Role of streptococcal T antigens in superficial skin infection

Abstract

FCT region genes of Streptococcus pyogenes encode surface proteins that include fibronectin- and collagen-binding proteins and the serological markers known as T antigens, some of which give rise to pilus-like appendages. It remains to be established whether FCT region surface proteins contribute to virulence by in vivo models of infection. In this study, a highly sensitive and ecologically relevant humanized mouse model was used to measure superficial skin infection. Three genes encoding FCT region surface proteins essential for T-serotype specificity were inactivated. Both the Deltacpa and DeltaprtF2 mutants were highly attenuated for virulence when topically applied to the skin following exponential growth but were fully virulent when delivered in stationary phase. In contrast, the DeltafctA mutant was virulent at the skin, regardless of its initial growth state. Immunoblots of cell extracts revealed anti-FctA-reactive, ladder-like polymers characteristic of streptococcal pili. In addition, FctA formed a heteropolymer with the putative collagen-binding protein Cpa. The DeltafctA mutant showed a loss in anti-Cpa-reactive polymers, whereas anti-FctA-reactive polymers were reduced in the Deltacpa mutant. The findings suggest that both FctA and Cpa are required for pilus formation, but importantly, an intact pilus is not essential for Cpa-mediated virulence. Although it is an integral part of the T-antigen complex, the fibronectin-binding protein PrtF2 is not covalently linked to the FctA- and Cpa-containing heteropolymer derived from cell extracts. The data provide direct evidence that streptococcal T antigens function as virulence factors in vivo, but they also reveal that a pilus-like structure is not essential for the most common form of streptococcal skin disease.

FIG. 1.

FCT region of strain ALAB49. The FCT region of ALAB49 is defined by boundaries of highly conserved ORFs encoding a putative chaperonin at the left flank, which is closest to the origin of replication, and a hypothetical protein at the right flank (5). Four of the FCT region loci encode (putative) cell wall-anchored surface proteins: cpa (encoding collagen-binding protein), fctA (also known as orf100 in M-type 3 strains, orf80 in M-type 5 strains, and eftLSLA in M-type 12 strains), fctB (also known as orf102 in M-type 3 strains, orf82 in M-type 5 strains, and orf2 in M-type 49 strains), and prtF2 (encoding fibronectin-binding protein of the fbaB lineage). The genes nra and msmR encode transcriptional regulators, sipA2 encodes a putative signal peptidase, and srtC2 encodes a sortase.

FIG. 2.

Immunoreactivity of T-typing sera to FCT region proteins. Immunoblots of recombinant GST-fusion polypeptides (5 μg per lane) were reacted with anti-T3 (α-T3), anti-T13, anti-TB3264, and anti-GST sera. Based on gel migration, the estimated sizes for GST fusion polypeptides rCpa, rFctA, rPrtF2, and rFctB are 76, 57, 121, and 42 kDa, respectively (GST alone is 26 kDa). The gel migration of rPrtF2 (amino acids 38 to 698) is slower than that expected based on the predicted amino acid sequence.

FIG. 3.

Roles of FCT region genes in virulence at the skin. Bacteria grown to either mid-logarithmic (filled symbols) or stationary (open symbols) phase in broth culture were used to inoculate scratched human skin engrafted on SCID mice. The inoculum dose (log₁₀ CFU) is depicted on the x axis. The net change (increase or decrease) in log₁₀ CFU recovered from a graft at biopsy, relative to the inoculum dose, is shown on the y axis. Each data point represents an inoculated skin graft. Bacterial inocula are indicated for wt ALAB49 and the Δcpa, ΔfctA, and ΔprtF2 mutants. Statistically significant differences (P values for two-tailed unpaired t test) between the net change in graft-recovered CFU for inocula prepared to the logarithmic versus stationary phase of growth are shown. The statistical significances of differences between the net change in graft-recovered CFU for wt ALAB49 versus each of the mutants, for the two inoculum conditions, are reported in the text. All data comparisons that were statistically significant by the unpaired t test were also significant by the Mann-Whitney U test, and vice versa (data not shown). NS, not significant.

FIG. 4.

Immunoblots of mutanolysin extracts from wt ALAB49 and mutants. Mutanolysin extracts of bacteria grown to mid-logarithmic (L) or stationary (S) phase were subjected to SDS-PAGE under reducing conditions. Immunoblots were incubated with rabbit antisera raised to rCpa, rFctA, or rPrtF2. For anti-FctA (α-FctA), one immunoblot was overdeveloped (lower left) in order to illustrate the presence of laddered pilus-like structures in the Δcpa mutant. A second anti-FctA immunoblot (lower right) was underdeveloped, allowing for resolution of the polymeric ladder observed in wt ALAB49 and the ΔprtF2 and ΔspeB mutants. The anti-PrtF2-reactive band migrated slower than for the size predicted for the mature PrtF2 monomeric form (77 kDa), similar to previous observations with cell-extracted and recombinant PrtF2 proteins (30, 44). Densitometry measures for selected lanes are presented in Table 1.

FIG. 5.

Cpa and FctA form a heteropolymer. Mutanolysin extracts from wt ALAB49 grown 16 h at 30°C were loaded onto three lanes and subjected to SDS-PAGE and electrotransfer; the blot was split down the middle of the center lane and the two halves incubated with antiserum raised to rCpa or rFctA polypeptide.

FIG. 6.

Lack of effect of growth phase on virulence of the ΔspeB mutant. Bacteria grown to either mid-logarithmic (filled symbols) or stationary (open symbols) phase in broth culture were used to inoculate scratched human skin engrafted on SCID mice. Bacterial inocula consist of wt ALAB49 (circles) or the ΔspeB mutant (squares). The inoculum dose (log₁₀ CFU) is depicted on the x axis. The net change (increase or decrease) in log₁₀ CFU recovered from a graft at biopsy, relative to the inoculum dose, is shown on the y axis. Each data point represents an inoculated skin graft. Statistical analysis is described in the Fig. 3 legend and reported in the text. Data on the ΔspeB mutant inoculated at stationary phase were previously reported (37).

FIG. 7.

Secreted cysteine proteinase activity of mutants. Secreted cysteine proteinase activity due to SpeB, present in culture broth supernatants, was measured for wt ALAB49 and mutants by using the azocasein substrate assay. The mean average OD₆₀₀ for all cultures was 0.646 ± 0.007, indicating that bacterial cell density was highly even across all samples. Data shown are compiled from two separate experiments, each performed in triplicate. The P value was calculated using the unpaired t test (two tailed).