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The role of autophagy in the intracellular survival of Campylobacter concisus.

Burgos-Portugal JA, Mitchell HM, Castaño-Rodríguez N, Kaakoush NO - FEBS Open Bio (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

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.

No MeSH data available.


Related in: MedlinePlus

Relative intracellular percentage of C. concisus UNSWCD following autophagy inhibition and induction. Errors are presented as Standard Error of the Mean (SEM) based on a minimum of four biological replicates. 3-MA: 3-methyladenine; CQD: 50 μM chloroquine diphosphate; rapamycin: 200 nM rapamycin.
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f0005: Relative intracellular percentage of C. concisus UNSWCD following autophagy inhibition and induction. Errors are presented as Standard Error of the Mean (SEM) based on a minimum of four biological replicates. 3-MA: 3-methyladenine; CQD: 50 μM chloroquine diphosphate; rapamycin: 200 nM rapamycin.

Mentions: The intracellular levels of C. concisus UNSWCD 6 h post-infection were similar to previous results obtained for this strain [19,20], with the intracellular percentage (number of internalized bacteria/number of bacteria added) of the bacterium being 0.44 ± 0.04% (Fig. 1), and the total number of intracellular bacteria being 5.1 × 105 ± 1.0 × 105 CFU ml−1 within the 5 × 105 host cells seeded onto the plate. This level of invasion is similar to levels of invasion observed for C. jejuni[26]. Upon addition of the autophagy inhibitor 3-MA at a concentration of 5 mM, the intracellular level of C. concisus UNSWCD increased approximately 2.2-fold. The addition of 10 mM 3-MA showed an even greater increase (approximately 3.5-fold, intracellular percentage: 1.54 ± 0.08%) in intracellular bacteria (Fig. 1). These findings were confirmed by employing alternative autophagy inhibitors, whereby the intracellular levels of C. concisus UNSWCD increased 2.0 and 2.8-fold upon inhibition of autophagy with 10 nM bafilomycin A-1 and 100 nM wortmannin, respectively (Fig. 1). Statistical analysis performed through a One-way ANOVA with a Dunnett’s post hoc test showed a statistically significant increase (P < 0.05) for 5 and 10 mM 3-MA, 10 nM bafilomycin A-1 and 100 nM wortmannin when compared to the non-treated control.


The role of autophagy in the intracellular survival of Campylobacter concisus.

Burgos-Portugal JA, Mitchell HM, Castaño-Rodríguez N, Kaakoush NO - FEBS Open Bio (2014)

Relative intracellular percentage of C. concisus UNSWCD following autophagy inhibition and induction. Errors are presented as Standard Error of the Mean (SEM) based on a minimum of four biological replicates. 3-MA: 3-methyladenine; CQD: 50 μM chloroquine diphosphate; rapamycin: 200 nM rapamycin.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048850&req=5

f0005: Relative intracellular percentage of C. concisus UNSWCD following autophagy inhibition and induction. Errors are presented as Standard Error of the Mean (SEM) based on a minimum of four biological replicates. 3-MA: 3-methyladenine; CQD: 50 μM chloroquine diphosphate; rapamycin: 200 nM rapamycin.
Mentions: The intracellular levels of C. concisus UNSWCD 6 h post-infection were similar to previous results obtained for this strain [19,20], with the intracellular percentage (number of internalized bacteria/number of bacteria added) of the bacterium being 0.44 ± 0.04% (Fig. 1), and the total number of intracellular bacteria being 5.1 × 105 ± 1.0 × 105 CFU ml−1 within the 5 × 105 host cells seeded onto the plate. This level of invasion is similar to levels of invasion observed for C. jejuni[26]. Upon addition of the autophagy inhibitor 3-MA at a concentration of 5 mM, the intracellular level of C. concisus UNSWCD increased approximately 2.2-fold. The addition of 10 mM 3-MA showed an even greater increase (approximately 3.5-fold, intracellular percentage: 1.54 ± 0.08%) in intracellular bacteria (Fig. 1). These findings were confirmed by employing alternative autophagy inhibitors, whereby the intracellular levels of C. concisus UNSWCD increased 2.0 and 2.8-fold upon inhibition of autophagy with 10 nM bafilomycin A-1 and 100 nM wortmannin, respectively (Fig. 1). Statistical analysis performed through a One-way ANOVA with a Dunnett’s post hoc test showed a statistically significant increase (P < 0.05) for 5 and 10 mM 3-MA, 10 nM bafilomycin A-1 and 100 nM wortmannin when compared to the non-treated control.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

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.

No MeSH data available.


Related in: MedlinePlus