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. 2016 Apr 15:6:24322.
doi: 10.1038/srep24322.

Ciliate Paramecium is a natural reservoir of Legionella pneumophila

Kenta Watanabe  1   2 , Ryo Nakao  3 , Masahiro Fujishima  4   5 , Masato Tachibana  6 , Takashi Shimizu  1   2 , Masahisa Watarai  1   2
Affiliations

Ciliate Paramecium is a natural reservoir of Legionella pneumophila

Kenta Watanabe et al. Sci Rep. .

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.

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Figures

Figure 1
Figure 1. L. pneumophila Philadelphia-1 establishes endosymbiosis in P. caudatum RB-1.
(a) Intracellular localization of Philadelphia-1 (GFP) and E. coli (AsRed2) in RB-1 at 30 min, 6 h, 24 h, and 48 h after infection. Bacteria were simultaneously added to RB-1 at an MOI of 10000. Scale bar represents 100 μm. (b) Bacteria and each dextran were added to RB-1 according to this schedule. LCVs containing each dextran were observed by confocal laser scanning microscopy 2 h and 24 h after infection (c), and percentages of ELCV and LLCV are shown with the total of all LCVs being 100% (d). Arrowheads point to LLCV, which are LCVs positive for both Texas Red- and Cascade Blue-conjugated dextrans. Scale bar represents 30 μm.
Figure 2
Figure 2. Infection of L. pneumophila Ofk308 is required to exhibit the cytotoxicity toward RB-1.
(a) RB-1 was infected with L. pneumophila Phi-1, Ofk308, and Bnt314 at MOIs of 10, 100, and 1000. RB-1 was treated with culture supernatants of Phi-1, Ofk308, and Bnt314 (b), or with killed bacteria (c). Cont., no infection. Relative RB-1 survival rates are indicated, with Cont. being defined as 100%. Data are averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences, compared with Cont., are indicated by asterisks (*P < 0.01).
Figure 3
Figure 3. lefA mutant has no cytotoxicity toward RB-1.
(a) RB-1 was infected with Phi-1, Ofk308, the lefA mutant (lefA), and the lefA complemental strain (lefA/lefA+) at MOIs of 1000. Cont., no infection. Relative RB-1 survival rates are indicated, with Cont. being defined as 100%. (b) Cell shapes of RB-1 infected with each strain of L. pneumophila 2 h, 24 h, and 48 h after infection. Scale bar represents 30 μm. (c) Number of bacteria per RB-1 cell. Data are averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared with lefA (a) or Phi-1 (c) are indicated by asterisks (*P < 0.01).
Figure 4
Figure 4. Expression of lefA in Ofk308 and Bnt314 is upregulated within RB-1.
(a) Each strain of L. pneumophila was used to infect RB-1 for 10 to 60 min, and RNA samples were collected from the bacteria. Expression of lefA was determined by real-time PCR. The fold increase of lefA was normalized to 16S rRNA; the expression levels are represented relative to a sample obtained 10 min after infection with Phi-1. (b) Each strain was incubated at 25 °C for 10 to 60 min without RB-1, and then RNA samples were collected. The expression was determined as described above. Statistically significant differences compared to Phi-1 are indicated by asterisks (*P < 0.01).
Figure 5
Figure 5. The maturation of host LCVs containing Ofk308 is inhibited.
(a) LCV maturation 30 min after infection was evaluated with LysoTracker. Bacteria were added to RB-1 at an MOI of 10000. White arrows point to LysoTracker-positive LCVs (or phagosomes containing E. coli). Black arrows point LysoTracker-negative LCVs. Scale bar represents 100 μm. (b) Relative LysoTracker-positive LCVs percentages are shown, with the total of all LCVs being 100%. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared to Phi-1 are indicated by asterisks (*P < 0.01).
Figure 6
Figure 6. Concanamycin A treatment affects the acidification and enlargement of host LCVs formed by infection of Ofk308.
RB-1 was infected with Ofk308 at an MOI of 10000 and pHrodo-conjugated dextran was added simultaneously (a) without or (b) with concanamycin A (CMA). White arrows point to pHrodo-negative LCVs. Black arrows point to enlarged LCVs. Scale bar represents 100 μm. (c) Relative pHrodo-positive LCVs percentages are shown, with the total of all LCVs being 100%. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences are indicated by asterisks (*P < 0.01).
Figure 7
Figure 7. lefA mutant fails to grow in human monocytic THP-1 cells.
Infected THP-1 cells were cultured for 2, 6, 12, and 24 h. Data are the averages of triplicate samples from three identical experiments, and error bars represent standard deviations. Statistically significant differences compared to Phi-1 are indicated by asterisks (*P < 0.01).
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