RESUMEN
Periodontitis is a progressive inflammatory disease that affects roughly half of American adults. Colonization of the oral cavity by the Gram-negative bacterial pathogen Porphyromonas gingivalis is a key event in the initiation and development of periodontal disease. Adhesive surface structures termed fimbriae (pili) mediate interactions of P. gingivalis with other bacteria and with host cells throughout the course of disease. The P. gingivalis fimbriae are assembled via a novel mechanism that involves proteolytic processing of lipidated precursor subunits and their subsequent polymerization on the bacterial surface. Given their extracellular assembly mechanism and central roles in pathogenesis, the P. gingivalis fimbriae are attractive targets for anti-infective therapeutics to prevent or treat periodontal disease. Here we confirm that conserved sequences in the N and C termini of the Mfa1 fimbrial subunit protein perform critical roles in subunit polymerization. We show that treatment of P. gingivalis with peptides corresponding to the conserved C-terminal region inhibits the extracellular assembly of Mfa fimbriae on the bacterial surface. We also show that peptide treatment interferes with the function of Mfa fimbriae by reducing P. gingivalis adhesion to Streptococcus gordonii in a dual-species biofilm model. Finally, we show that treatment of bacteria with similar peptides inhibits extracellular polymerization of the Fim fimbriae, which are also expressed by P. gingivalis These results support a donor strand-based assembly mechanism for the P. gingivalis fimbriae and demonstrate the feasibility of using extracellular peptides to disrupt the biogenesis and function of these critical periodontal disease virulence factors.
Asunto(s)
Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Porphyromonas gingivalis/fisiología , Biopelículas , Escherichia coli/metabolismo , Proteínas Fimbrias/genética , Porphyromonas gingivalis/citología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: The gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells. RESULTS: We report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation. CONCLUSION: Taken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions.