Cationic antimicrobial peptides disrupt the Streptococcus pyogenes ExPortal

Although they possess a well‐characterized ability to porate the bacterial membrane, emerging research suggests that cationic antimicrobial peptides (CAPs) can influence pathogen behaviour at levels that are sublethal. In this study, we investigated the interaction of polymyxin B and human neutrophil peptide (HNP‐1) with the human pathogen Streptococcus pyogenes. At sublethal concentrations, these CAPs preferentially targeted the ExPortal, a unique microdomain of the S. pyogenes membrane, specialized for protein secretion and processing. A consequence of this interaction was the disruption of ExPortal organization and a redistribution of ExPortal components into the peripheral membrane. Redistribution was associated with inhibition of secretion of certain toxins, including the SpeB cysteine protease and the streptolysin O (SLO) cytolysin, but not SIC, a protein that protects S. pyogenes from CAPs. These data suggest a novel function for CAPs in targeting the ExPortal and interfering with secretion of factors required for infection and survival. This mechanism may prove valuable for the design of new types of antimicrobial agents to combat the emergence of antibiotic‐resistant pathogens.     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Nucleotide sequence of the streptococcin A-FF22 lantibiotic regulon: model for production of the lantibiotic SA-FF22 by strains of Streptococcus pyogenes

Streptococcin A-FF22 (SA-FF22) is a type AII linear lantibiotic produced by Streptococcus pyogenes strain FF22. Sequence analysis of an approximate 10 kb region of DNA showed it to contain nine open reading frames arranged in three operons responsible for regulation, biosynthesis and immunity of SA-FF22. This region is organized similarly to the Lactococcus lactis lacticin 481 region, however, unlike lacticin 481, a two-component regulatory system is essential for SA-FF22 production. Located immediately downstream of the scn region is a putative transposase gene, the presence of which supports earlier data that indicated a mobile nature to this region.     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.     

Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms

Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Identification of Streptococcus salivarius by PCR and DNA probe

AIMS: To establish species-specific PCR and DNA probe methods for Streptococcus salivarius and to clarify the distribution of dextranase in oral isolates of Strep. salivarius. METHODS AND RESULTS: A pair of PCR primers and a DNA probe were designed based on the nucleotide sequence of the dextranase gene of Strep. salivarius JCM5707. Both the PCR primer and the DNA probe specifically detected Strep. salivarius but none of the other oral streptococci (23 strains of 13 species). The primer and the probe were capable of detecting 1 pg and 1 ng of the genomic DNA, respectively, purified from Strep. salivarius JCM5707. All oral isolates (130 strains from 12 subjects) of Strep. salivarius from human saliva were positive by both methods. CONCLUSION: The present PCR and DNA probe methods are highly specific to Strep. salivarius and are useful for the its detection and identification of this bacterium. The dextranase widely distributes among oral isolates of Strep. salivarius. Significance and Impact of the Study: The DNA sequence of a dextranase gene present in the genome of Strep. salivarius is useful as the target DNA of the species-specific PCR and DNA probe.     

Streptococcus pyogenes cytolysin‐mediated translocation does not require pore formation by streptolysin O

Bacterial toxin injection into the host cell is required for the virulence of numerous pathogenic bacteria. Cytolysin‐mediated translocation (CMT) of Streptococcus pyogenes uses streptolysin O (SLO) to translocate the S. pyogenes nicotinamide adenine dinucleotide‐glycohydrolase (SPN) into the host cell cytosol, resulting in the death of the host cell. Although SLO is a pore‐forming protein, previous studies have shown that pore formation alone is not sufficient for CMT to occur. Thus, the role and requirement of the SLO pore remains unclear. In this study, we constructed various S. pyogenes strains expressing altered forms of SLO to assess the importance of pore formation. We observed that SLO mutants that are unable to form pores retain the ability to translocate SPN. In addition, SPN translocation occurs after inhibition of actin polymerization, suggesting that CMT occurs independently of clathrin‐mediated endocytosis. Moreover, despite the ability of mutants to translocate SPN, their cytotoxic effect requires SLO pore formation.     

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.     

乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

Intra- and interspecies variation among Bari-1 elements of the melanogaster species group.

We have investigated the distribution of sequences homologous to Bari-1, a Tc1-like transposable element first identified in Drosophila melanogaster, in 87 species of the Drosophila genus. We have also isolated and sequenced Bari-1 homologues from D. simulans, D. mauritiana, and D. sechellia, the species constituting with D. melanogaster the melanogaster complex, and from D. diplacantha and D. erecta, two phylogenetically more distant species of the melanogaster group. Within the melanogaster complex the Bari-1 elements are extremely similar to each other, showing nucleotide identity values of at least 99.3%. In contrast, Bari-1-like elements from D. diplacantha and D. erecta are on average only 70% similar to D. melanogaster Bari-1 and are usually defective due to nucleotide deletions and/or insertions in the ORFs encoding their transposases. In D. erecta the defective copies are all located in the chromocenter and on chromosome 4. Surprisingly, while D. melanogaster Bari-1 elements possess 26-bp inverted terminal repeats, their D. diplacantha and D. erecta homologues possess long inverted terminal repeats similar to the terminal structures observed in the S elements of D. melanogaster and in several other Tc1-like elements of different organisms. This finding, together with the nucleotide and amino acid identity level between D. diplacantha and D. erecta elements and Bari-1 of D. melanogaster, suggests a common evolutionary origin and a rapid diversification of the termini of these Drosophila Tc1-like elements.     

Cloning of the gene encoding Streptococcin A-FF22, a novel lantibiotic produced by Streptococcus pyogenes, and determination of its nucleotide sequence.

Streptococcin A-FF22 (SA-FF22) is a lantibiotic produced by Streptococcus pyogenes FF22. The nucleotide sequence of the SA-FF22 structural gene (scnA) was determined and shown to encode a 51-amino-acid prepeptide. The proteolytic processing site of the SA-FF22 prepeptide differs from that which characterizes other type A lantibiotics.     

Nitrate-dependent activation of the Dif signaling pathway of Myxococcus xanthus mediated by a NarX-DifA interspecies chimera

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.     

Intra- and interspecies relatedness ofYersinia pestis by DNA hybridization and its relationship toYersinia pseudotuberculosis

The biochemical characteristics ofYersinia pestis are presented and compared with those ofY. pseudotuberculosis. Motility at 28°C, urease, fermentation of rhamnose, and growth rate on nutrient agar are the best means of separating these organisms. DNA hybridization studies demonstrated thatY. pestis strains are 90% or more interrelated and thatY. pestis andY. pseudotuberculosis are indistinguishable by DNA relatedness. On the basis of DNA data and biochemical and antigenic similarity, these organisms should be treated as two separate subspecies of the same species.Y. pseudotuberculosis was described beforeY. pestis and therefore has priority.Y. pseudotuberculosis subsp.pseudotuberculosis andY. pseudotuberculosis subsp.pestis are recommended as new designations forY. pseudotuberculosis andY. pestis. For medical purposes,Y. pestis andY. pseudotuberculosis can and should continue to be used.     

Function of the pyruvate oxidase-lactate oxidase cascade in interspecies competition between Streptococcus oligofermentans and Streptococcus mutans

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H(2)O(2) production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H(2)O(2) in the earlier growth phase and log phase, while Lox mainly contributed to H(2)O(2) production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H(2)O(2) can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H(2)O(2) production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H(2)O(2). In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H(2)O(2) formation so as to win the interspecies competition.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and Gram-negative bacteria. Streptococcus pyogenes, an important human pathogen that can survive and grow in human blood, has developed mechanisms to escape the attack by these peptides. Thus, protein H and M1 protein, two surface proteins of the highly pathogenic S. pyogenes AP1 strain, bind full-length beta(2)GPI and thereby prevent the processing of beta(2)GPI by proteases from polymorphonuclear neutrophils (PMNs) into antibacterial peptides. In addition, protein H and M1 protein, released from the bacterial cell wall by PMN-derived proteases, bind to, and inhibit the activity of, beta(2)GPI-derived antibacterial peptides. Taken together, the data suggest that the interaction between the streptococcal proteins and beta(2)GPI or beta(2)GPI-derived peptides presents a novel mechanism to resist an antibacterial attack by beta(2)GPI-cleavage products.