Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence - PubMed (original) (raw)

. 2002 Jul 23;99(15):10078-83.

doi: 10.1073/pnas.152298499. Epub 2002 Jul 16.

Gail L Sylva, Kent D Barbian, Benfang Lei, Jessica S Hoff, Nicole D Mammarella, Meng-Yao Liu, James C Smoot, Stephen F Porcella, Larye D Parkins, David S Campbell, Todd M Smith, John K McCormick, Donald Y M Leung, Patrick M Schlievert, James M Musser

Affiliations

Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence

Stephen B Beres et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares approximately 1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A(2) (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

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Figures

Figure 1

Figure 1

GAS phages. (A) Schematic of GAS core genome and phage integration sites. Shown is the core genome (≈1.7 Mbp) derived by deleting phage sequence from the genome sequence of strains SF370 (serotype M1), MGAS8232 (serotype M18), and MGAS315 (serotype M3). Phage integration sites are indicated with triangles that are shaded to match the source GAS strain. Stacked triangles indicate that the phages are integrated at the same chromosomal site. The numbers in each triangle are designations that refer to the clockwise order of the phages. The six rRNA operons that are conserved in all three genomes are shown in green. (B) Relationships among GAS phages. Phage sequences present in the three GAS genomes were aligned with

clustalw

(

http://inn-prot.weizmann.ac.il/software

) and an unrooted tree was generated with the

drawtree

application in

phylip

(

http://evolution.genetics.washington.edu/phylip.html

). Phage size (kb) and proven or putative virulence factors encoded by each phage are indicated. Phages that are integrated at the same chromosomal location are color coded in B. mf2, mitogenic factor 2; mf3, mitogenic factor 3; mf4, mitogenic factor 4; sda, streptodornase alpha; sdn, streptodornase.

Figure 2

Figure 2

Amino acid alignment of Sla (streptococcal phospholipase A2 homologue) and textilotoxin subunits. The carboxy terminal 116 amino acid residues of Sla were aligned with the four subunits of textilotoxin with

clustalw

, using default parameters. Amino acid residues that match the consensus sequence are shaded in dark gray, and similar amino acid residues are shaded in light gray. The textilotoxin phospholipase A2 active site consensus sequence is shown in red, and conserved Cys residues are underlined.

Figure 3

Figure 3

SDS/PAGE and Western immunoblot analysis of rSla, rSpeK, and rSSA. (A) SDS/PAGE gel of purified recombinant proteins. The purified recombinant proteins were analyzed with a 15% polyacrylamide gel. (B) Representative Western immunoblot using sera obtained humans infected with serotype M3 strains of GAS. A, acute-phase sera; C, convalescent-phase sera.

Figure 4

Figure 4

Mitogenic activity of rSpeK. (A) Human peripheral blood mononuclear cells were incubated with purified rSpeK at 37°C for 3 days. [3H]thymidine was added to the medium, cells were harvested after 24 h, and cpm were determined by scintillation counting. Experiments done in quadruplicate, and error bars indicate the standard error of the mean. TSST-1, a known superantigen, was used as a positive control. (B) T cell TCR Vβ stimulation by rSpeK. PBMC from five human donors were studied. The percentage of T cells expressing the listed Vβ TCRs are shown. Only the Vβs with statistically significant stimulation (as determined by the Student's t test; P < 0.01) are shown.

Figure 5

Figure 5

PLA2 activity of rSla. PLA2 activity was assayed with a commercially available kit that measures the hydrolysis of phospholipids at the _sn_-2 position. The assay uses the 1,2-dithio analog of diheptanoyl phosphatidylcholine as a chromogenic substrate. Bee venom PLA2 was used as positive control (data not shown) and rSpeK was used as negative control.

Figure 6

Figure 6

Hypothesis to explain the emergence of a new, unusually virulent subclone of serotype M3 GAS. Under this hypothesis, all isolates shown are multilocus enzyme electrophoretic type 2 (8). Relatively early in the 20th century, an ancestral strain acquired phage Φ315.2, a T12-like phage that encodes SSA (13). Subsequently, a single nucleotide mutation resulting in a single amino acid replacement (Val106Ile) produced the SpeA3 allele (16) encoded by phage Φ315.5. This _ssa_-positive, _speA3_-positive strain gained phage Φ315.4 that encodes Sla and SpeK, and disseminated widely in the mid/late 1980s. The hypothesis is based on data presented in this paper and published previously (8, 13, 14, 16, 28).

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