Functional characterization of oligopeptidases from oral Capnocytophaga

Abstract

Periodontal diseases have a complex, mixed-species bacterial etiology. Established ‘periodontopathogenic’ bacteria such as Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola express high levels of various peptidolytic (protein/peptide-cleaving) enzymes that play key roles in periodontal disease pathogenesis. ‘Trypsin-like’ activities, i.e. the cleavage of peptide chains after arginine or lysine residues, is one such activity. Trypsin-like activities are mediated by several different enzyme families including Oligopeptidase B (OpdB), a member of the S9A family of serine peptidases. The opportunistic pathogen Capnocytophaga gingivalis, exhibits high levels of peptidolytic activities, unlike commensal species of oral Capnocytophaga bacteria, such as Capnocytophaga ochracea. Interestingly, C. gingivalis encodes two OpdB homologues (Cg-OpdB1, Cg-OpdB2) as well as two other closely-related S9A serine peptidases (Cg-POP1, Cg-POP2), which are predicted to function as prolyl oligopeptidases (POP): enzymes that hydrolyze peptide chains after proline residues. Here, I studied the biochemical functions of Cg-OpdB1, Cg- OpdB2, Cg-POP1, Cg-POP2, and compared and contrasted their respective activities against OpdB and POP homologues encoded by other oral bacteria including T. denticola (Td-OpdB, Td-POP), T. forsythia (Tf-OpdB), C. ochracea (Co-OpdB, Co-POP), and Porphyromonas catoniae (Pc-OpdB). The arginine and lysine-specific peptidolytic activities of Cg-OpdB2 were notably higher than those of Cg-OpdB1, and the four other OpdB homologues characterized. Transcriptional and cell phenotype analysis revealed that C. gingivalis and the other Capnocytophaga taxa that encoded two opdB genes had higher cellular trypsin-like activities than Capnocytophaga species encoding one opdB gene. The biochemical activities of Td-POP and Co-POP were analyzed in detail. Td-POP showed higher prolyl oligopeptidase activities than Co-POP. Both Td-POP and Co-POP also cleaved peptide chains after alanine residues, albeit at considerably lower efficiencies. Cg-POP1 and Cg-POP2 exhibited strong autoproteolytic activities, efficiently cleaving-off N-terminal affinity tags, hindering their biochemical characterization. Results from transcriptional and cell phenotype analysis indicated that C. gingivalis and other Capnocytophaga taxa that encoded two pop homologues exhibited higher prolyl oligopeptidase activities. The function of OpdB and POP were explored by constructing individual ΔopdB and Δpop (deletion) mutants in C. ochracea. Results revealed the pop gene was solely responsible for prolyl oligopeptidase activities in C. ochracea. The opdB gene was not responsible for all arginine and lysine-specific oligopeptidase activities, implying other peptidases, putatively aminopeptidases, contributed to trypsin-like activities in C. ochracea. I explored the genomic distributions, phylogeny and genetic contexts of OpdB and POP homologues across the phylum Bacteroidetes. Cg-OpdB2 shared (predicted) evolutionary history with OpdB homologues present in other Capnocytophaga species, whilst Cg-OpdB1 was more closely related to OpdB present in T. denticola, Porphyromonas and Tannerella taxa. The POP proteins exhibited a more complex phylogenetic pattern. C. gingivalis was a notable outlier in the genus Capnocytophaga, in terms of its genomic arrangements of pop and opdB homologues. In conclusion, taxa within the phylum Bacteroidetes exhibit notable differences in their respective distributions of opdB and pop gene homologues, and these oligopeptidases exhibit notable variations in their respective biochemical activities. Taken together, the results presented in this thesis greatly add to our general understanding of the biochemical functions of S9 serine peptidases in oral bacteria.