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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

Visualization of the co-localization of C. concisus with autophagosomes using confocal microscopy. (A) Untreated Caco-2 cells, (B–J) Caco-2 cells infected with C. concisus UNSWCD. LC3B was stained in red, C. concisus was stained in green. C. concisus UNSWCD was found to aggregate and adhere to Caco-2 cells, internalize into Caco-2 cells, and co-localize with the LC3B antibody (B–J).
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f0020: Visualization of the co-localization of C. concisus with autophagosomes using confocal microscopy. (A) Untreated Caco-2 cells, (B–J) Caco-2 cells infected with C. concisus UNSWCD. LC3B was stained in red, C. concisus was stained in green. C. concisus UNSWCD was found to aggregate and adhere to Caco-2 cells, internalize into Caco-2 cells, and co-localize with the LC3B antibody (B–J).

Mentions: Autophagosomes were detected through the presence of LC3B in Caco-2 cells distributed within the cytoplasm and near the membrane regions of the cells (Fig. 4A), indicating that autophagy is an active process that normally occurs within Caco-2 cells. Inhibition of the autophagy process using 3-MA at 5 and 10 mM resulted in downregulation of autophagosome formation in Caco-2 cells (Fig. S1). In contrast, the density of LC3B detected following induction of autophagosomes with 50 μM CQD was higher than that observed in the Caco-2 cells without chemical treatment (Fig. S1), consistent with the activity of these compounds. Following infection of Caco-2 cells with C. concisus UNSWCD, the bacterium was found to co-localize with LC3B (Fig. 4B–I). Moreover, some cells were found to be hyperinfected by C. concisus UNSWCD (Fig. 4F, J). Calculation of the co-localization coefficient for the C. concisus antibody and the LC3B antibody showed the coefficient to range from 0.54 to 0.90, indicating that significant co-localization between the two antibodies existed. The fact that C. concisus UNSWCD interacts with the autophagosome, provides further evidence that autophagy is involved in the intracellular survival of C. concisus within host cells, and raises the possibility that C. concisus could employ autophagy to survive intracellularly. A similar observation has been made with C. jejuni, with the bacterium co-localizing with GFP-LC3-labeled structures consistent with autophagosomes [28]. In a recent study, Lam et al. reported that Listeria monocytogenes co-localizes with LC3 [34], a finding that led them to suggest that this co-localization gives rise to spacious Listeria-containing phagosomes, membrane-bound compartments that harbor slow-growing bacteria associated with persistent infection [34].


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)

Visualization of the co-localization of C. concisus with autophagosomes using confocal microscopy. (A) Untreated Caco-2 cells, (B–J) Caco-2 cells infected with C. concisus UNSWCD. LC3B was stained in red, C. concisus was stained in green. C. concisus UNSWCD was found to aggregate and adhere to Caco-2 cells, internalize into Caco-2 cells, and co-localize with the LC3B antibody (B–J).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Visualization of the co-localization of C. concisus with autophagosomes using confocal microscopy. (A) Untreated Caco-2 cells, (B–J) Caco-2 cells infected with C. concisus UNSWCD. LC3B was stained in red, C. concisus was stained in green. C. concisus UNSWCD was found to aggregate and adhere to Caco-2 cells, internalize into Caco-2 cells, and co-localize with the LC3B antibody (B–J).
Mentions: Autophagosomes were detected through the presence of LC3B in Caco-2 cells distributed within the cytoplasm and near the membrane regions of the cells (Fig. 4A), indicating that autophagy is an active process that normally occurs within Caco-2 cells. Inhibition of the autophagy process using 3-MA at 5 and 10 mM resulted in downregulation of autophagosome formation in Caco-2 cells (Fig. S1). In contrast, the density of LC3B detected following induction of autophagosomes with 50 μM CQD was higher than that observed in the Caco-2 cells without chemical treatment (Fig. S1), consistent with the activity of these compounds. Following infection of Caco-2 cells with C. concisus UNSWCD, the bacterium was found to co-localize with LC3B (Fig. 4B–I). Moreover, some cells were found to be hyperinfected by C. concisus UNSWCD (Fig. 4F, J). Calculation of the co-localization coefficient for the C. concisus antibody and the LC3B antibody showed the coefficient to range from 0.54 to 0.90, indicating that significant co-localization between the two antibodies existed. The fact that C. concisus UNSWCD interacts with the autophagosome, provides further evidence that autophagy is involved in the intracellular survival of C. concisus within host cells, and raises the possibility that C. concisus could employ autophagy to survive intracellularly. A similar observation has been made with C. jejuni, with the bacterium co-localizing with GFP-LC3-labeled structures consistent with autophagosomes [28]. In a recent study, Lam et al. reported that Listeria monocytogenes co-localizes with LC3 [34], a finding that led them to suggest that this co-localization gives rise to spacious Listeria-containing phagosomes, membrane-bound compartments that harbor slow-growing bacteria associated with persistent infection [34].

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