John O. Cisar

Coaggregation-Mediated Interactions of Streptococci and Actinomyces Detected in Initial Human Dental Plaque

Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria. These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque. To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips. In intrageneric coaggregation, streptococci such as Streptococcus gordonii DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as Streptococcus oralis 34. To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against S. gordonii DL1 (anti-DL1). These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque. Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains. In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci. Cells reactive with antibody against type 2 fimbriae of Actinomyces naeslundii T14V (anti-type-2) were much less frequent than either subset of streptococci. However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells. These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo. Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.

Identification and Characterization of hsa, the Gene Encoding the Sialic Acid-Binding Adhesin of Streptococcus gordonii DL1

ABSTRACT Oral colonization by Streptococcus gordonii, an important cause of subacute bacterial endocarditis, involves bacterial recognition of sialic acid-containing host receptors. The sialic acid-binding activity of this microorganism was previously detected by bacterium-mediated hemagglutination and associated with a streptococcal surface component identified as the Hs antigen. The gene for this antigen (hsa) has now been cloned in Escherichia coli, and its expression has been detected by colony immunoblotting with anti-Hs serum. Mutants of S. gordonii containing hsa inactivated by the insertion of an erythromycin resistance gene or deletion from the chromosome were negative for Hs-immunoreactivity, bacterium-mediated hemagglutinating activity, and adhesion to α2-3-linked sialoglycoconjugates. The deletion in the latter mutants was complemented by plasmid-borne hsa, resulting in Hs antigen production and the restoration of cell surface sialic acid-binding activity. The hsa gene encodes a 203-kDa protein with two serine-rich repetitive regions in its 2,178-amino-acid sequence. The first serine-rich region occurs within the amino-terminal region of the molecule, between different nonrepetitive sequences that may be associated with sialic acid binding. The second serine-rich region, which is much longer than the first, is highly repetitive, containing 113 dodecapeptide repeats with a consensus sequence of SASTSASVSASE. This long repetitive region is followed by a typical gram-positive cell wall anchoring region at the carboxyl-terminal end. Thus, the predicted properties of Hsa, which suggest an amino-terminal receptor-binding domain attached to the cell surface by a molecular stalk, are consistent with the identification of this protein as the sialic acid-binding adhesin of S. gordonii DL1.

Characterization of a Streptococcus sp.-Veillonella sp. Community Micromanipulated from Dental Plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.

Functional Analysis of the Streptococcus gordonii DL1 Sialic Acid-Binding Adhesin and Its Essential Role in Bacterial Binding to Platelets

ABSTRACT Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of Streptococcus gordonii DL1 was previously associated with the hsa gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an hsa chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing hsa constructs encoding Hsa that lacked either the N- or C-terminal portion of SR2. In contrast, hsa constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by S. gordonii DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of hsa. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction.

Sortase-Catalyzed Assembly of Distinct Heteromeric Fimbriae in Actinomyces naeslundii

Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.

Salivary Receptors for the Proline-rich Protein-binding and Lectin-like Adhesins of Oral Actinomyces and Streptococci

Colonization of the tooth surface by actinomyces and viridans group streptococci involves the attachment of these bacteria to adsorbed salivary components of the acquired enamel pellicle. The hypothesis that this attachment depends on specific adhesins has now been assessed from the binding of bacteria with well-defined adhesive properties to blots of SDS-PAGE-separated parotid and submandibular-sublingual (SM-SL) saliva. Streptococcus sanguis and type 2 fimbriated Actinomyces naeslundii, which bound terminal sialic acid and Galβ1-3GalNAc, respectively, recognized only a few SM-SL salivary components, primarily MG2. In contrast, type 1 fimbriated A. naeslundii and S. gordonii, which bound purified proline-rich proteins (PRPs), recognized several other components from both SM-SL and parotid saliva. Significantly, bacteria that lacked PRP-binding and the lectin-like activities detected by binding to MG2 failed to bind any immobilized salivary component. These findings suggest the involvement of specific adhesins in bacterial recognition of many adsorbed salivary proteins and glycoproteins.

The Actinomyces oris type 2 fimbrial shaft FimA mediates co-aggregation with oral streptococci, adherence to red blood cells and biofilm development.

Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin‐like activity associated with the Actinomyces oris type 2 fimbria, a surface structure assembled by sortase (SrtC2)‐dependent polymerization of the shaft and tip fimbrillins, FimA and FimB respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 fimbriae composed only of FimA, like the wild type co‐aggregated strongly with receptor‐bearing streptococci, agglutinated with sialidase‐treated red blood cells, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 fimbriae and were non‐adherent in each of these assays. Plasmid‐based expression of the deleted gene in respective mutants restored adherence to wild‐type levels. These findings uncover the importance of the lectin‐like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation.

Molecular Cloning and Characterization of Genes for Shigella sonnei Form I O Polysaccharide: Proposed Biosynthetic Pathway and Stable Expression in a Live Salmonella Vaccine Vector

ABSTRACT The gene region for biosynthesis of Shigella sonnei form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of Salmonella-based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS630. A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS91 and IS630) and wzz upstream of the form I coding region and a fragment of aqpZ and additional full or partial IS elements (i.e., IS629, IS91, and IS911) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS91 from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated Shigella and Escherichia coli extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated Salmonella enterica serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent S. sonnei challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis.

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

By combining X‐ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor‐dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG‐like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram‐positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell–cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram‐positive fimbriae.

Identification of Polymorphonuclear Leukocyte and HL-60 Cell Receptors for Adhesins of Streptococcus gordonii and Actinomyces naeslundii

ABSTRACT Interactions of oral streptococci and actinomyces with polymorphonuclear leukocytes (PMNs), mediated by sialic acid- and Gal/GalNAc-reactive adhesins, respectively, result in activation of the PMNs and thereby may contribute to the initiation of oral inflammation. Sialidase treatment of PMNs or HL-60 cells abolished adhesion ofStreptococcus gordonii but was required for adhesion ofActinomyces naeslundii. The same effects of sialidase were noted for adhesion of these bacteria to a major 150-kDa surface glycoprotein of either PMNs or undifferentiated HL-60 cells and to a 130-kDa surface glycoprotein of differentiated HL-60 cells. These glycoproteins were both identified as leukosialin (CD43) by immunoprecipitation with a specific monoclonal antibody (MAb). Adhesion of streptococci and actinomyces to a 200-kDa minor PMN surface glycoprotein was also detected by bacterial overlay of untreated and sialidase-treated nitrocellulose transfers, respectively. This glycoprotein was identified as leukocyte common antigen (CD45) by immunoprecipitation with a specific MAb. CD43 and CD45 both possess extracellular mucinlike domains in addition to intracellular domains that are implicated in signal transduction. Consequently, the interactions of streptococci and actinomyces with the mucinlike domains of these mammalian cell surface glycoproteins result not only in adhesion but, in addition, may represent the initial step in PMN activation by these bacteria.