Structure-Function Characterization of Streptococcus intermedius Surface Antigen Pas

Abstract

Streptococcus intermedius, an oral commensal bacterium, is found at various sites, including subgingival dental plaque, purulent infections, and cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization, and infection. Among the 19 genomically annotated cell wall-attached surface proteins on S. intermedius, Pas is an adhesin that belongs to the antigen I/II (AgI/II) family. Here, we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of V^Pas and C₁₂₃^Pas show high similarity with AgI/II of Streptococcus mutans. V^Pas hosts a conserved metal binding site, and likewise, the C₁₂₃^Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine-rich domains (SRCRs), and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa, the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas-deficient mutant (Δpas) displayed significant reduction in dual-biofilm formation with C. albicans. In similar studies with P. aeruginosa, Pas did not mediate the biofilm formation with either the acute isolate (PAO1) or the chronic isolate (FRD1). However, the sortase A-deficient mutant (ΔsrtA) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. IMPORTANCE Streptococcus intermedius, an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and cystic fibrosis lungs. In this study, we have (i) determined the crystal structure of the V and C regions of Pas; (ii) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the bloodstream from the oral cavity; (iii) characterized Pas's high-affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (iv) most importantly, shown that these surface proteins on the oral commensal S. intermedius enhance biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa.

Keywords: Candida albicans; Streptococcus intermedius; protein structure-function; surface antigens.

FIG 1

Primary structures of Pas, AgI/II, SspB, and GbpC. Each of these proteins has very similar domain arrangements except for GbpC, which only has the V region. The extents of the Pas constructs FL^Pas, V^Pas, and C₁₂₃^Pas used in this study are shown in dashed lines on the top.

FIG 2

V domain. (A) The crystal structure of V^Pas has a conserved metal binding site coordinated by S535, N537, and E544. This site is occupied by a Mg²⁺, as opposed to Ca²⁺ ion observed in crystal structures of V^AgI/II, V^GpbC, and V^SspB. (B) The superposition of V^Pas (gray), V^AgI/II (pink), V^GpbC (blue), and V^SspB (green) is shown, where three regions (1, 2, and 3) show maximal variations. (C) Structure-based sequence alignment of V^Pas, V^AgI/II, V^GpbC, and V^SspB where the metal binding site is indicated by orange dots, and the boxed regions are the loop regions that hover over the putative binding site on V^Pas, V^AgI/II and V^GpbC.

FIG 3

C domain. (A) Crystal structure of C₁₂₃^Pas with each domain highlighted. The insets show the two isopeptide bonds in C₁^Pas and C₂^Pas and the two calcium binding sites in C₁₂₃^Pas. (B) Superposition of C₁₂₃^Pas (gray), C₁₂₃^AgI/II (pink), and C₁₂₃^SspB (green) shows that they are very similar; however, C₁^Pas shows large deviations. (C) Structure-based protein sequence alignment of C₁₂₃^Pas, C₁₂₃^AgI/II, and C₁₂₃^SspB is shown.

FIG 4

S. intermedius adherence to fibrinogen. S. intermedius binding to fibrinogen coated on a plate is not mediated by Pas but is greatly impacted by a knockout of sortase A.

FIG 5

S. intermedius monospecies biofilm. The bottom panels for S. intermedius, which is grown with C. albicans culture supernatant, exhibited more robust biofilm than the top panels with the same strain (hexidium iodide red stain). S. intermedius strains were grown in THB, diluted at a ratio of 1:2,000 into 1 ml fresh TSB medium, and grown for 24 h at 5% CO₂ and 37°C under static conditions.

FIG 6

Biofilm studies, crystal violet staining. (A) In the monospecies biofilm, there was a significant reduction (54%) in biofilm formation with Δpas, and when complemented, this phenotype was mostly (79%) restored. The sortase A mutant also displayed significant reduction (71%) in biofilm formation. (B) The dual-species study showed less reduction with the Pas knockout (31%) and was fully recovered by the complemented strain. (C) The S. intermedius with C. albicans supernatant has similar reduction to the dual species biofilm. (D) Pas and sortase mediate different interactions with acute and chronic strains of P. aeruginosa. PAO1, acute P. aeruginosa; FRD1, chronic P. aeruginosa. Biofilms were grown in Todd Hewitt with 20 mM glucose for 16 h and incubated at 37°C with 5% CO₂. (E) Sortase A knockout of S. intermedius also reduces biofilm with three species biofilm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All statistics were analyzed by one-way ANOVA using GraphPad Prism 9.

FIG 7

Dual-species biofilm. S. intermedius and C. albicans biofilm was assessed using hexidium iodide (S. intermedius) or calcofluor white (C. albicans). S. intermedius strains with C. albicans were grown in THB and were diluted each at a ratio of 1:2,000 into 1 ml fresh TSB medium and grown for 24 h at 5% CO₂ and 37°C under static conditions.