Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.

Selected strains of Streptococcus equi subsp. zooepidemicus produce a bacteriocin-like inhibitory substance called zoocin A. Zoocin A has bacteriolytic activity against closely related species of streptococci, including the etiological agents of dental caries and Streptococcus pyogenes. Zoocin A functions to hydrolyse the peptidoglycan of bacterial cell walls. Producer-cell immunity to zoocin A is encoded by zif, the zoocin A immunity factor. The zooA-zif gene locus is chromosomally encoded and flanked by two transposon-like sequences with significant similarity to the lss-lif locus encoding lysostaphin in Staphylococcus simulans. This suggested the possibility that the zooA-zif locus was acquired by S. equi subsp. zooepidemicus through a horizontal gene transfer event. Studies conducted to examine this hypothesis were unable to establish the mechanism by which zooA-zif was acquired. Examination of 24 S. equi subsp. zooepidemicus strains using random amplified polymorphic DNA analysis and pulse field gel electrophoresis showed that the species was highly heterogeneous. Examination of the 24 strains using Southern hybridisation revealed only three S. equi subsp. zooepidemicus strains contained the zooA and zif genes; however the partial open reading frames (flaR and rgg-like) flanking zooA and zif appeared to be conserved in all tested strains. Long template PCR was used to examine this chromosomal area containing the zooA and zif genes in 24 S. equi subsp. zooepidemicus strains. These results, along with comparison of sequence data from two zoocin A producing strains, and analysis of the sequenced S. equi subsp. zooepidemicus H70 and S. equi subsp. equi 4047 genomes showed the area surrounding the zooA-zif locus to be one of considerable genetic variation substantiating the hypothesis that zooA-zif were acquired via horizontal gene transfer. Studies on zoocin A production suggested that zooA expression was under carbon catabolite regulation, (CCR). In the Gram-positive bacteria CCR, is mediated by the carbon catabolite control protein (CcpA). CcpA is a DNA binding protein that can negatively regulate transcription of its target gene by binding to a catabolite-responsive element (CRE). This study reports the regulation of zif and zooA expression in response to cellular growth on different carbon sources, the presence of a ccpA-like gene in the genome of S. equi subsp. zooepidemicus, the presence of a CRE sequence in the promoter region of zooA, and the effect of a ccpA deletion on zooA expression. Zoocin A activity, zooA and zif mRNA transcript levels, and zooA and zif reporter gene activities were monitored during growth of S. equi subsp. zooepidemicus 4881 in a pH-controlled batch culture system. Zoocin A titres were maximal when the bacteria were grown on a starch-based medium and minimal when grown on a glucose-based medium. Northern hybridization showed that zif was constitutively expressed during growth regardless of the carbon source available. In contrast, zooA was transcribed at low levels in cells grown on a glucose-based medium but at much higher levels in cells grown on a starch-based medium. Use of a zooA promoter-chloramphenicol acetyl transferase (CAT) reporter demonstrated that zooA expression increased 17-fold in cells grown on a starch-based medium. Use of a zif promoter-CAT reporter showed that in cells grown on a starch-based medium zif was transcribed approximately 4 times as strongly as in cells grown on glucose. Transcription of zif was always much less than that of zooA. A ccpA deletion mutant appeared to exhibit complete de-repression of zooA under all growth conditions. Together these data showed that zoocin A expression was regulated in response to the carbon source on which the bacteria were grown.

Würzburg, Universiẗat, Diss., 2007.

Würzburg, Universiẗat, Diss., 2007.

The important human pathogen Streptococcus pyogenes causes both mild infections such as pharyngitis and impetigo but also severe life threatening invasive infections.  Specific antibodies (IgG) recognize pathogens and are important mediators for pathogen clearance by the immune defence. S.ipyogenes expresses a highly effective and specific IgG endopeptidase called IdeS (immunoglobulin degrading enzyme of S.ipyogenes). IdeS rescues bacteria from opsonising IgG by cleavage of IgG generating two fragments F(ab´)2 and ½Fc. Moreover, IdeS block ROS production by neutrophils. In this thesis I have studied (i) allelic variants of IdeS and their biological potential, (ii) consequences of ½Fc production for host-pathogen interactions and (iii) IdeS processing by streptococcal and neutrophil proteases. When investigating the allelic variants of IdeS we could show that in respect to IgG degradation and inhibition of ROS production the allelic variants where indistinguishable, however the allelic variant of serotype M28 appears to be an unique exception as this protein was deficient in IgG cleavage but still inhibited ROS production. Further, the ½Fc fragments produced when IgG is cleaved by IdeS were shown to prime human neutrophils and under ex vivo experimental conditions this increased the bactericidal activity of the neutrophils. Finally, we made the interesting finding that IdeS is N-terminally processed by neutrophil proteases and by the streptococcal protease SpeB, but retain enzymatic activity and was less immunogenic compared to the full length protein.

Thèse de doctorat : Génétique Moléculaire : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 36-44).

Disputats, Københavns Universitet.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 64-73).

Thesis (Ph. D.)--Marshall University, 2002. / Title from document title page. Document formatted into pages; contains x, 147 p. Includes abstract. References at the end of each chapter.