An in vitro model for studying the contributions of the Streptococcus mutans glucan-binding protein A to biofilm structure.

The method described here for analyzing biofilms was sensitive enough to allow the detection of differences formed by pure cultures of S. mutans or a GbpA knockout strain. Other strains have also been tested, and the differences in biofilm structure were sometimes even more extensive (data not shown). The advantages of this method are that it is quick, inexpensive, and adaptable to almost any laboratory setting. The constant rotation of the cultures, which was employed to simulate salivary flow, appears to be a critical element for establishing biofilm differences. An analysis of protein profiles confirmed that the biofilm bacteria were metabolically distinct from the planktonic phase bacteria. For the strains tested, the variations in biofilm architecture could be visualized with or without magnification. Staining of the bacteria was not required, though we typically stained the biofilms with either crystal violet or Schiff's reagent. Altogether, this in vitro method for generating biofilms allowed the evaluation of visual, quantitative (confocal microscopy), and functional (antimicrobial susceptibility) differences. We have employed these methods in a reductionist approach to understanding the contribution of individual proteins to dental plaque development. These methods may also be useful in the screening of mutants that would be of greatest for testing in multispecies biofilms, animal models, or more complex biofilm models.

The TprK protein of Treponema pallidum is periplasmic and is not a target of opsonic antibody or protective immunity.

The finding that Treponema pallidum, the syphilis spirochete, contains 12 orthologs of the Treponema denticola outer membrane major sheath protein has engendered speculation that members of this T. pallidum repeat (Tpr) family may be similarly surface exposed. In this regard, the TprK protein was reported to be a target of opsonic antibody and protective immunity and subject to immunologically driven sequence variation. Despite these findings, results from our previous analyses of treponemal outer membranes in concert with computer-based predictions for TprK prompted us to examine the cellular location of this protein. TprK-alkaline phosphatase fusions expressed in Escherichia coli demonstrate that TprK contains a signal peptide. However, opsonophagocytosis assays failed to indicate surface exposure of TprK. Moreover, results from three independent methodologies, i.e., (a) indirect immunofluorescence analysis of agarose-encapsulated organisms, (b) proteinase K treatment of intact spirochetes, and (c) Triton X-114 phase partitioning of T. pallidum conclusively demonstrated that native TprK is entirely periplasmic. Consistent with this location, immunization with the recombinant protein failed to induce either protective immunity or select for TprK variants in the rabbit model of experimental syphilis. These findings challenge the notion that TprK will be a component of an efficacious syphilis vaccine.

Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence and promotes in vivo accumulation of recombinations between the glucosyltransferase B and C genes.

Glucan-binding protein A (GbpA) of Streptococcus mutans has been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenic gbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutans was grown in the presence of [3H] thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. The gbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB and gtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.