Contribution of exogenous genetic elements to the group A Streptococcus metagenome

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is approximately 1.9 Mb in size, with approximately 10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics.     

Complete genome of acute rheumatic fever-associated serotype M5 Streptococcus pyogenes strain manfredo

Comparisons of the 1.84-Mb genome of serotype M5 Streptococcus pyogenes strain Manfredo with previously sequenced genomes emphasized the role of prophages in diversification of S. pyogenes and the close relationship between strain Manfredo and MGAS8232, another acute rheumatic fever-associated strain.     

Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Background  

The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).     

Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.     

Typing of group A streptococci isolated from urban population of different social status and age

Pulse-electrophoresis, sequencing of emm genes coding protein M and PCR analysis of speA, speB, and speC genes were used for characterization of group A streptococci (GAS) isolated in different years in Moscow and Tuapse mostly from children and military staff. It has been shown that epidemic process of streptococcal infection caused by GAS in Moscow is based on circulation of many independent clones of Streptococcus pyogenes. Obtained data on complex typing of S. pyogenes would be useful for study of molecular epidemiology of diseases caused by GAS and improvement of epidemiologic surveillance.     

Genetic organisation of the M protein region in human isolates of group C and G streptococci: two types of multigene regulator-like (mgrC) regions

In addition to beta-haemolytic streptococci belonging to Lancefield group A (Streptococcus pyogenes, GAS), human isolates of group C (GCS) and group G (GGS) streptococci (S. dysgalactiae subsp. equisimilis) have been implicated as causative agents in outbreaks of purulent pharyngitis, of wound infections and recently also of streptococcal toxic shock-like syndrome. Very little is known about the organisation of the genomic region in which the emm gene of GCS and GGS is located. We have investigated the genome sequences flanking the emm gene in GCS by sequencing neighbouring fragments obtained by inverse PCR. Our sequence data for GCS strains 25287 and H46A revealed two types of arrangement in the emm region, which differ significantly from the known types of mga regulon in GAS. We named this segment of the genome mgrC (for multigene regulon-like segment in group C streptococci). In strains belonging to the first mgrC type (prototype strain 25287) the emm gene is flanked up-stream by mgc, a gene that is 61% identical to the mga gene of GAS. A phylogenetic analysis of the deduced protein sequences showed that Mgc is related to Mga proteins of various types of GAS but forms a distinct cluster. Downstream of emm, the mgrC sequence region is bordered by rel. This gene encodes a protein that functions in the synthesis and degradation of guanosine 3',5' bipyrophosphate (ppGpp) during the stringent regulatory response to amino acid deprivation. In the second mgrC type (prototype strain H46A), the genes mgc and emm are arranged as in type 1. But an additional ORF (orf) is inserted in opposite orientation between emm and rel. This orf shows sequence homology to cpdB, which is present in various microorganisms and encodes 2',3' cyclo-nucleotide 2'-phosphodiesterase. PCR analysis showed that these two mgrC arrangements also exist in GGS. Our sequence and PCR data further showed that both types of mgrC region in GCS and GGS are linked via rel to the streptokinase region characterised recently in strain H46A. A gene encoding C5a peptidase, which is present at the 3' end of the mga regulon in GAS, was not found in the mgrC region identified in the GCS and GGS strains investigated here.     

MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.     

Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49.

The antigenically variant M protein of Streptococcus pyogenes enhances virulence by promoting resistance to phagocytosis. The serum opacity factor (OF), produced by a subset of M serotypes, is also antigenically variant, and its antigenic variability exactly parallels that of M protein. OF-positive and OF-negative streptococci are also phenotypically distinguishable by a number of other criteria. In order to study the differences between OF-positive and OF-negative streptococci, we cloned and sequenced the type 49 M protein gene (emm49), the first to be cloned from an OF-positive strain. This gene showed evolutionary divergence from the OF-negative M protein genes studied previously. Furthermore, emm49 was part of a gene family, in contrast to the single-copy nature of previously characterized M protein genes.     

Expression of different group A streptococcal M proteins in an isogenic background demonstrates diversity in adherence to and invasion of eukaryotic cells

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.