methods. Amino acid sequence of PLBP peptides is similar to substrate binding proteins of ABC transporters in other Gram-positive cocci.