Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

Response of fatty acid synthesis genes to the binding of human salivary amylase by Streptococcus gordonii.

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.

Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.

Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3(-) mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.

Role of hydrogen peroxide in competition and cooperation between Streptococcus gordonii and Actinomyces naeslundii.

In dental plaque alpha-haemolytic streptococci, including Streptococcus gordonii, are considered beneficial for oral health. These organisms produce hydrogen peroxide (H(2)O(2)) at concentrations sufficient to kill many oral bacteria. Streptococci do not produce catalase yet tolerate H(2)O(2). We recently demonstrated that coaggregation with Actinomyces naeslundii stabilizes arginine biosynthesis in S. gordonii. Protein arginine residues are sensitive to oxidation by H(2)O(2). Here, the ability of A. naeslundii to protect S. gordonii against self-produced H(2)O(2) was investigated. Coaggregation with A. naeslundii enabled S. gordonii to grow in the absence of arginine, and promoted survival of S. gordonii following growth with or without added arginine. Arginine-replete S. gordonii monocultures contained 20-30 microM H(2)O(2) throughout exponential growth. Actinomyces naeslundii did not produce H(2)O(2) but synthesized catalase, removed H(2)O(2) from coaggregate cultures and decreased protein oxidation in S. gordonii. On solid medium, S. gordonii inhibited growth of A. naeslundii; exogenous catalase overcame this inhibition. In coaggregate cultures, A. naeslundii cell numbers were >90% lower than in monocultures after 24 h. These results indicate that coaggregation with A. naeslundii protects S. gordonii from oxidative damage. However, high cell densities of S. gordonii inhibit A. naeslundii. Therefore, H(2)O(2) may drive these organisms towards an ecologically balanced community in natural dental plaque.

Interaction of salivary alpha-amylase and amylase-binding-protein A (AbpA) of Streptococcus gordonii with glucosyltransferase of S. gordonii and Streptococcus mutans.

BACKGROUND: Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation by Streptococcus gordonii and Streptococcus mutans. The alpha-amylase-binding protein A (AbpA) of S. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs of S. gordonii and S. mutans. RESULTS: The addition of salivary alpha-amylase to culture supernatants of S. gordonii precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by S. gordonii (Gtf-G). rAbpA was expressed from an inducible plasmid, purified from Escherichia coli and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both S. gordonii and S. mutans. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of S. mutans Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A S. gordonii abpA-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva. CONCLUSION: The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization.

O-mannosylation in Candida albicans enables development of interkingdom biofilm communities.

Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. IMPORTANCE In the human mouth, microorganisms form communities known as biofilms that adhere to the surfaces present. Candida albicans is a fungus that is often found within these biofilms. We have focused on the mechanisms by which C. albicans becomes incorporated into communities containing bacteria, such as Streptococcus. We find that impairment of early stage addition of mannose sugars to C. albicans hyphal filament proteins deleteriously affects their subsequent performance in mediating formation of polymicrobial biofilms. Our analyses provide new understanding of the way that microbial communities develop, and of potential means to control C. albicans infections.

Collagen-binding streptococcal surface proteins influence the susceptibility of biofilm cells to endodontic antimicrobial solutions.

INTRODUCTION: Previous studies have indicated that the antimicrobial efficacy of endodontic irrigants may be diminished in the presence of patient tissues and fluids. With Streptococcus gordonii as a model microorganism, we used a genetic approach to investigate the hypothesis that bacterial surface proteins with collagen-binding abilities may function to protect biofilm cells from antiseptics commonly used in root canal treatment. METHODS: S. gordonii strain DL1 or isogenic mutant strains with deletions of genes encoding collagen-binding surface proteins were grown in microtiter plates to form 8-hour biofilms. Planktonic cells were aspirated, and the remaining biofilm cells were buffer-washed and then incubated with either pH-adjusted buffer or potentially protective solutions of type I collagen, serum, or saliva. Biofilms were rewashed, pulsed with sodium hypochlorite, chlorhexidine digluconate, or BioPure MTAD, and then rewashed. Fresh medium was added, and survivor cell growth was monitored for 24 hours. RESULTS: Buffer-treated biofilm cells of all 3 strains were similarly killed by sodium hypochlorite, chlorhexidine digluconate, and MTAD. Collagen, serum, and saliva significantly protected strain DL1 from all 3 antiseptics compared with buffer-treated cells (P ≤ .0004). However, preincubation with collagen, serum, or saliva left both mutant strain biofilms significantly more susceptible to all 3 antiseptics than were respectively treated strain DL1 biofilms (P ≤ .005). CONCLUSIONS: Interactions of S. gordonii surface proteins with collagen or similar components in serum and saliva may play roles in protecting biofilm cells from endodontic antiseptics. Elucidating molecular mechanisms underlying bacterial resistance to antimicrobials may facilitate the development of more effective treatments.

Streptococcus gordonii collagen-binding domain protein CbdA may enhance bacterial survival in instrumented root canals ex vivo.

INTRODUCTION: The surface-associated collagen-binding protein Ace of Enterococcus faecalis has been implicated as a virulence factor that contributes to bacterial persistence in endodontic infections. The purpose of this study was to determine if proteins with amino acid sequence similarity to Ace found in more abundant oral streptococci could play a similar role in potentially enhancing endodontic infections. METHODS: A Streptococcus gordonii gene similar to ace was identified by genome sequence searches in silico. An isogenic derivative of strain DL1 with a disruption in the identified gene was constructed by allelic replacement. Parent and mutant strains were characterized for their ability to bind immobilized collagen type 1 in a microtiter plate-binding assay. Survival of the strains in a human tooth ex vivo-instrumented root canal model was compared by inoculating canals with parental or mutant bacteria and determining the colony-forming units (CFUs) recovered at various time points over a 12-day period. RESULTS: The S. gordonii gene, encoding a protein with a conserved collagen-binding domain similar to that of Ace, was designated cbdA. The cbdA-deficient cells were less able to bind collagen type 1 than parental cells (P < .0001). Genetic complementation of the cbdA-deficient strain restored the collagen-binding phenotype. By day 12, significantly fewer (P = .03) cbdA-deficient than parental CFUs were recovered from instrumented canals. CONCLUSIONS: A gene encoding a putative collagen-binding protein was identified in S. gordonii. Fewer S. gordonii cbdA-deficient cells survived ex vivo compared with parental cells, suggesting that collagen-binding proteins may contribute to the persistence of oral streptococci in instrumented root canals.