Proper protein folding is critical to cellular function. Disulfide bond-forming machines that facilitate proper protein folding are well recognized in eukaryotes and Gram-negative bacteria. Disulfide bond formation contributes to the overall protein folding process, stabilizing structures and protecting against degradation. In Gram-negative bacteria, this process occurs in the oxidizing periplasmic space and is required a pair of oxidoreductase enzymes DsbA and DsbB. In contrast, little is known about oxidative protein folding in single- membrane Gram-positive bacteria, which are not considered to have periplasms. Specifically, how protein precursors translocated across the cytoplasmic membrane by the general secretion Sec translocon in an unfolded state manage to fold correctly is poorly understood. Recent findings of oxidoreductase-encoding genes in the genome of actinobacteria and Vitamin K epoxide reductase in Mycobacterium tuberculosis, considered as a functional homolog of Escherichia coli DsbB, offer some clue to an oxidative folding mechanism in these organisms. Therefore, our laboratory recently began to investigate this fundamental problem using an experimental model in Actinomyces oris, an actinobacterium known to play an important role in the formation of oral biofilms or dental plaque. By structural analysis, we identified disulfide bonds in FimA of A. oris. FimA is the fimbrial shaft required for biofilm formation and interspecies interactions. We demonstrated that the C-terminal disulfide bond of FimA is essential for fimbrial assembly and biofilm formation. More recently, we revealed that disruption of a disulfide bond in coaggregation factor CafA eliminates A. oris coaggregation with Streptococcus oralis. To find additional factors that affect interspecies interactions, we performed a large-scale screen with a Tn5 transposon mutant library in A. oris and identified coaggregation- defective mutants mapped to genes potentially encoding various components of an oxidative protein folding pathway. By using a multidisciplinary approach that combines genetics, biophysics, biochemistry, crystallography, mass spectrometry, cell-based assays, and models of dental caries and bacterial infection, we aim to elucidate the mechanism of oxidative protein folding in A. oris, to determine the conservation of this pathway in other actinobacteria, and to explore preventive strategies for dental caries and bacterial infections.
This study examines a pathway of how Gram-positive pathogens maintain proper folding of virulence factors during their assembly and explores strategies to block this pathway. A clear understanding of these aspects will provide new targets for anti-infective therapies and prevention of oral biofilm-associated diseases.
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