Enhanced Biofilm Formation and Loss of Capsule Synthesis: Deletion of a Putative Glycosyltransferase inPorphyromonas gingivalis

Abstract

Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity (ϳ61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased autoaggregation. Our results indicate that we have identified a gene involved in capsularpolysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.The clinical importance of dental plaque and its accessibility for in vivo research makes it one of most studied and bestunderstood biofilm communities. It is now well documented that gram-negative anaerobic bacteria play a significant role in the development of periodontitis, with Porphyromonas gingivalis being implicated as one of the major players in the progression of this chronic disease (10,19,30,61). A number of factors are associated with the virulence of the organism, including a variety of proteases, endotoxins, and collegenases and the production of surface structures, such as fimbriae and capsular polysaccharide (38). This opportunistic pathogen also attaches to and invades human epithelial cells (18,53,56,57), connective tissue, and endothelial cells (15,17,55), and invasion has been shown to be mediated by the expression of fimbriae and a variety of surface adhesins. It is evident that the growth of P. gingivalis within the subgingival plaque is central to the disease process; however, although there have been numerous studies of the pathogenicity of the organism and its interactions with other organisms within the biofilm community, little is known about the molecular-genetic basis of biofilm formation in P. gingivalis.Research on oral biofilms has focused on determining the spatial organization and complex development of plaque by examining the succession of organisms in the growing biofilm. By monitoring the development of the microbial community on the tooth surface after professional cleaning, studies have demonstrated that the early colonizers are primarily grampositive organisms and the late, o...

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

“…Unless otherwise stated, all chemicals were obtained from Sigma. Plasmids were constructed in E. coli DH5a or BL21DE3 and then electroporated into P. gingivalis strain W83 using previously described methods (AlbertiSegui et al, 2010;Davey & Duncan, 2006). P1 transduction, using hup alleles provided by Oberto et al (2009), was performed as described by Thomason et al (2007) to isolate the various hup mutants.…”

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