Dental plaque represents one of the most complex microbial communities or biofilms known to afflict man. Oral biofilm-related diseases - dental carries, gingivitis and periodontitis - devastate a large human population and continue to pose a huge economic burden due to the lack of effective therapies. The long-term goal of this project is to elucidate the basic mechanisms of oral biofilm development and identify key players that may be attractive targets for developing drugs and vaccines. The development of dental plaque begins with the attachment of early bacterial colonizers to the tooth enamel, generating an adhesive matrix that then attracts the intermediate and late colonizers. Actinomyces is a key early colonizer that plays a prominent role in biofilm development by virtue of its ability to directly interact not only with the tooth surface but also with a number of both early and intermediate colonizers. Therefore, our studies have focused on dissecting the adhesive principles, i.e. fimbriae and other surface proteins, specifically involved in these interactions ad the mechanism of their assembly on the bacterial surface. During the past grant period, we succeeded in developing a facile new gene disruption technology for Actinomyces oris, and through it, identified the key components of two distinct fimbriae that are pivotal in the aforementioned cell-cell interactions. We showed that the tip fimbrillin FimQ serves dual functions, facilitating the assembly of type 1 fimbriae and directly mediating bacterial adherence to salivary proline-rich proteins known to coat the tooth surface. In contrast, the shaft fimbrilli FimA of the type 2 fimbriae mediates the receptor polysaccharide-dependent coaggregation with oral streptococci, adherence to erythrocytes and biofilm development. Structural studies revealed two adhesive IgG-like modules of FimA essential for its multivalent functions. Significantly, we showed that polymerization of these fimbrillins into fimbrial polymers requires their cognate fimbriae-specific sortase, a conserved transpeptidase in Gram-positive bacteria. The resulting polymers are anchored to the cell wall by the housekeeping sortase SrtA, which is also essential for the cell wall anchoring of many surface proteins with a cell wall sorting signal One of these, AcaF, is found to play a significant role in bacterial coaggregation. Most importantly, we discovered that inactivation of SrtA greatly perturbs bacterial morphology accompanied with abnormal cell wall and septa. Thus, the sortase machinery is a key player of bacterial pathogenesis and fitness in A. oris. Driven by these major advancements and several new hypotheses, the continuation proposal has three major aims: (1) Uncover the physiological function and regulation of sortase SrtA in cell surface homeostasis, (2) Dissect the molecular interactions of fimbrillins and non-fimbrial surface proteins with various cellular receptors, (3) Further elucidate the fundamental mechanisms of fimbrial assembly in Actinomyces, delineate the distinct mechanism of fimbrial assembly mediated by the tip fimbrillin FimQ, and identify trans- acting factors required for sortase-mediated fimbrial assembly.
Our studies focus on characterization of surface structures of oral bacteria Actinomyces that play an important role in the formation of oral biofilms or dental plaque. The novel findings emerging from these studies show great promise for the development of new classes of inhibitors which block the assembly of surface structures as one of the strategies for preventing oral biofilm-associated diseases.
|Chang, Chungyu; Amer, Brendan R; Osipiuk, Jerzy et al. (2018) In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking. Proc Natl Acad Sci U S A 115:E5477-E5486|
|Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E et al. (2018) Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii. J Bacteriol 200:|
|Wu, Chenggang; Al Mamun, Abu Amar Mohamed; Luong, Truc Thanh et al. (2018) Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC. MBio 9:|
|Wittchen, Manuel; Busche, Tobias; Gaspar, Andrew H et al. (2018) Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation. BMC Genomics 19:82|
|Juárez-Vázquez, Ana Lilia; Edirisinghe, Janaka N; Verduzco-Castro, Ernesto A et al. (2017) Evolution of substrate specificity in a retained enzyme driven by gene loss. Elife 6:|
|Sanchez, Belkys C; Chang, Chungyu; Wu, Chenggang et al. (2017) Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris. MBio 8:|
|Luong, Truc Thanh; Reardon-Robinson, Melissa E; Siegel, Sara D et al. (2017) Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris. J Bacteriol 199:|
|Siegel, Sara D; Reardon, Melissa E; Ton-That, Hung (2017) Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope. Curr Top Microbiol Immunol 404:159-175|
|Echelman, Daniel J; Alegre-Cebollada, Jorge; Badilla, Carmen L et al. (2016) CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks. Proc Natl Acad Sci U S A 113:2490-5|
|Wu, Chenggang; Reardon-Robinson, Melissa Elizabeth; Ton-That, Hung (2016) Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant. Methods Mol Biol 1440:109-22|
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