Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315

Abstract

The genome of the highly virulent group A Streptococcus (GAS) serotype M3 strain MGAS315 has six prophages that encode six proven or putative virulence factors. We examined prophage induction and expression of prophage-encoded virulence factors by this strain under in vitro conditions inferred to approximate in vivo conditions. Coculture of strain MGAS315 with Detroit 562 (D562) human epithelial pharyngeal cells induced the prophage encoding streptococcal pyrogenic exotoxin K (SpeK) and extracellular phospholipase A(2) (Sla) and the prophage encoding streptodornase (Sdn). Increased gene copy numbers after induction correlated with increased speK, sla, and sdn transcript levels. Although speK and sla are located contiguously in prophage Phi315.4, these genes were transcribed independently. Whereas production of immunoreactive SpeK was either absent or minimal during coculture of GAS with D562 cells, production of immunoreactive Sla increased substantially. In contrast, despite a lack of induction of the prophage encoding speA during coculture of GAS with D562 cells, the speA transcript level and production of immunoreactive streptococcal pyrogenic exotoxin A (SpeA) increased. Exposure of strain MGAS315 to hydrogen peroxide, an oxidative stressor, induced the prophage encoding mitogenic factor 4 (MF4), and there was a concomitant increase in the mf4 transcript. All prophages of strain MGAS315 that encode virulence factors were induced during culture with mitomycin C, a DNA-damaging agent. However, the virulence factor gene transcript levels and production of the encoded proteins decreased after mitomycin C treatment. Taken together, the results indicate that a complex relationship exists among environmental culture conditions, prophage induction, and production of prophage-encoded virulence factors.

FIG. 1.

Electron microscopy of phage particles purified from strain MGAS315. The bacteria were treated with mitomycin C and centrifuged at 141,000 × g. Phage particles were suspended in buffer and negatively stained with uranyl acetate. Magnification = ×300,000.

FIG. 2.

PCR amplification of virulence factor genes present in culture supernatants of GAS. PCR was conducted with purified phage DNA templates obtained from strain MGAS315 cultured with PR-THY medium (control) (A), PR-THY medium plus mitomycin C (B), or PR-THY medium plus hydrogen peroxide (0.5 mM) (C). The genes that were amplified are listed above the lanes. The speB gene was used as a negative control to confirm that there was no contaminating GAS chromosomal DNA in the phage preparations. speA, streptococcal pyrogenic exotoxin A; ssa, streptococcal superantigen; mf4, mitogenic factor 4; speK, streptococcal pyrogenic exotoxin K; sla, phospholipase A₂; sdn, streptodornase; speB, streptococcal pyrogenic exotoxin B.

FIG. 3.

Induction of phages during strain MGAS315 coculture with pharyngeal epithelial cells. PCR was conducted with purified phage DNA templates obtained from strain MGAS315 cultured in MEM (A) or MEM plus D562 cells (B). The genes that were amplified are listed above the lanes. The speB gene was used as a negative control to confirm that there was no contaminating GAS chromosomal DNA in the phage preparations. speA, streptococcal pyrogenic exotoxin A; ssa, streptococcal superantigen; mf4, mitogenic factor 4; speK, streptococcal pyrogenic exotoxin K; sla, phospholipase A₂; sdn, streptodornase; speB, streptococcal pyrogenic exotoxin B.

FIG. 4.

Western immunoblot analysis of prophage-encoded secreted proteins made by MGAS315 during culture with mitomycin C. Proteins present in the culture supernatant were precipitated, separated by SDS-12% PAGE, and transferred to nitrocellulose. The membranes were probed with primary rabbit antibody (1:10,000) specific for each protein and secondary antibody (1:3,000) conjugated to horseradish peroxidase, and reactivity was visualized with chemiluminescent reagents. The treatments were as follows: PR-THY medium and mitomycin C (MMC).

FIG. 5.

Western immunoblot analysis of prophage-encoded secreted proteins made by MGAS315 during coculture with D562 epithelial cells. Proteins present in the culture supernatant were concentrated, separated by SDS-12% PAGE, and transferred to nitrocellulose. The membranes were probed with primary rabbit antibody (1:10,000) specific for each protein and secondary antibody (1:3,000) conjugated to horseradish peroxidase, and reactivity was visualized with chemiluminescent reagents.

FIG. 6.

Transcript levels of prophage-encoded genes in strain MGAS315 during culture with mitomycin C, hydrogen peroxide, and D562 epithelial cells relative to transcript levels under control conditions. Gene names are listed on the y axis. Relative transcript levels of genes after exposure to mitomycin C (black), hydrogen peroxide (red), and D562 epithelial cell coculture (blue) were normalized to the transcript level of proS, a constitutively expressed endogenous gene (10, 27). The mean transcript levels (± standard errors of the mean) of the prophage-encoded genes under experimental conditions are expressed as differences (n-fold) relative to control conditions.

FIG. 7.

RT-PCR analysis of speK and sla transcription. RNA purified from strain MGAS315 cocultured with D562 epithelial cells was reverse transcribed into cDNA, and PCR was conducted with primers specific for speK and sla. The amplicons generated were speK (1), sla (2), and speK, sla, and the intergenic region (3). The template used was cDNA synthesized from RNA purified from strain MGAS315 cocultured with D562 epithelial cells (A) or purified chromosomal DNA (control) (B). The stem-loop structure (not to scale) is located in the intergenic region (472 bp), 116 nucleotides upstream of the sla start codon.