PepO is a protein involved in Streptococcus sanguinis evasion to complement immunity

Objectives: To investigate the role of PepO in S. sanguinis susceptibility to complement-mediated immunity.
Methods: An isogenic pepO mutant was obtained in strain SK36 by double cross-over recombination with a null allele and compared to parent strain. Then, 8 clinical isolates S. sanguinis strains (6 from oral and 2 from blood sites) were assessed in all assays. Deposition of C3b (major opsonin of the complement system) were compared among all strains. Briefly, strains were incubated with 20% human serum (30 min; 37°C) or PBS, and surface C3b probed with antibody FITC-conjugated anti-human C3b (1:300). Levels of bound C3b and (MFI) were measured by flow cytometry. Transcriptional activities of pepO were compared by RT-qPCR among all strains.
Results: Mean MFIs of surface C3b increased by 97% in pepO mutant (C3b: 1,091 ± 122), compared to parent strain SK36 (C3b: 409.53 ± 24) (p<0.05 Kruskal Wallis). Interestingly, isolates with higher MFIs of surface C3b (SK678: 1,234.59 ± 98.45; SK72: 676.50 ± 32.65; SK115: 665.84 ± 23.67) showed lower pepO expression (0.00 ± 0.00; 3.95 ± 0.18; 1.24 ± 0.39, respectively). Consistently, strains with lower C3b deposition (SK49: 127.90 ± 8.98; SK330: 360.90 ± 32.65) showed higher pepO expression (6.42 ± 0.58; 20.16 ± 0.87, respectively) (Pearson correlation: r -0.5853, p<0.05).
Conclusions: The presence and expression of pepO is inverse related with the deposition of C3b on the surface of S. sanguinis, which suggests PepO as an important protein that mediates evasion to complement immunity.