title: The role of autophagy in the intracellular survival of Campylobacter concisus.
abstract: Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Burgos-Portugal
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Mitchell
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Castaño-Rodríguez
authors.affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
authors.forename: Nadeem O
authors.initials: NO
authors.lastname: Kaakoush
keywords: Autophagy; CCV, Campylobacter-containing vacuole; CD, Crohn’s disease; CQD, chloroquine diphosphate; Campylobacter concisus; Ct, threshold cycle; IBD, inflammatory bowel diseases; Intracellular survival; MOI, Multiplicity of Infection; Microscopy; Plasmid; TEM, transmission electron microscopy