UCLA

Bacteria utilize proteins at their surface for a multitude of processes including adhesion, biofilm formation, motility, and virulence. Thus, understanding the biogenesis and surface display of these factors is instrumental in our understanding of bacterial pathogenesis and virulence mechanisms. Within this dissertation we describe the identification and characterization of a newly identified peptide which is functionally conserved amongst Actinobacteria and serves to modulate anchoring of proteins to the cell wall through modulation of membrane homeostasis of the housekeeping sortase. In the oral cavity associated bacterial species, Actinomyces oris, we identified a small peptide consisting of 52 amino acids which is encoded directly downstream of the gene encoding the housekeeping sortase SrtA. Henceforth we refer to this peptide as SafA for Sortase Associated Factor A. Firstly, through bioinformatic analysis we found that nearly all Actinobacteria encode a SafA homolog immediately downstream of their respective housekeeping sortase genes, with the exception of Bifidobacterium dentium in which the genome does not contain a separate SafA reading frame, but rather the C-terminus of the housekeeping sortase harbors a domain homologous to SafA in A. oris. In A. oris we found that deletion of safA results in phenotypes consistent with deletion of the housekeeping sortase itself, which include the formation of abnormally long pili as detected by electron microscopy and the failure of A. oris to interact with another oral bacterial species Streptococcus oralis. Cellular fractionation and immunoblotting revealed that in the absence of SafA, SrtA is cleaved and released into the extracellular milieu. While software predictions did not identify a signal peptide sequence in SrtA, manual amino acid sequence, sequence analysis did in fact reveal that SrtA contains a tripartite domain consistent with a type I signal peptide sequence and a predicted cleavage site between A56 and S57. Edman degradation amino acid sequencing confirmed this cleavage site and mutational analysis revealed that the signal peptidase LepB2 is responsible for this observed cleavage of SrtA. To elucidate how SafA protects SrtA from cleavage we utilized a Bacterial Adenylate Cyclase Two-Hybrid system which demonstrated that SafA and SrtA directly interact. Furthermore, we identified a three amino acid domain in SafA consisting of FPW residues which is essential for mediating this interaction. Finally, we found that ectopic expression of SafA from A. oris, Corynebacterium diphtheriae, and Corynebacterium matruchotii rescued the aforementioned functional defects of the safA mutant of A. oris, thus supporting the conclusion that SafA is both functionally and evolutionarily conserved. The findings described herein demonstrate a new paradigm for the modulation surface protein display in Actinobacteria. The conservation of SafA across Actinobacteria coupled with the essential role for sortases in mediating anchoring of pili and key virulence factors provides a unique target and opportunity to inhibit the virulence of Actinobacteria species.