Purification and characterization of streptolysin O from Streptococcus pyogenes.

1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.     

Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene

A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed.     

Growth of Streptococcus pyogenes and streptolysin O production in complex and synthetic media

A comparative study of the growth of a highly haemolytic group A streptococcal strain in Trypticase-yeast extract medium and in a chemically defined medium was undertaken. Appreciable growth was obtained in the latter medium with the release of significant amounts (120 haemolytic units) of streptolysin O. This indicates that toxinogenic factors present only in the peptones of complex media are not essential for biosynthesis and release of this cytolytic toxin, as is the case for many bacterial toxins. NADase was also released in the synthetic medium. Bacterial cell mass, growth rate, and streptolysin O production were threefold higher in the complex medium. The effects of various carbohydrates on streptolysin O production in complex medium are investigated. No repression of toxin formation by glucose was observed. The relationship between growth and toxinogenesis in streptococci and in other toxinogenic bacteria is discussed.     

Chemokine production by rat macrophages stimulated with streptolysin O from Streptococcus pyogenes

The contribution of streptolysin O (SLO) from Streptococcus pyogenes to neutrophil infiltration in inflammatory lesions was determined by production of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 and -3, and macrophage inflammatory protein (MIP)-1alpha by rat macrophages stimulated with SLO in culture. Active SLO induced the production of CINCs and MIP-1alpha in dose- and time-dependent manners. These inductions were ascertained by chemokine mRNA expression in macrophages. Streptolysin S was without effect. The SLO-cholesterol complex induced the chemokine production in proportion to the residual hemolytic activity of the complex. In addition, the effects of SLO on the chemokine production were confirmed by the injection of active SLO into the preformed air pouch on the back of rats. The infiltration of neutrophils into the pouch fluid (exudate) increased steadily with a lag phase of about 2 hr. The major chemokine found in exudates was MIP-1alpha but not CINCs. In this study, it became clear that active SLO, but not the inactive one, contributed to the production of MIP-1alpha and CINCs in the conditioned medium and in exudates.     

Insertional inactivation of the major autolysin gene of Streptococcus pneumoniae.

The lytA gene encoding the major pneumococcal autolysin (N-acetylmuramoyl-L-alanine amidase) was inactivated by inserting the 2-kilobase MspI fragment of pE194 containing the staphylococcal ermC gene. Stable autolysis-deficient (Lyt-) mutants and their isogenic Lyt+ parents were used in experiments designed to test possible physiological functions of the amidase. No autolysis could be induced in the mutants grown at 37 degrees C by deoxycholate, by incubation in stationary phase, or by treatment with penicillin. On the other hand, the Lyt- mutants exhibited normal growth rates and yields and normal adaptive responses during shifts from one growth temperature or nutritional condition to another. There was no evidence for impeded cell separation (chain formation). Colonies of Lyt- insertional mutants produced normal hemolytic zones on blood agar; they showed normal (high) levels of competence for genetic transformation. Lyt- mutants were also able to produce type 3 and 6 capsular polysaccharides, and such strains showed the same degree of virulence in mice as did the isogenic Lyt+ parent. The physiological function(s) of the amidase remains a puzzle.     

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.     

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

Insertional inactivation by Tn3701 of pIP964 hemolysin expression in Enterococcus faecalis

Abstract Transposition of the conjugative mobile element (Tn 3701 ) of Etreptococcus pyogenes strain A454 onto the Enterococcus feacalis hemolysin plasmid pIP964 modified the expression of the genes involved in hemolysin production. By hemolysis complementation tests the genes coding for two components required for the hemolytic activity expression were found to be located on the Eco RI E and G fragments of pIP964.     

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.     

Insertional inactivation of the tomato polygalacturonase gene

The site-selected insertion (SSI) procedure was used to generate insertional knockout mutations in the gene for tomato polygalacturonase (PG), a critical enzyme in fruit ripening. Previously, it had been shown that the Dissociation (Ds) elements in a select group of tomato plants frequently inserted into PG, at least in somatic tissues. DNA isolated from pollen produced by progeny of these plants was screened by SSI to identify plants likely to transmit the insertions in PG to progeny. These results identified one family as likely candidate for yielding germinally transmitted insertions. Four thousand progeny were screened and five were found containing germinally transmitted Ds insertions in PG, one of which contained two Ds insertions in PG. The Ds elements were stabilized by genetically removing the transposase and four of the five insertions were recovered as homozygous in the next generation. Enzymatic analysis of fruit from these individuals demonstrated that there was at least a 1000-fold reduction in polygalacturonase levels in those plants bearing Ds insertions in PG exons. Individuals with modified PG sequences due to the sequence footprint, resulting from excision of the element, were identified using the single-strand conformational polymorphism (SSCP) method. Enzymatic analysis of fruit from a plant homozygous for one such excision allele showed a significant reduction in polygalacturonase activity. Since there is no transgenic material left in PG, this demonstrates the ability to modify a gene of commercial value in planta and subsequently removing all transgenic material.