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Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

Hellinga JR, Garduño RA, Kormish JD, Tanner JR, Khan D, Buchko K, Jimenez C, Pinette MM, Brassinga AK - Microbiologyopen (2015)

Bottom Line: A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila.While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen.Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2.

No MeSH data available.


Related in: MedlinePlus

Formation of Legionella-containing vacuoles (LCVs) in Caenorhabditis elegans tissue and fluids. Representative DIC still micrographic images of spherical structures containing rod-shaped and/or coccoid shaped bacteria similar to LCVs in N2 nematodes fed live Lp02. (A and C) Spherical structures (indicated by white arrow) containing rod-shaped bacteria in the pseudocoelomic cavity (PC) of a nematode 2 days p.i. Note that the distinct structure of the intestine (I), and the distinct U-loop structure of the gonad arm (GA) (distal part is shown) containing the developing oocytes (not shown), the spermatheca (not shown) and embryos (E) (one is shown). Scale bar is 5 μm. (B) Magnification of inset box in (A). Scale bar is 2 μm. (D) Spherical structure (indicated by white arrow) containing coccoid-shaped bacteria within the gonadal tissue (GT) of a nematode 6 days p.i. Note the rod-shaped bacteria in the intestinal lumen (IL). Scale bar is 2 μm. Still images in (A–C) are taken from Video S1, and still image in (D) taken from Video S2.
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fig02: Formation of Legionella-containing vacuoles (LCVs) in Caenorhabditis elegans tissue and fluids. Representative DIC still micrographic images of spherical structures containing rod-shaped and/or coccoid shaped bacteria similar to LCVs in N2 nematodes fed live Lp02. (A and C) Spherical structures (indicated by white arrow) containing rod-shaped bacteria in the pseudocoelomic cavity (PC) of a nematode 2 days p.i. Note that the distinct structure of the intestine (I), and the distinct U-loop structure of the gonad arm (GA) (distal part is shown) containing the developing oocytes (not shown), the spermatheca (not shown) and embryos (E) (one is shown). Scale bar is 5 μm. (B) Magnification of inset box in (A). Scale bar is 2 μm. (D) Spherical structure (indicated by white arrow) containing coccoid-shaped bacteria within the gonadal tissue (GT) of a nematode 6 days p.i. Note the rod-shaped bacteria in the intestinal lumen (IL). Scale bar is 2 μm. Still images in (A–C) are taken from Video S1, and still image in (D) taken from Video S2.

Mentions: In addition to the bacterial colonization of the intestinal lumen, vacuoles containing nonmotile rod-shaped bacteria (presumably Lp02) were observed in the pseudocoelomic cavity first appearing 2 days postinfection (Fig.2A–C; Video S1A–C). Interestingly, vacuoles containing motile coccoid-shaped bacteria were also observed in the pseudocoelomic cavity as well as in the gonadal tissue in abundance by 6–7 days postinfection (Fig.2D; Video S2). It should be noted that these vacuoles containing bacterial forms were not previously observed in the Brassinga et al. (2010) study most likely due to high fluorescent protein levels obscuring or hindering vacuole formation. Furthermore, these vacuoles containing bacterial forms, in particular those vacuoles containing motile coccoid-shaped bacteria are morphologically similar to LCVs formed in Legionella-infected protozoa when nearing completion of its developmental lifecycle (Video S3). Vacuoles containing motile coccoid-shaped bacteria were also observed in the uterus and in the vulval region, but not in the vulval muscles, in the range of 4–7 days postinfection (data not shown). Furthermore, these bacteria-filled vacuoles were commonly observed in the fluidic material and/or viscera extruded from the vulva entry (Video S4). It should be noted that while great care was taken when preparing the colonized nematodes for imaging, extrusion of the material was not due to egg-laying since these animals no longer contained fertilized embryos at this time point in development, but appears to be a part of the pathologic process due to excessive fluid retention in the pseudocoelom of the infected host as noted in the Brassinga et al. (2010) study. Fluid extrusion from the vulva was frequently observed in dying nematodes on assay plates or in situ when mounted for imaging. In summary, formation of these vacuoles containing bacterial forms are specific to nematodes colonized with live Lp02 as these vacuoles were not observed in nematodes fed heat-killed Lp02 or in nematodes fed live and heat-killed OP50 (data not shown).


Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

Hellinga JR, Garduño RA, Kormish JD, Tanner JR, Khan D, Buchko K, Jimenez C, Pinette MM, Brassinga AK - Microbiologyopen (2015)

Formation of Legionella-containing vacuoles (LCVs) in Caenorhabditis elegans tissue and fluids. Representative DIC still micrographic images of spherical structures containing rod-shaped and/or coccoid shaped bacteria similar to LCVs in N2 nematodes fed live Lp02. (A and C) Spherical structures (indicated by white arrow) containing rod-shaped bacteria in the pseudocoelomic cavity (PC) of a nematode 2 days p.i. Note that the distinct structure of the intestine (I), and the distinct U-loop structure of the gonad arm (GA) (distal part is shown) containing the developing oocytes (not shown), the spermatheca (not shown) and embryos (E) (one is shown). Scale bar is 5 μm. (B) Magnification of inset box in (A). Scale bar is 2 μm. (D) Spherical structure (indicated by white arrow) containing coccoid-shaped bacteria within the gonadal tissue (GT) of a nematode 6 days p.i. Note the rod-shaped bacteria in the intestinal lumen (IL). Scale bar is 2 μm. Still images in (A–C) are taken from Video S1, and still image in (D) taken from Video S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Formation of Legionella-containing vacuoles (LCVs) in Caenorhabditis elegans tissue and fluids. Representative DIC still micrographic images of spherical structures containing rod-shaped and/or coccoid shaped bacteria similar to LCVs in N2 nematodes fed live Lp02. (A and C) Spherical structures (indicated by white arrow) containing rod-shaped bacteria in the pseudocoelomic cavity (PC) of a nematode 2 days p.i. Note that the distinct structure of the intestine (I), and the distinct U-loop structure of the gonad arm (GA) (distal part is shown) containing the developing oocytes (not shown), the spermatheca (not shown) and embryos (E) (one is shown). Scale bar is 5 μm. (B) Magnification of inset box in (A). Scale bar is 2 μm. (D) Spherical structure (indicated by white arrow) containing coccoid-shaped bacteria within the gonadal tissue (GT) of a nematode 6 days p.i. Note the rod-shaped bacteria in the intestinal lumen (IL). Scale bar is 2 μm. Still images in (A–C) are taken from Video S1, and still image in (D) taken from Video S2.
Mentions: In addition to the bacterial colonization of the intestinal lumen, vacuoles containing nonmotile rod-shaped bacteria (presumably Lp02) were observed in the pseudocoelomic cavity first appearing 2 days postinfection (Fig.2A–C; Video S1A–C). Interestingly, vacuoles containing motile coccoid-shaped bacteria were also observed in the pseudocoelomic cavity as well as in the gonadal tissue in abundance by 6–7 days postinfection (Fig.2D; Video S2). It should be noted that these vacuoles containing bacterial forms were not previously observed in the Brassinga et al. (2010) study most likely due to high fluorescent protein levels obscuring or hindering vacuole formation. Furthermore, these vacuoles containing bacterial forms, in particular those vacuoles containing motile coccoid-shaped bacteria are morphologically similar to LCVs formed in Legionella-infected protozoa when nearing completion of its developmental lifecycle (Video S3). Vacuoles containing motile coccoid-shaped bacteria were also observed in the uterus and in the vulval region, but not in the vulval muscles, in the range of 4–7 days postinfection (data not shown). Furthermore, these bacteria-filled vacuoles were commonly observed in the fluidic material and/or viscera extruded from the vulva entry (Video S4). It should be noted that while great care was taken when preparing the colonized nematodes for imaging, extrusion of the material was not due to egg-laying since these animals no longer contained fertilized embryos at this time point in development, but appears to be a part of the pathologic process due to excessive fluid retention in the pseudocoelom of the infected host as noted in the Brassinga et al. (2010) study. Fluid extrusion from the vulva was frequently observed in dying nematodes on assay plates or in situ when mounted for imaging. In summary, formation of these vacuoles containing bacterial forms are specific to nematodes colonized with live Lp02 as these vacuoles were not observed in nematodes fed heat-killed Lp02 or in nematodes fed live and heat-killed OP50 (data not shown).

Bottom Line: A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila.While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen.Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2.

No MeSH data available.


Related in: MedlinePlus