• Multispecies oral biofilms studied at the single community level as a model system for spatiotemporal development of biofilms and interspecies interactions

      Chalmers, Natalia I.; Kolenbrander, Paul E.; Bavoil, Patrik M. (2008)
      Oral biofilms are multispecies communities that are important in the development of the two most prevalent oral diseases---dental caries and periodontal disease. The primary initial colonizers of human enamel are streptococci, veillonellae and actinomyces. Bacteria of these genera coaggregate (cell-cell interactions between genetically distinct bacteria). The streptococci can be classified into two groups: those with receptor polysaccharides (RPS) and those with adhesins that recognize RPS. RPS mediates streptococcal intrageneric coaggregation. Besides coaggregation, veillonellae interact with streptococci metabolically by using lactic acid produced by streptococci. The hypothesis tested in this study is that coaggregation mediated interactions between streptococci and veillonellae are important for the development of oral biofilms in vivo. To translate naturally occurring interactions from undisturbed dental plaque to multispecies in vitro communities, a simple community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by immunofluorescence with quantum-dot-conjugated antibodies and micromanipulated from the tooth surface. Characterization of the application of quantum-dot primary immunofluorescence was accomplished and was essential for the success of this approach. An antibody-unreactive streptococcus invisible during micromanipulation was also obtained. The streptococci were identified as Streptococcus oralis and Streptococcus gordonii. The veillonellae could not be cultured; however, immunoreactive veillonellae cells were present in the original mixture and a Veillonella 16S-rRNA gene sequence was amplified. This sequence was similar to Veillonella sp. PK1910. The two streptococci coaggregated by an RPS-dependent mechanism, and both coaggregated with Veillonella sp. PK1910. Veillonella sp. PK1910 was used as a surrogate in in vitro community reconstruction. In vitro the two streptococcal isolates and Veillonella sp. PK1910 grew on saliva as the sole nutritional source and formed interdigitated multispecies clusters. Veillonella sp. PK1910 grew only in biofilms where streptococci were present. To map the spatial relationship between these species in vivo we used a novel approach integrating immunofluorescence and fluorescence in situ hybridization. Reconstruction in vitro of a community composed of Veillonella sp. PK1910, S. oralis, and S. gordonii demonstrates the vital role of RPS-mediated coaggregation in initial colonization of multispecies communities. The integrated in vitro - in vivo approach serves as a new paradigm for the study of oral biofilms and their development.
    • Remodeling of the Chlamydia psittaci inclusion by the type III secreted, BAR domain-containing inclusion membrane protein IncA

      Phillips, Daniel Austin; Bavoil, Patrik M. (2016)
      Chlamydia species cause infections in humans that range from prevalent, often asymptomatic genital infections caused by Chlamydia trachomatis to relatively rare, potentially life-threatening zoonotic infections caused by avian Chlamydia psittaci. All Chlamydia spp. encode integral membrane proteins (Incs) that are type III-secreted through the injectisome from the bacterial cell into the host-derived inclusion membrane where they interact with host proteins. These include Incs of C. trachomatis that interact with membrane curvature-inducing BAR-domain sorting nexins (SNX), potentially disrupting retromer-mediated endosome-to-Golgi trafficking to benefit the pathogen. Eukaryotic BAR domain proteins interact with bacterial proteins, but prokaryotic BAR domain proteins have yet to be confirmed. I propose that IncA/Cps, the first BAR protein described in a prokaryote, specifically contributes to the remodeling of the C. psittaci inclusion membrane to form a) IncA-laden retromer-like tubules extending outward into the cytosol, and/or b) folds and tubular extensions extending inward into the inclusion lumen. IncA of C. psittaci (IncA/Cps) contains a predicted SNX-BAR-like domain that is absent in the C. trachomatis ortholog. Comparative sequence analysis of IncA orthologs across Chlamydia spp. suggests that the SNX-BAR-like domain of IncA/Cps was acquired by convergent evolution. IncA/Cps BAR-mediated in vitro tubulation of liposomes was observed. Transfected HEK293 cells expressed filamentous/tubular structures laden with IncA/Cps. These observations suggest that IncA/Cps has membrane remodeling activity. During C. psittaci infection, IncA/Cps localized to the inclusion membrane and to nocodazole-sensitive retromer-like tubules extending from the inclusion membrane into the host cytosol. IncA-specific immunofluorescence staining of the C. psittaci inclusion displayed a characteristic irregular configuration with concave pits and folds extending into the inclusion lumen, sharply contrasting the characteristic ovoid shape of the C. trachomatis inclusion. In addition, abundant luminal IncA/Cps staining did not colocalize with growing chlamydiae and was sensitive to host sphingolipid biosynthesis inhibition. This suggests that luminal IncA/Cps is structurally continuous with the inclusion membrane. Inward extensions may enhance inclusion membrane contact-dependent growth of C. psittaci and eventually infectious progeny per infected cell. The higher infectious load may contribute to C. psittaci-induced severe pathologies and high transmission rates between birds and to humans.