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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

Morphological differentiation of internalized Legionella pneumophila bacterial forms. Representative transmission electron microscopy images were obtained from samples of nematodes fed L. pneumophila Lp02 for 6 days. (A and B) Internalized bacterial forms (white arrow) within intestinal cells (IC) with microvilli (MV) on the apical surface supported by the terminal web (TW). Note that the bacterial membrane is more defined due to progressive formation of multiple layers. (C) Irregular- shaped transitional bacterial forms (white arrow) inside an IC next to the gonadal tissue (GT) (i.e., gonad arm). (D) Presence of a well-defined Legionella-containing vacuoles, containing irregular-shaped transitional forms and membrane whorls (indicated by white arrow), in a GT cell of the gonad arm. Membrane whorls most likely represent eukaryotic membrane fragments. Scale bar is 500 nm for all panels.
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fig08: Morphological differentiation of internalized Legionella pneumophila bacterial forms. Representative transmission electron microscopy images were obtained from samples of nematodes fed L. pneumophila Lp02 for 6 days. (A and B) Internalized bacterial forms (white arrow) within intestinal cells (IC) with microvilli (MV) on the apical surface supported by the terminal web (TW). Note that the bacterial membrane is more defined due to progressive formation of multiple layers. (C) Irregular- shaped transitional bacterial forms (white arrow) inside an IC next to the gonadal tissue (GT) (i.e., gonad arm). (D) Presence of a well-defined Legionella-containing vacuoles, containing irregular-shaped transitional forms and membrane whorls (indicated by white arrow), in a GT cell of the gonad arm. Membrane whorls most likely represent eukaryotic membrane fragments. Scale bar is 500 nm for all panels.

Mentions: The embedded Lp02 bacteria appear to disrupt the underlying terminal web, a strong cytoskeletal network consisting largely of intermediate filaments anchoring the microvilli (McGhee 2007), gaining access to the intestinal cell (Fig.7C). Indeed, single internalized bacterial forms with typical gram-negative ultrastructure features are observed within a tight vacuole in the intestinal cell (Fig.7D). Furthermore, single and grouped transitional bacterial forms are also observed to be internalized within tight vacuoles in intestinal cells (Fig.8A and B). Transitional bacterial forms are intermediate forms prior to the formation of cyst forms and are characterized to feature wavy well-defined membranes incipient of a thick layer, and may sometimes feature PHBA inclusions. Adjacent to and in the gonadal tissue, tight vacuoles containing multiple irregular shaped bacterial forms with multiple membrane laminations were observed to be morphologically similar to LCVs containing differentiating L. pneumophila cyst forms in protozoa and nonphagocytic cells as reported elsewhere (Fig.8C and D) (Faulkner and Garduño 2002; Berk et al. 2008). This suggests that replication occurred within the vacuole until nutritional sources are depleted triggering the process of replicative forms transitioning into cyst forms. Taken together, these ultrastructural observations suggest that L. pneumophila Lp02 appears to have the ability to transverse the intestinal epithelium into the intestinal cell from the colonized intestinal tract as well as differentiate to cyst forms. It should be noted that despite repeated attempts, it was not possible to identify Lp02 bacteria within the C. elegans tissue on the basis of immunogold labeling due to the thickness of the tissue in the fixed sections of colonized nematodes. Conversely, Lp02 bacteria within the intestinal tract were readily immunogold labeled due to the lack of interfering tissue when sectioning the colonized nematodes in a longitudinal manner (Figure S1). Thus, analyses of the ultrastructural features of bacterial forms via TEM imaging constituted the best available approach to identify the multiphasic forms of L. pneumophila.


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)

Morphological differentiation of internalized Legionella pneumophila bacterial forms. Representative transmission electron microscopy images were obtained from samples of nematodes fed L. pneumophila Lp02 for 6 days. (A and B) Internalized bacterial forms (white arrow) within intestinal cells (IC) with microvilli (MV) on the apical surface supported by the terminal web (TW). Note that the bacterial membrane is more defined due to progressive formation of multiple layers. (C) Irregular- shaped transitional bacterial forms (white arrow) inside an IC next to the gonadal tissue (GT) (i.e., gonad arm). (D) Presence of a well-defined Legionella-containing vacuoles, containing irregular-shaped transitional forms and membrane whorls (indicated by white arrow), in a GT cell of the gonad arm. Membrane whorls most likely represent eukaryotic membrane fragments. Scale bar is 500 nm for all panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554460&req=5

fig08: Morphological differentiation of internalized Legionella pneumophila bacterial forms. Representative transmission electron microscopy images were obtained from samples of nematodes fed L. pneumophila Lp02 for 6 days. (A and B) Internalized bacterial forms (white arrow) within intestinal cells (IC) with microvilli (MV) on the apical surface supported by the terminal web (TW). Note that the bacterial membrane is more defined due to progressive formation of multiple layers. (C) Irregular- shaped transitional bacterial forms (white arrow) inside an IC next to the gonadal tissue (GT) (i.e., gonad arm). (D) Presence of a well-defined Legionella-containing vacuoles, containing irregular-shaped transitional forms and membrane whorls (indicated by white arrow), in a GT cell of the gonad arm. Membrane whorls most likely represent eukaryotic membrane fragments. Scale bar is 500 nm for all panels.
Mentions: The embedded Lp02 bacteria appear to disrupt the underlying terminal web, a strong cytoskeletal network consisting largely of intermediate filaments anchoring the microvilli (McGhee 2007), gaining access to the intestinal cell (Fig.7C). Indeed, single internalized bacterial forms with typical gram-negative ultrastructure features are observed within a tight vacuole in the intestinal cell (Fig.7D). Furthermore, single and grouped transitional bacterial forms are also observed to be internalized within tight vacuoles in intestinal cells (Fig.8A and B). Transitional bacterial forms are intermediate forms prior to the formation of cyst forms and are characterized to feature wavy well-defined membranes incipient of a thick layer, and may sometimes feature PHBA inclusions. Adjacent to and in the gonadal tissue, tight vacuoles containing multiple irregular shaped bacterial forms with multiple membrane laminations were observed to be morphologically similar to LCVs containing differentiating L. pneumophila cyst forms in protozoa and nonphagocytic cells as reported elsewhere (Fig.8C and D) (Faulkner and Garduño 2002; Berk et al. 2008). This suggests that replication occurred within the vacuole until nutritional sources are depleted triggering the process of replicative forms transitioning into cyst forms. Taken together, these ultrastructural observations suggest that L. pneumophila Lp02 appears to have the ability to transverse the intestinal epithelium into the intestinal cell from the colonized intestinal tract as well as differentiate to cyst forms. It should be noted that despite repeated attempts, it was not possible to identify Lp02 bacteria within the C. elegans tissue on the basis of immunogold labeling due to the thickness of the tissue in the fixed sections of colonized nematodes. Conversely, Lp02 bacteria within the intestinal tract were readily immunogold labeled due to the lack of interfering tissue when sectioning the colonized nematodes in a longitudinal manner (Figure S1). Thus, analyses of the ultrastructural features of bacterial forms via TEM imaging constituted the best available approach to identify the multiphasic forms of L. pneumophila.

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