Alexander N. Suvorov

Complete genome sequence of an M1 strain of Streptococcus pyogenes

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial “molecular mimicry” of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

Genome Sequence of a Nephritogenic and Highly Transformable M49 Strain of Streptococcus pyogenes

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.

Construction of a Streptococcus pyogenes recA mutant via insertional inactivation, and cloning and sequencing of the complete recA gene

To facilitate future genetic studies with Streptococcus pyogenes (Sp), a recA mutant (Rec11) was constructed using a streptococcal integration vector carrying a PCR-derived internal recA fragment. The insertion of the plasmid in the mutant chromosome was identified by Southern hybridization. Resistance to UV and the ability to accept linear DNA transformation by Rec11 were greatly decreased, confirming its RecA phenotype. Using the PCR-derived fragment as a probe, we cloned and sequenced the complete Sp recA gene, which is highly homologous to the recA of S. pneumoniae and Lactococcus lactis.

The Streptococcus pyogenes Genome Sequencing Project

Advances in genome analysis have allowed unprecedented progress to be made in understanding the complete gene structure and organization of a pathogenic organism as evidenced by recent reports of the entire DNA sequence of organisms such as Haemophilus influenzae and Mycoplasma pneumoniae (4, 5). In this communication, we report on the progress to determine the complete nucleotide sequence of an M1 strain of Streptococcus pyogenes. Strain SF370 is a well characterized M1 strain that was originally isolated from a wound infection and possesses the same RFLP pattern found in strains associated with severe invasive disease. As a prelude to genomic sequencing, the nucleotide sequence of the SF370 emm gene was determined and found to be identical to that of Lancefield strain T1/19/8. This strain is lysogenic for a speC-containing, but not a speA-containing bacteriophage. The complete sequence of the T12 bacteriophage containing the speA gene has recently been determined and is reported elsewhere (6). No other extrachromosomal elements were known to exist in this strain, although sequencing has revealed that additional temperate phages or defective phages are probably present. A physical map of SF370 has been constructed utilizing three different restriction enzymes (SmaI, SfiI, and SgrAI), and its genome size has been estimated to be 1920 kb, with thirty six genes identified to date on the genetic map (7).

Analysis of Pathogenic Group B Streptococci by Pulsed Field Gel Electrophoresis

Group B streptococci (GBS) is the major cause of newborn mortality (1). Several GBS genes encoding for alpha, beta antigens, C5a peptidase which are potentially involved in virulence have been determined, cloned and sequenced (2,3,4,6). However, their impact in pathogenicity and their location on the chromosome is not yet known. In order to investigate the relationship of GBS serotype with chromosomal location of the genes more detailed we performed the pulsed field gel electrophoresis (PFGE) genome analysis of 20 GBS strains belonging to serotypes I, II and III.

Immune complex binding Streptococcus pyogenes type M12/emm12 in experimental glomerulonephritis

In a rabbit model, we have previously reported evidence for a pathogenic role of streptococcal IgG Fc-binding proteins (IgGFcBP) in poststreptococcal glomerulonephritis (PSGN). These proteins, of the M protein family, were shown to trigger anti-IgG production and enhance renal deposition of IgG and/or immune complexes (ICs), with resulting activation of complement and cytokine cascades. In the present study, type M12/emm12, group A streptococci (GAS) were found often to bind artificial ICs, viz. peroxidase-anti-peroxidase rabbit IgG (PAP) or tetanus toxoid-anti-tetanus human IgG (TAT), rather than monomeric IgG. Animals injected with each of four IC binding clinical isolates (from patients with scarlet fever or PSGN) showed pronounced inflammatory and degenerative glomerular changes, morphologically similar to human PSGN, with membrane thickening and IgG and complement C3 deposition, as well as secretion of IL-6 and TNF-α by mesangial and endothelial cells. In contrast, non-binding strains (two from asymptomatic carriers and one from a PSGN case) failed to trigger any renal changes. Only the IC binding strains induced elevated titres of anti-IgG. Though the streptococcal binding component(s) has not been demonstrated, the selective binding of ICs by type M12/emm12 strains appears important for the well-known, marked nephritogenic potential of this GAS type.

Summary of the Round Table Discussion on Genome Structure and Regulation of Gene Expression in Streptococci and Enterococci

The significant progress made in recent years in the areas of streptoccocal genome structure and function was highlighted by the following topical contributions

Chromosomal Analysis of Group A Streptococci by Pulsed Field Gel Electrophoresis

According to contemporary serological classification all streptococci can be distinguished from one another by the antigenic diversity of their cell wall carbohydrate (3). The group A streptococci (GAS) can be further differentiated by the antigenic features of cell surface localized M protein. However, it is unclear to what extent this classification reflects the real genetic correlation between the streptococcal strains belonging to the same or even different serological groups. Using pulsed field gel electrophoresis (PFGE) as a tool for analysis of large chromosomal fragments of streptococci, we have constructed the first physical and genetic map of a GAS strain belonging to serotype M1 (4). More than 30 different DNA markers were localized on a circular chromosomal map. In the present study we have accomplished a PFGE analyses of 10 GAS strains belonging to different serotypes.