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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

Scanning electron microscopy images of Caco-2 cells without (A) and with chloroquine diphosphate treatment (B, C). Following treatment of Caco-2 cells with 50 μM CQD, cellular protrusions/blebs (indicated by an asterisk) were induced on the apical membrane surface of Caco-2 cells.
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f0015: Scanning electron microscopy images of Caco-2 cells without (A) and with chloroquine diphosphate treatment (B, C). Following treatment of Caco-2 cells with 50 μM CQD, cellular protrusions/blebs (indicated by an asterisk) were induced on the apical membrane surface of Caco-2 cells.

Mentions: CQD plays a more complex role in the cell having been associated with stimulation of autophagosome formation. It has also been shown to accumulate inside lysosomes leading to inhibition of lysosomal enzymes [31]. Addition of CQD resulted in complete bacterial clearance, a finding that is supported by a study by Oelschlaegear et al. which found a reduction in the intracellular survival of C. jejuni, E. coli, Citrobacter freundii and S. Typhimurium upon addition of CQD [32]. The authors of this study suggested that the most likely cause of this was the intracellular accumulation of CQD to bactericidal concentrations [32]. To determine if this was the case we investigated the effect of CQD on the viability of C. concisus UNSWCD. While this compound resulted in a decrease in bacterial numbers of up to 1.2 log (25 μM CQD), 1.7 log (50 μM CQD) and 2.8 log (100 μM CQD), it did not result in bacterial clearance. Moreover, while the viability of the host cells exposed to 50 μM CQD for 14 h significantly decreased to 75.2 ± 2.0% when compared to the non-induced control (98.8 ± 0.6%), this could not fully explain the lack of intracellular bacteria in Caco-2 cells exposed to CQD. To investigate this further, we visualized the Caco-2 cells exposed to 50 μM CQD using scanning electron microscopy, and found cellular protrusions (blebs) on the apical membrane surface (Fig. 3B, C) that were not present in the negative control (Fig. 3A). The regions surrounding these protrusions showed a significant decrease in microvilli abundance (Fig. 3B, C). These findings are supported by a study by Fan et al. which reported exposure to high concentrations of CQD to result in vacuolation and blebbing in A549 lung cancer cells [33]. Thus, although cellular alterations and toxicity as a consequence of CQD exposure will result in a significant decrease in intracellular levels of C. concisus, the effects that CQD exerts on autophagy are also likely to be involved in the complete bacterial clearance observed.


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)

Scanning electron microscopy images of Caco-2 cells without (A) and with chloroquine diphosphate treatment (B, C). Following treatment of Caco-2 cells with 50 μM CQD, cellular protrusions/blebs (indicated by an asterisk) were induced on the apical membrane surface of Caco-2 cells.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0015: Scanning electron microscopy images of Caco-2 cells without (A) and with chloroquine diphosphate treatment (B, C). Following treatment of Caco-2 cells with 50 μM CQD, cellular protrusions/blebs (indicated by an asterisk) were induced on the apical membrane surface of Caco-2 cells.
Mentions: CQD plays a more complex role in the cell having been associated with stimulation of autophagosome formation. It has also been shown to accumulate inside lysosomes leading to inhibition of lysosomal enzymes [31]. Addition of CQD resulted in complete bacterial clearance, a finding that is supported by a study by Oelschlaegear et al. which found a reduction in the intracellular survival of C. jejuni, E. coli, Citrobacter freundii and S. Typhimurium upon addition of CQD [32]. The authors of this study suggested that the most likely cause of this was the intracellular accumulation of CQD to bactericidal concentrations [32]. To determine if this was the case we investigated the effect of CQD on the viability of C. concisus UNSWCD. While this compound resulted in a decrease in bacterial numbers of up to 1.2 log (25 μM CQD), 1.7 log (50 μM CQD) and 2.8 log (100 μM CQD), it did not result in bacterial clearance. Moreover, while the viability of the host cells exposed to 50 μM CQD for 14 h significantly decreased to 75.2 ± 2.0% when compared to the non-induced control (98.8 ± 0.6%), this could not fully explain the lack of intracellular bacteria in Caco-2 cells exposed to CQD. To investigate this further, we visualized the Caco-2 cells exposed to 50 μM CQD using scanning electron microscopy, and found cellular protrusions (blebs) on the apical membrane surface (Fig. 3B, C) that were not present in the negative control (Fig. 3A). The regions surrounding these protrusions showed a significant decrease in microvilli abundance (Fig. 3B, C). These findings are supported by a study by Fan et al. which reported exposure to high concentrations of CQD to result in vacuolation and blebbing in A549 lung cancer cells [33]. Thus, although cellular alterations and toxicity as a consequence of CQD exposure will result in a significant decrease in intracellular levels of C. concisus, the effects that CQD exerts on autophagy are also likely to be involved in the complete bacterial clearance observed.

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