The distributions and biochemical activities of calpain-family cysteine peptidases in oral actinomyces

Abstract

Calpains are a large and diverse family of calcium ion-dependent cysteine peptidases. They contain a conserved catalytic triad of Cys-His-Asn residues. Their function in bacteria remains largely unknown, as only a single bacterial calpain homologue, the thiol protease (Tpr) from Porphyromonas gingivalis has been characterized to date. Previous bioinformatic analysis has revealed that calpain peptidase homologues were present in several species (phylotypes) of oral Actinomyces bacteria. Actinomyces are normal inhabitants of the oral cavity but may also cause serious opportunistic infections, such as actinomycosis. In this thesis, I investigated the biochemical activities and biophysical properties of two calpain peptidase homologues respectively encoded by Actinomyces israelii and Actinomyces gerencseriae. In my first study, the biochemical activities and biophysical properties of the calpain-like cysteine peptidase encoded by Actinomyces israelii DSM 43320 (AiCP) were characterized in vitro. Three point-mutated forms of AiCP were constructed (Cys-226-Ser, Tyr-227-Phe and Asn-436-Asp), and their respective activities were studied in parallel. AiCP preferentially hydrolyzed peptides (peptide-like substrates) containing two consecutive basic residues: arginine (Arg) or lysine (Lys), optimally cleaving to the C-terminal side of ‘-Arg-Arg-’ motifs. It also cleaved the protein casein. The three point-mutated AiCP proteins exhibited similar hydrolytic activities. In the presence of calcium ions, the initially-expressed AiCP protein underwent autolytic (i.e. proteolytic self-digesting) processing to form two major fragments. The general location of the primary digestion site was identified using mass spectrometry. The Cys-226-Ser and Asn-436-Asp mutant forms of AiCP did not undergo self-digestion, but the Tyr-227-Phe mutant did. In my second study, the transcription of three calpain peptidase homologues encoded by Actinomyces gerencseriae ATCC 23860 (AgCPa, AgCPb, AgCPc) was analyzed by qRT-PCR. Under nutrient-limiting conditions, the three calpain homologue genes were up-regulated compared to during cultivation under nutrient-replete conditions. The biochemical activities and biophysical properties of AgCPa were characterized in vitro. AgCPa exhibited a preference for the hydrolysis of peptides containing ‘-Arg-Arg-’, ‘-Gly-Gly-Arg-’, or ‘-Ala-Phe-Lys-’ motifs; cleaving to the C-terminal side of these peptide motifs. Peptidolytic activities were strictly-dependent on calcium ions. The hydrolytic activities of AgCPa against the fluorogenic peptide-like substrate H-Arg-Arg-AMC were notably higher than those of AiCP. In the presence of calcium ions, two major fragments and one intermediate product were produced via the autolytic self-processing of the initially-expressed AgCPa protein. The Cys-226-Ser point-mutated form of AgCPa was constructed, expressed and characterized. The Cys-226-Ser mutant AgCPa protein did not undergo autolytic processing. In my final study, bioinformatic analyses were performed to investigate the distributions of calpain-like peptidases in Actinomyces taxa, and to analyze their domain structure. Detailed ‘BLAST’ searches followed by conserved domain database (CDD) searches led to the identification of 67 putatively-functional calpain peptidase homologues in the genus Actinomyces. The majority of the Actinomyces calpain peptidases lack identifiable signal peptide and thus may be cytoplasmic. In conclusion, calpain-like peptidases are widely distributed in Actinomyces taxa. Two calpain peptidases were functionally-characterized. Results presented in the thesis greatly expand our understanding of the biochemical activities of bacterial calpain peptidases, and lay the foundation for future studies.