Biochemical activities of actinopain peptidases from actinomyces bacteria

Abstract

Calcium-dependent cysteine peptidases that belong to family C2 in the MEROPS database, are known as ‘Calpains’. These peptidases are widely distributed in eukaryotes, where their biochemical functions have been well studied. However, they are also present in bacteria, where their respective functions remain largely unexplored. To date, only the Thiol protease (Tpr) from Porphyromonas gingivalis has been functionally characterized and reported in the scientific literature.

In this thesis, the biochemical activities of several different calpain homologues encoded by oral Actinomyces bacteria were characterized. Bacteria belonging to the genus Actinomyces (and Schaalia) are abundant within the oral cavity, where they are generally associated with good oral health. However, some species of Actinomyces have been associated with oral diseases including dentin caries, and rare but serious infections called actinomycosis.

The taxonomic distributions and phylogenetic relationships between calpain peptidase homologues within Phylum Actinobacteria were analyzed. Results indicated calpains were widespread in taxa within the Actinomyces and Schaalia genera, with many strains encoding multiple different calpain homologues. Notably, the type strains of Actinomyces gerencseriae and Actinomyces viscosus encode 4 and 3 calpain homologues respectively (AgCP1-4, AvCP1-3).

The in vitro biochemical activities and proteolytic self-processing activities of AgCP1, AgCP2, and AgCP3 were investigated. AgCP1 lacked detectable self-digestion and hydrolytic activities. AgCP2 and AgCP3 both underwent proteolytic self-digestion in the presence of calcium ions forming several lower molecular weight protein products. AgCP3 hydrolyzed fluorogenic peptide substrates e.g. Z-Arg-Arg-AMC and Ala-Phe-Lys-AMC ca. 30-fold faster than AgCP2 (Z = benzyloxycarbonyl; AMC = 7amino-4-methylcoumarin). Both AgCP2 and AgCP3 cleaved peptide substrates at the C-terminal side of basic peptide motifs, e.g. ‘-Arg-Arg-’, ‘-Gly-Arg-’, ‘-Phe-Lys-’, ‘Phe-Arg’. Various single-site amino acid mutants of AgCP3 were created and biochemically characterized, including the cysteine, histidine and asparagine catalytic triad residues. All mutants retained catalytic activities to varying degrees, but some lost their self-processing activities.

The biochemical activities of wild-type AvCP3 and the C213A active-site mutant (AvCP3-C213A) were investigated. Wild-type AvCP3 exhibited self-processing activities similar to those of AgCP3, while AvCP3-C213A lacked autolytic activities. Transcriptional analysis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that all four calpain homologues in A. gerencseriae, and all three calpains in A. viscosus were expressed during anaerobic cultivation in nutritionally replete media. The peptidolytic activities of freshly-prepared cell extracts of diverse Actinomyces and Schaalia strains (including A. gerencseriae, A. israelii and A.viscosus) were investigated. Overall peptidolytic activities were generally low, and activities indicative of calpain peptidases were not detected.

In the final part of my thesis, initial investigations into the biochemical activities of a calpain homologue from Schaalia odontolytica, putative dipeptidyl peptidase (DAP2) and ‘papain-like’ C1 peptidase proteins from Actinomyces gerencseriae, and proline iminopeptidase (PIP) and U32 peptidases from Parvimonas micra, were conducted. These were selected for comparative purposes.

In conclusion, the results presented in this thesis greatly add to our general understanding of the biochemical activities and properties of calpain family proteins species of oral Actinomyces bacteria. Future studies are required to establish the biological functions of bacterial calpain homologues, which remain enigmatic.