Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence.

Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.