In vivo acquisition of prophage in Streptococcus pyogenes

Genomic analysis of 12 Streptococcus pyogenes genomes representing six different serotypes reveals that they are poly-lysogenized, with as many as seven separate phage genomes (some of which are defective). Sequence alignments of these genomes (excluding incorporated prophage) have revealed that they are approximately 90% conserved, indicating that their diversity and disease capacity might be phage related. However, because S. pyogenes are only found in humans, how are new phages acquired? In vitro and in vivo experiments show that efficient phage transfer from donor to recipient streptococci occurs in the presence of mammalian cells. This suggests that, through evolution, phage have devised a system whereby progeny phage are induced and transferred to host streptococci at a site where host organisms are more prevalent.     

Phage A25-mediated transfer induction of a prophage in Streptococcus pyogenes

Summary The group A streptococcal strain 56188 used as standard donor in transduction with the virulent phage A25 is lysogenic for a phage called P56188. By using specific antiphage sera it is shown that A25 lysates obtained from 56188 contain a fraction of about 10^-4 phenotypically A25 but genotypically P56188 particles. A25-mediated transduction of prophage P56188 is measured by scoring plaques produced by transfer induction on 5004, a lysogenic strain unable to support the growth of A25. Data are obtained suggesting that A25 can also transduce a prophage carried by strain T25₃.Prophage P5004 present in 5004 is found to interfere with the propagation of A25 but does not seem to exert its action by directing extensive degradation of A25 DNA. Lysogenization of SM27 with P5004 leads to dramatically decreased burst sizes of A25, associated with the loss of its ability to plaque on this strain. Furthermore, P5004 lysogens of SM27 yield fewer streptomycin resistant transductants than their parent but gain the ability to serve as donors in A25-mediated transduction. A comparison of the burst size and the yield of transducing particles of A25 on various lysogenic and nonlysogenic hosts suggests that interfering with A25 growth is a widespread property of streptococcal prophages, which might favour processes leading to the formation of transducing A25 particles.     

Surface interactome in Streptococcus pyogenes

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.     

Mutagenesis and mechanism-based inhibition of Streptococcus pyogenes Glu-tRNAGln amidotransferase implicate a serine-based glutaminase site

The absence of Gln-tRNA synthetase in certain bacteria necessitates an alternate pathway for the production of Gln-tRNA(Gln): misacylated Glu-tRNA(Gln) is transamidated by a Gln-dependent amidotransferase (Glu-AdT) via catalysis of Gln hydrolysis, ATP hydrolysis, activation of Glu-tRNA(Gln), and aminolysis of activated tRNA by Gln-derived NH(3). As observed for other Gln-coupled amidotransferases, substrate binding, Gln hydrolysis, and transamidation by Glu-AdT are tightly coordinated [Horiuchi, K. Y., Harpel, M. R., Shen, L., Luo, Y., Rogers, K. C., and Copeland, R. A. (2001) Biochemistry 40, 6450-6457]. However, Glu-AdT does not employ an active-site Cys nucleophile for Gln hydrolysis, as is common in all other glutaminases: some Glu-AdT lack Cys, but all contain a conserved Ser (Ser176 in the A subunit of Streptococcus pyogenes Glu-AdT) within a sequence signature motif of Ser-based amidases. Our current results with S. pyogenes Glu-AdT support this characterization of Glu-AdT as a Ser-based glutaminase. Slow-onset (approximately 50 M(-1) s(-1)), tight-binding (t(1/2) > 2.5 h for complex dissociation), Gln-competitive inhibition of the Glu-tRNA(Gln)/ATP-independent glutaminase activity of Glu-AdT by gamma-Glu boronic acid is consistent with engagement of a Ser nucleophile in the glutaminase active site. Conversion to rapidly reversible, yet still potent (K(i) = 73 nM) and Gln-competitive, inhibition under full transamidation conditions mirrors the coupling between Gln hydrolysis and aminolysis reactions during productive transamidation. Site-directed replacement of Ser176 by Ala abolishes glutaminase and Gln-dependent transamidase activities of Glu-AdT (>300-fold), but retains a wild-type level of NH(3)-dependent transamidation activity. These results demonstrate the essentiality of Ser176 for Gln hydrolysis, provide additional support for coordinated coupling of Gln hydrolysis and transamidase transition states during catalysis, and validate glutaminase-directed inhibition of Glu-AdT as a route for antimicrobial chemotherapy.     

Persistence of Streptococcus pyogenes in stationary-phase cultures

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.     

Fluoride modifies adhesion of Streptococcus pyogenes

Streptococcus pyogenes grown in the presence of subinhibitory concentrations of sodium fluoride had a diminished ability, compared to control cells, to adhere to buccal cells, collagen, fibronectin, and laminin. In addition, sodium fluoride was a competitive inhibitor of streptococcal adhesion to collagen and fibronectin, but not laminin. It is suggested that sodium fluoride may be useful in therapy or prophylaxis in infections involving group A streptococci.     

Antigenic diversity of IgA receptors in Streptococcus pyogenes

Abstract In a previous study, group A and group B streptococcal IgA receptors were shown to differ serologically, in agreement with their known structural unrelatedness. The present study was undertaken to serologically compare the IgA binding epitopes of group A streptococcal strains representing various serotypes by the use of antisera to this species. It was found that blocking antibodies occurred in antisera to IgA binding but not to non-binding strains and that binding of IgA to a streptococcal strain was generally blocked by antiserum to the homologous type. However, cross-testing of a panel of 11 IgA binding strains, representing various M and T serotypes, with 10 different antisera to group A streptococci, demonstrated that IgA receptors were inhibited to a highly variable degree and that inhibition patterns were unique for each type. Comparing solubilized IgA receptors of various strains in immunoblot experiments, a variation in the molecular mass, between approximately 35 and 45 kDa, emerged. The IgA binding epitopes, analogous to protective sites of streptococcal M-protein, thus exhibited hypervariability which may suggest that IgA binding also plays a key role for evading host immune defence mechanisms.