Novel Models of Streptococcus canis Colonization and Disease Reveal Modest Contributions of M-Like (SCM) Protein

Abstract

Streptococcus canis is a common colonizing bacterium of the urogenital tract of cats and dogs that can also cause invasive disease in these animal populations and in humans. Although the virulence mechanisms of S. canis are not well-characterized, an M-like protein, SCM, has recently identified been as a potential virulence factor. SCM is a surface-associated protein that binds to host plasminogen and IgGs suggesting its possible importance in host-pathogen interactions. In this study, we developed in vitro and ex vivo blood component models and murine models of S. canis vaginal colonization, systemic infection, and dermal infection to compare the virulence potential of the zoonotic S. canis vaginal isolate G361 and its isogenic SCM-deficient mutant (G361∆scm). We found that while S. canis establishes vaginal colonization and causes invasive disease in vivo, the contribution of the SCM protein to virulence phenotypes in these models is modest. We conclude that SCM is dispensable for invasive disease in murine models and for resistance to human blood components ex vivo, but may contribute to mucosal persistence, highlighting a potential contribution to the recently appreciated genetic diversity of SCM across strains and hosts.

Keywords: M protein; Streptococcus canis; innate immunity; vaginal colonization; virulence factor.

Figure 1

SCM deficiency minimally impacts S. canis growth, hemolytic activity, and biofilm formation. Growth curves of S. canis G361 or G361Δscm in Todd-Hewitt broth (THB, (A)) or RPMI-1640 (RPMI, (B)) as measured by optical density (OD₆₀₀). (C) Biofilm formation of S. canis G361 or G361Δscm or GAS 5448 or 5448Δemm1 in THB or RPMI quantified by SYTO 13 fluorescence and expressed as the percent fluorescence of the WT strain. (D) Representative images (two per condition) of S. canis G361 or G361Δscm biofilms grown for 48 h in THB or RPMI and stained with SYTO 13. Symbols represent individual experimental replicates (A–C) with lines indicating interquartile ranges. Representative images are of independent experimental replicates, scale bar = 200 μm (D). Data were analyzed by two-way ANOVA with Sidak’s multiple comparisons post-test (A–C). *, p < 0.05.

Figure 2

SCM does not alter S. canis survival, reactive oxygen species release, cytokine production, nor induce antigenic activity in human sera. (A) Percent survival of S. canis G361 or G361Δscm after 30 min of exposure to canine DH82 macrophages, MOI = 1. (B) Reactive oxygen species production by DH82 macrophages infected with S. canis G361 or G361Δscm, MOI = 10, and normalized to uninfected cells. (C) Percent survival of S. canis G361 or G361Δscm after 30 min of exposure to human THP-1 differentiated macrophages, MOI = 1. (D) THP-1 cells were infected with S. canis G361 or G361Δscm, MOI = 1, and cell supernatant added to HEK-Blue cells. Alkaline phosphatase activity was measured colorimetrically at OD620 and background signal (uninfected cell supernatant) was deducted. Fold IL-1β release was calculated versus GAS across four independent experiments. (E) Percent survival of S. canis G361 or G361Δscm after 30 or 60 min of infection in human whole blood. (F) Percent survival of S. canis G361 or G361Δscm after 30 or 60 min of infection in isolated human neutrophils, MOI = 1. (G) Quantification of human IgG titers, expressed as relative fluorescent units (RFU), for a purified truncated form of SCM (n = 20 donors) via modified ELISA using diluted human sera, positive control: recombinant human IgG, negative control: buffer only. Symbols represent independent experimental replicates (A–D), biological replicates ((E), n = 6/group, (F), n = 5/group), or the results of one independent experiment (G), performed twice independently), with lines indicating medians and interquartile ranges. Data were analyzed by Wilcoxon matched-pairs signed rank test (A), two-way ANOVA with Sidak’s multiple comparisons post-test (B,E,F), or Friedman test with Dunn’s multiple comparisons test (C,D) and determined not significant.

Figure 3

S. canis is highly virulent in mouse models of systemic and dermal infection, yet SCM does not contribute to virulence in these models. (A) Percent survival of S. canis G361 or G361Δscm after 30 min of infection in murine whole blood collected from CD1 mice. (B) CD1 male and female mice were infected subcutaneously with 1 × 10⁸ CFU of WT S. canis G361 or G361Δscm and skin lesion size measured daily. (C) Representative image of skin lesions three days post subcutaneous infection with WT S. canis G361 (left) or G361Δscm (right). (D) CD1 male and female mice were infected intraperitoneally with 5  ×  10⁷ CFU of WT S. canis G361, G361Δscm, or S. pyogenes 5448 and survival monitored over 3 days. Symbols represent biological replicates ((A), n = 5/group, (B), n = 20/group, and (D), n = 10-21/group) with lines indicating medians and interquartile ranges (A,B) or percentage survival (D). Data were analyzed by Wilcoxon matched-pairs signed rank test (A), two-way ANOVA with Sidak’s multiple comparisons post-test (B) or Log rank Mantel-Cox test (D) and determined not significant.

Figure 4

S. canis adheres to vaginal epithelial cells and persists in a murine model of vaginal colonization, and SCM confers a fitness advantage in this environment. (A) Percent adherence of S. canis G361, G361Δscm, or GBS COH1 to VK2 cells after 30 min of infection, MOI = 1. CD1 female mice were vaginally administered 1  ×  10⁷ CFU of WT S. canis G361, G361Δscm, or WT GBS COH1, or PBS as a control. (B) Mice were vaginally swabbed daily, and the levels of bacterial CFU recovered from swabs are shown. (C) Cells collected from day 3 post-inoculation were analyzed for surface markers via flow cytometry. Total cell counts of each population recovered on the swabs are shown. (D) Vaginal epithelial tissues were fixed, sectioned, and stained with H&E. Histological examination revealed keratinized epithelium (top images) and neutrophil infiltration (bottom images) similarly across treatment groups. Magnification = 200X. (E) CD1 female mice were vaginally administered 1  ×  10⁷ CFU each of WT S. canis G361 and G361Δscm in competition. Mice were vaginally swabbed daily, and the levels of bacterial CFU recovered from swabs are shown. Symbols represent biological replicates ((B), n = 18/group, (C), n = 12–18/group, and (E), n = 20/group) or the means of four independent experimental replicates (B), performed in technical duplicate) with lines indicating medians and interquartile ranges. Dotted line in (B,E) indicates limit of detection. Data were analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons test (A), two-way ANOVA with Sidak’s multiple comparisons post-test (B,C) or Wilcoxon matched-pairs signed rank test (E). ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, not significant.