Reduced virulence of group A streptococcal Tn916 mutants that do not produce streptolysin S

Abstract

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn916 insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn916 donor strain Enterococcus faecalis CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn916 insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated sagA, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus -1.16 g; P < 0.05) and had fewer necrotic lesions (0 versus 7; P = 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus -0.66 g; P < 0.05) and produced fewer necrotic lesions (1 versus 8; P = 0.001). Revertants of the mutants in which Tn916 had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn916 mutations, are markedly less virulent than their isogenic parents.

FIG. 1

Southern hybridization analysis of HindIII restriction digests of genomic DNA probed with tetM. (A) Hemolytic wild-type T18P (lane 1) does not hybridize with the tetM probe. Nonhemolytic transconjugants SB1-4, SB2-9, SB30-2, SB1-9, SB5-9, SB6-9, SB7-9, SB1-1, and SB8-2 (lanes 2 to 10, respectively) all contain at least one copy of Tn916 and hybridize with the tetM probe. Isolates in lanes 2, 7, and 9 possess more than one Tn916 insertion. All lanes possess two bands hybridizing with the tetM probe corresponding to approximately 6.5 and 14 kb. The Tn916 donor strain CG110 (lane 11) contains several copies of Tn916. (B) Hemolytic wild-type MGAS166s (lane 1) does not hybridize with the tetM probe. The nonhemolytic transconjugants SBNH1, SBNH3, SBNH4, SBNH5, SBNH6, SBNH7, and SBNH8 (lanes 2 to 8, respectively) all possess at least one copy of Tn916. Isolates in lanes 3, 4, 7, and 8 possess more than one Tn916 insertion. Isolates in all lanes possess two bands of similar size, approximately 14 and 7.5 kb. The migration of molecular size standards (1-kb ladder) is indicated on the left.

FIG. 2

Nucleotide sequence and protein translation of sagA. A 390-bp region of genomic DNA from MGAS166s corresponding to the chromosomal point of insertion of Tn916 (▿) is indicated. The putative consensus elements of the sagA ORF and the putative 53-amino-acid translation product are shown. S.D., Shine-Dalgarno consensus sequence.

FIG. 3

Total RNA extracted from mutant SBNH5 (lanes 2 to 7) and wild-type MGAS166s (lanes 8 to 13) was quantified, standardized, blotted, and probed with a PCR amplicon of sagA labeled with α-³²P. Lane 1, 0.16- to 1.77-kb RNA standard; lane 2, SBNH5 RNA harvested at mid-log phase; lanes 3 to 7, SBNH5 RNA at 2, 4, 6, 8, and 10 h post-mid-log phase, respectively; lane 8, MGAS166s RNA harvested at mid-log phase; lanes 9 to 13, MGAS166s RNA at 2, 4, 6, 8, and 10 h post-mid-log phase, respectively. The mutant strain is devoid of any transcripts from sagA, while the wild type contains sagA transcripts at all time points.

FIG. 4

Comparisons of mean weight changes 24 h after infection with wild-type strains (MGAS166s and T18Ps) and the respective isogenic nonhemolytic mutants (SBNH5 and SB30-2). Animals infected with nonhemolytic mutants of each wild type gained weight, in contrast to the marked weight loss caused by infection with the parent strains.

FIG. 5

Photographs of hairless SKH1 mice 24 h after infection with 10⁶ CFU of either the SLS-producing wild-type MGAS166s (A) or the SLS-deficient Tn916 mutant SBNH5 (B). A well-demarcated zone of induration with centralized necrosis is visible on the right flank of the mouse infected with MGAS166s. No necrosis was seen in mice infected with SBNH5.

FIG. 6

Tissue biopsies from euthanized mice which were infected with 10⁶ CFU of either the SLS-producing wild-type MGAS166s (A) or the SLS-deficient Tn916 mutant SBNH5 (B). The tissue section in panel A demonstrates acute inflammation with edema and tissue necrosis. The tissue depicted in panel B does not show evidence of necrosis, and the inflammation is markedly reduced compared with that in panel A. Tissue samples were stained with hematoxylin and eosin, and the final magnification is approximately ×17.