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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

Elevated levels of germline apoptosis in the Caenorhabditis elegans gonadal tissue are dependent on the Legionella pneumophila Type IV Dot/Icm system. Number of corpse (i.e., apoptotic) cells counted per gonad (n = 20–30) in ced-1::gfp (MD701) and RNAi-treated (ced-3) ced-1::gfp (MD701) nematodes fed wild-type Lp02, Δdot/icm, ΔdotA, ΔsdhA, and ΔsidF mutant strains as well as S. Typhimurium SL1344. Bars are mean values with SEM. P values obtained by unpaired two-tailed student’s test with Welch’s correction; *P < 0.0001.
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fig06: Elevated levels of germline apoptosis in the Caenorhabditis elegans gonadal tissue are dependent on the Legionella pneumophila Type IV Dot/Icm system. Number of corpse (i.e., apoptotic) cells counted per gonad (n = 20–30) in ced-1::gfp (MD701) and RNAi-treated (ced-3) ced-1::gfp (MD701) nematodes fed wild-type Lp02, Δdot/icm, ΔdotA, ΔsdhA, and ΔsidF mutant strains as well as S. Typhimurium SL1344. Bars are mean values with SEM. P values obtained by unpaired two-tailed student’s test with Welch’s correction; *P < 0.0001.

Mentions: In C. elegans, susceptibility to Lp02 is influenced by innate immune responses governed by the p38 MAPK and insulin-like DAF-2 signaling pathways as loss-of-function mutant nematodes exhibited an immunocompromised phenotype (Brassinga et al. 2010). Activation of the p38 MAPK pathway, functionally orthologous to the macrophage MAPK pathway, triggers the activation of the downstream germline apoptosis (i.e., programmed cell death [PCD]) pathway (Aballay and Ausubel 2001; Kinchen and Hengartner 2005; Gartner et al. 2008). As key components of the core apoptotic machinery CED-9/CED-4/CED-3 are homologous to the mammalian BCL2/APAF-1/CASPASE, it was previously determined that a functional L. pneumophila Dot/Icm system bestowed an antiapoptotic effect on the PCD pathway (Brassinga et al. 2010). However, the identity of the Dot/Icm-mediated substrates that interacted with the PCD pathway was not known. In L. pneumophila-infected macrophages, caspase-mediated apoptosis is blocked by the interaction of effector molecule SidF with proapoptotic proteins BNIP3 and Bcl-rambo (Banga et al. 2007). To determine if the substrate SidF is involved in inhibiting apoptosis mediated by the PCD pathway in C. elegans, apoptotic cells were enumerated in transgenic Plim-7ced-1::gfp nematodes fed wild-type Lp02 and ΔsidF mutant strains. CED-1, a transmembrane receptor expressed on the surface of somatic sheath cells, is essential for the engulfment of apoptotic germ cells and thus translational fused CED-1:GFP provides a convenient visual marker of apoptotic cells for enumeration (Hedgecock et al. 1983; Conradt and Horvitz 1999; Schumacher et al. 2005). Because SdhA has been shown to maintain the integrity of the vacuole rather than interact with the macrophage apoptosis pathway (Creasey and Isberg 2012), the ΔsdhA strain was included as a negative control substrate as it is expected that the apoptotic cell levels will not be altered in comparison to levels achieved in nematodes colonized with Lp02. As an additional control, nematodes with RNAi-mediated downregulation of the caspase ced-3 were included to verify that apoptosis was activated by CED-3. As previously reported in Brassinga et al. (2010), apoptotic cell levels were significantly elevated in Plim-7ced-1::gfp nematodes fed the ΔdotA strain that features a dysfunctional Dot/Icm system (Fig.6). Apoptotic cell levels were likewise elevated in Plim-7ced-1::gfp nematodes fed the Δdot/icm strain in which the icm/dot loci are genetically deleted (Fig.6). The absence of the substrate SidF, but not SdhA, marginally increased apoptotic cell levels suggesting that other Dot/Icm substrate(s) may interact directly with CED-3 to inhibit activation of apoptosis (Fig.6).


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)

Elevated levels of germline apoptosis in the Caenorhabditis elegans gonadal tissue are dependent on the Legionella pneumophila Type IV Dot/Icm system. Number of corpse (i.e., apoptotic) cells counted per gonad (n = 20–30) in ced-1::gfp (MD701) and RNAi-treated (ced-3) ced-1::gfp (MD701) nematodes fed wild-type Lp02, Δdot/icm, ΔdotA, ΔsdhA, and ΔsidF mutant strains as well as S. Typhimurium SL1344. Bars are mean values with SEM. P values obtained by unpaired two-tailed student’s test with Welch’s correction; *P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: Elevated levels of germline apoptosis in the Caenorhabditis elegans gonadal tissue are dependent on the Legionella pneumophila Type IV Dot/Icm system. Number of corpse (i.e., apoptotic) cells counted per gonad (n = 20–30) in ced-1::gfp (MD701) and RNAi-treated (ced-3) ced-1::gfp (MD701) nematodes fed wild-type Lp02, Δdot/icm, ΔdotA, ΔsdhA, and ΔsidF mutant strains as well as S. Typhimurium SL1344. Bars are mean values with SEM. P values obtained by unpaired two-tailed student’s test with Welch’s correction; *P < 0.0001.
Mentions: In C. elegans, susceptibility to Lp02 is influenced by innate immune responses governed by the p38 MAPK and insulin-like DAF-2 signaling pathways as loss-of-function mutant nematodes exhibited an immunocompromised phenotype (Brassinga et al. 2010). Activation of the p38 MAPK pathway, functionally orthologous to the macrophage MAPK pathway, triggers the activation of the downstream germline apoptosis (i.e., programmed cell death [PCD]) pathway (Aballay and Ausubel 2001; Kinchen and Hengartner 2005; Gartner et al. 2008). As key components of the core apoptotic machinery CED-9/CED-4/CED-3 are homologous to the mammalian BCL2/APAF-1/CASPASE, it was previously determined that a functional L. pneumophila Dot/Icm system bestowed an antiapoptotic effect on the PCD pathway (Brassinga et al. 2010). However, the identity of the Dot/Icm-mediated substrates that interacted with the PCD pathway was not known. In L. pneumophila-infected macrophages, caspase-mediated apoptosis is blocked by the interaction of effector molecule SidF with proapoptotic proteins BNIP3 and Bcl-rambo (Banga et al. 2007). To determine if the substrate SidF is involved in inhibiting apoptosis mediated by the PCD pathway in C. elegans, apoptotic cells were enumerated in transgenic Plim-7ced-1::gfp nematodes fed wild-type Lp02 and ΔsidF mutant strains. CED-1, a transmembrane receptor expressed on the surface of somatic sheath cells, is essential for the engulfment of apoptotic germ cells and thus translational fused CED-1:GFP provides a convenient visual marker of apoptotic cells for enumeration (Hedgecock et al. 1983; Conradt and Horvitz 1999; Schumacher et al. 2005). Because SdhA has been shown to maintain the integrity of the vacuole rather than interact with the macrophage apoptosis pathway (Creasey and Isberg 2012), the ΔsdhA strain was included as a negative control substrate as it is expected that the apoptotic cell levels will not be altered in comparison to levels achieved in nematodes colonized with Lp02. As an additional control, nematodes with RNAi-mediated downregulation of the caspase ced-3 were included to verify that apoptosis was activated by CED-3. As previously reported in Brassinga et al. (2010), apoptotic cell levels were significantly elevated in Plim-7ced-1::gfp nematodes fed the ΔdotA strain that features a dysfunctional Dot/Icm system (Fig.6). Apoptotic cell levels were likewise elevated in Plim-7ced-1::gfp nematodes fed the Δdot/icm strain in which the icm/dot loci are genetically deleted (Fig.6). The absence of the substrate SidF, but not SdhA, marginally increased apoptotic cell levels suggesting that other Dot/Icm substrate(s) may interact directly with CED-3 to inhibit activation of apoptosis (Fig.6).

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