Streptococcus mutans is the etiological agent of dental caries costing billions of dollars annually in associated health care. Its acidogenic and aciduric (acid tolerant) properties result in pathogenesis. Of the physiological characteristics leading to disease, cytoplasmic membrane composition plays an integral role. Acid tolerance is mediated in large part by a membrane associated F1 Fo ATPase that pumps protons out of the cytoplasm. We demonstrated in S. mutans the dispensability of the signal recognition particle (SRP) co-translational protein translocation pathway, previously believed to be essential in every eukaryotic, archaeal and prokaryotic cell implying the existence of a back-up mechanism for membrane biogenesis and stress tolerance in this organism. Bacterial YidC is a member of the universally conserved Oxa1 /YidC/Alb3 (mitochondria/ bacterial/ chloroplast) family of membrane-associated chaperones and insertases. We identified two YidC orthologs in S. mutans. Yeast mitochondrial Oxa1 has a charged cytoplasmic tail and demonstrated ribosome binding and co-translational translocation functions. S. mutans YidC2 has a similar charged tail. Like SRP disruption, elimination of YidC2 results in stress (acid,osmotic, oxidative) sensitivity including decreased membrane associated ATPase activity in S. mutans. Interestingly, S. mutans YidC2 and YidC1 both can functionally replace E. coli YidC, including F1 Fo ATPase membrane insertion; however, in S. mutans elimination of YidC1 has no effect on acid or stress tolerance indicating organism specific differences in the context of the homologous secretion machinery. To date simultaneous elimination of YidC2 and SRP components, or YidC1 and YidC2, from S. mutans has not been achieved. A conditional expression system using the inulin inducible, glucose repressible fruA promoter will be developed to assess the relative roles of YidC1 and YidC2 in S. mutans membrane biogenesis. Domain swapping experiments also will be performed to begin to dissect the respective roles of the two proteins. Biogenesis of the F1 Fo ATPase will be investigated specifically by optimizing a PMF (proton motive force) assay for use with S. mutans inverted membrane vesicles and measurement of quenching of 9-amino-6- chloro-2-methoxyacridne (ACMA). F1Fo ATPase activity will be confirmed using specific inhibitors, orthovanadate for P-type, and oligomycin to F-type ATPases. Lastly, co-immunoprecipitation and chemical crosslinking will evaluate YidC1 and YidC2 interactions with each other and other components of the protein secretion apparatus including the SecY-containing membrane translocon pore.
this research will further our understanding of S. mutans virulence mechanisms and suggest novel therapeutic targets. ? ? ?
| Palmer, Sara R; Crowley, Paula J; Oli, Monika W et al. (2012) YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans. Microbiology 158:1702-12 |
