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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

Transmission electron microscopy images of Caco-2 cells reflecting the initial stages of Campylobacter concisus UNSWCD infection. (A) Image shows characteristics of a Caco-2 cell. Microvilli are observed on the apical membrane surface, vacuolar compartments with no cellular material can be found between the apical membrane surface and the nucleus (Nu). (B) Image of a Caco-2 cell infected by C. concisus UNSWCD. Notable characteristics include lysosomal compartments, vacuoles and a Campylobacter-containing vacuole. (C) High magnification image of a Caco-2 cell vacuole. A granular compartment can be seen inside the vacuole. (D) High magnification image of two lysosomes showing a one layered membrane encapsulating dense granular material. (E) High magnification image of Caco-2 cell mitochondria and a densely stained bacterium. The mitochondria show a clear cristae lining and appear close to a bacterial cell, which may suggest a possible association between mitochondria and C. concisus UNSWCD in the formation and maturation of autophagosomes. (F) C. concisus UNSWCD within Caco-2 cells inside a Campylobacter-containing vacuole.
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f0030: Transmission electron microscopy images of Caco-2 cells reflecting the initial stages of Campylobacter concisus UNSWCD infection. (A) Image shows characteristics of a Caco-2 cell. Microvilli are observed on the apical membrane surface, vacuolar compartments with no cellular material can be found between the apical membrane surface and the nucleus (Nu). (B) Image of a Caco-2 cell infected by C. concisus UNSWCD. Notable characteristics include lysosomal compartments, vacuoles and a Campylobacter-containing vacuole. (C) High magnification image of a Caco-2 cell vacuole. A granular compartment can be seen inside the vacuole. (D) High magnification image of two lysosomes showing a one layered membrane encapsulating dense granular material. (E) High magnification image of Caco-2 cell mitochondria and a densely stained bacterium. The mitochondria show a clear cristae lining and appear close to a bacterial cell, which may suggest a possible association between mitochondria and C. concisus UNSWCD in the formation and maturation of autophagosomes. (F) C. concisus UNSWCD within Caco-2 cells inside a Campylobacter-containing vacuole.

Mentions: Further, the association between C. concisus infection and the autophagy process was visualized in more detail using TEM. Observation of the TEM images showed the bacterium to be densely stained and to have spiral shaped morphology (Fig. 5A, B). The dimensions of C. concisus UNSWCD were 4 μm long × 0.5 μm wide (Fig. 5A, B), which is consistent with previous reports on the size of the bacterium. An outer layer around the bacterium was identified (Fig. 5A, B), that may correspond to the formation of a capsule as has been shown in C. jejuni[35]. TEM images of Caco-2 cells not infected with the bacterium showed typical characteristics such as microvilli on the apical membrane surface and vacuolar compartments with no darkly stained material between the apical membrane surface and the nucleus (Fig. 6A). At the initial stages of infection with C. concisus UNSWCD, Caco-2 cells appeared more rounded than non-infected cells (Fig. 6B). Infected cells contained a higher number of vacuoles (Fig. 6B, C), lysosomes showing a one layered membrane encapsulating dense granular material (Fig. 6D), and internalized bacteria (densely stained) in close association with mitochondria (Fig. 6E). Moreover, some internalized C. concisus UNSWCD cells were found to be inside a vacuole (Fig. 6F), most likely to be a Campylobacter-containing vacuole [6]. This vacuole compartment may allow C. concisus UNSWCD to persist intracellularly, as this CCV was found closer to the basolateral end. Such a defensive mechanism to evade lysosomal degradation has been previously identified for C. jejuni[7]. More recently, Bouwman et al. have demonstrated that intracellular C. jejuni can reside in membrane-bound CD63-positive cellular compartments [36]. Thus, it would be of interest to determine if compartments containing C. concisus were also CD63 positive.


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)

Transmission electron microscopy images of Caco-2 cells reflecting the initial stages of Campylobacter concisus UNSWCD infection. (A) Image shows characteristics of a Caco-2 cell. Microvilli are observed on the apical membrane surface, vacuolar compartments with no cellular material can be found between the apical membrane surface and the nucleus (Nu). (B) Image of a Caco-2 cell infected by C. concisus UNSWCD. Notable characteristics include lysosomal compartments, vacuoles and a Campylobacter-containing vacuole. (C) High magnification image of a Caco-2 cell vacuole. A granular compartment can be seen inside the vacuole. (D) High magnification image of two lysosomes showing a one layered membrane encapsulating dense granular material. (E) High magnification image of Caco-2 cell mitochondria and a densely stained bacterium. The mitochondria show a clear cristae lining and appear close to a bacterial cell, which may suggest a possible association between mitochondria and C. concisus UNSWCD in the formation and maturation of autophagosomes. (F) C. concisus UNSWCD within Caco-2 cells inside a Campylobacter-containing vacuole.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0030: Transmission electron microscopy images of Caco-2 cells reflecting the initial stages of Campylobacter concisus UNSWCD infection. (A) Image shows characteristics of a Caco-2 cell. Microvilli are observed on the apical membrane surface, vacuolar compartments with no cellular material can be found between the apical membrane surface and the nucleus (Nu). (B) Image of a Caco-2 cell infected by C. concisus UNSWCD. Notable characteristics include lysosomal compartments, vacuoles and a Campylobacter-containing vacuole. (C) High magnification image of a Caco-2 cell vacuole. A granular compartment can be seen inside the vacuole. (D) High magnification image of two lysosomes showing a one layered membrane encapsulating dense granular material. (E) High magnification image of Caco-2 cell mitochondria and a densely stained bacterium. The mitochondria show a clear cristae lining and appear close to a bacterial cell, which may suggest a possible association between mitochondria and C. concisus UNSWCD in the formation and maturation of autophagosomes. (F) C. concisus UNSWCD within Caco-2 cells inside a Campylobacter-containing vacuole.
Mentions: Further, the association between C. concisus infection and the autophagy process was visualized in more detail using TEM. Observation of the TEM images showed the bacterium to be densely stained and to have spiral shaped morphology (Fig. 5A, B). The dimensions of C. concisus UNSWCD were 4 μm long × 0.5 μm wide (Fig. 5A, B), which is consistent with previous reports on the size of the bacterium. An outer layer around the bacterium was identified (Fig. 5A, B), that may correspond to the formation of a capsule as has been shown in C. jejuni[35]. TEM images of Caco-2 cells not infected with the bacterium showed typical characteristics such as microvilli on the apical membrane surface and vacuolar compartments with no darkly stained material between the apical membrane surface and the nucleus (Fig. 6A). At the initial stages of infection with C. concisus UNSWCD, Caco-2 cells appeared more rounded than non-infected cells (Fig. 6B). Infected cells contained a higher number of vacuoles (Fig. 6B, C), lysosomes showing a one layered membrane encapsulating dense granular material (Fig. 6D), and internalized bacteria (densely stained) in close association with mitochondria (Fig. 6E). Moreover, some internalized C. concisus UNSWCD cells were found to be inside a vacuole (Fig. 6F), most likely to be a Campylobacter-containing vacuole [6]. This vacuole compartment may allow C. concisus UNSWCD to persist intracellularly, as this CCV was found closer to the basolateral end. Such a defensive mechanism to evade lysosomal degradation has been previously identified for C. jejuni[7]. More recently, Bouwman et al. have demonstrated that intracellular C. jejuni can reside in membrane-bound CD63-positive cellular compartments [36]. Thus, it would be of interest to determine if compartments containing C. concisus were also CD63 positive.

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