title: RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.
abstract: Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.
authors.affiliation: The Center for Neuroscience and Cell Biology (CNC), Coimbra, Portugal; Biocant, Biotechnology Innovation Center, Cantanhede, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Cruz
authors.affiliation: Centre for Blood Research and Department of Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Pitter
authors.initials: P
authors.lastname: Huesgen
authors.affiliation: Vector-Borne Diseases Laboratories, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Riley
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland, United States of America.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.affiliation: The Center for Neuroscience and Cell Biology (CNC), Coimbra, Portugal; Biocant, Biotechnology Innovation Center, Cantanhede, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Faro
authors.affiliation: Centre for Blood Research and Department of Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.affiliation: Vector-Borne Diseases Laboratories, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, United States of America.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Martinez
authors.affiliation: The Center for Neuroscience and Cell Biology (CNC), Coimbra, Portugal; Biocant, Biotechnology Innovation Center, Cantanhede, Portugal.
authors.forename: Isaura
authors.initials: I
authors.lastname: Simões
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D019295:Computational Biology; D005434:Flow Cytometry; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020114:Rickettsia conorii / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D057055:Aspartic Acid Proteases