Encystment of Vermamoeba (Hartmannella) vermiformis: Effects of environmental conditions and cell concentration

Fouque, Emilie; Trouilhé, Marie-Cécile; Thomas, Vincent; Humeau, Philippe; Héchard, Yann

doi:10.1016/j.exppara.2014.03.029

Cited by 21 publications

(21 citation statements)

References 30 publications

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“…For encystment assay, both infected and uninfected V. vermiformis 172A strain were grown PAS-E. coli cultures, and adjusted at a concentration of 5.10 5 cells ml −1 in PAS, in 12-wells plates. Cells were incubated in Neff's encystment medium, and encystment rate was evaluated at 0 h, 3 h, 6 h, 9 h and 24 h by differential counting of trophozoites and mature cysts, as described previously (Fouque et al, 2014). Data represent mean +/− SD from three independent experiments in triplicates.…”

Section: Resultsmentioning

confidence: 99%

“…Supernatants were then discarded, and replaced with 1 ml of pre-warmed Neff's encystment buffer (0.1 M KCl, 8 mM MgSO4, 0.4 mM CaCl2, 1 mM NaHCO3, 20 mM ammediol [2-amino-2-methyl-1,3propanediol], pH 8.8). Encystment rate was evaluated at 0, 3, 6, 9 and 24 h by differential counting of total cells versus cells treated with 0.5% SDS (effectively lyses trophozoites, but not mature cysts) on FastRead counting chambers, as described previously (Fouque et al, 2014). The impact of TM6 bacteria infection on amoebae's growth rate was estimated, in 12-wells plates, by inoculating 5.10 2 cells in 1 ml of PAS enriched with E. coli at 5 × 10 8 ml −1 .…”

Section: Growth and Encystment Assaysmentioning

confidence: 99%

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Shedding light on microbial dark matter: a TM6 bacterium as natural endosymbiont of a free‐living amoeba

Delafont

Samba-Louaka

Bouchon

et al. 2015

Environ Microbiol Rep

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The TM6 phylum belongs to the so-called microbial dark matter that gathers uncultivated bacteria detected only via DNA sequencing. Recently, the genome sequence of a TM6 bacterium (TM6SC1) has led to suggest that this bacterium would adopt an endosymbiotic life. In the present paper, free-living amoebae bearing a TM6 strain were isolated from a water network. The amoebae were identified as Vermamoeba vermiformis and the presence of a TM6 strain was detected by polymerase chain reaction and microscopy. The partial sequence of its 16S rRNA gene showed this strain to be closely related to the sequenced TM6SC1 strain. These bacteria displayed a pyriform shape and were found within V. vermiformis. Therefore, these bacteria were named Vermiphilus pyriformis. Interactions studies showed that V. pyriformis was highly infectious and that its relation with V. vermiformis was specific and highly stable. Finally, it was found that V. pyriformis inhibited the encystment of V. vermiformis. Overall, this study describes for the first time an endosymbiotic relationship between a TM6 bacterium and a free-living amoeba in the environment. It suggests that other bacteria of the TM6 phylum might also be endosymbiotic bacteria and may be found in other free-living amoebae or other organisms.

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Section: Resultsmentioning

confidence: 99%

Section: Growth and Encystment Assaysmentioning

confidence: 99%

Shedding light on microbial dark matter: a TM6 bacterium as natural endosymbiont of a free‐living amoeba

Delafont

Samba-Louaka

Bouchon

et al. 2015

Environ Microbiol Rep

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“…To induce encystation, A. castellanii were incubated in encystation buffer (0.1 M KCl, 8 mM MgSO 4 , 0.4 mM CaCl 2 , 20 mM Tris (2-amino-2hydroxymethyl-1,3-propanediol), 1 mM NaHCO 3 , pH 8.8) at 30 °C for 24 h47.…”

Section: Methodsmentioning

confidence: 99%

Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii

Mengue¹,

Régnacq²,

Aucher³

et al. 2016

Sci Rep

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Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.

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“…Unfavorable environment conditions, such as high temperatures and pH, nutrient depletion, and osmotic stress, have been shown to induce encystation (41,42); thus, it is unclear why sGW incubation of A. polyphaga, A. castellanii, and V. vermiformis resulted in maintenance of the trophozoite form given the relatively low nutrient components in the sGW medium. For A. castellanii, the failure to encyst has been described previously by Stöhr, who reported that 5 to 8% of exponentially growing cells encysted after a 72 h of nutrient starvation compared to 70% of stationary-phase cells after 30 h of starvation (43).…”

Section: Discussionmentioning

confidence: 99%

Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

Buse

Ashbolt

2015

Appl Environ Microbiol

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bWater conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the waterbased pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log 10 CFU, P < 0.001) compared to controls (⌬0 to 0.1 log 10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. G ray water (GW) usage has proven to be both an economically and a logistically suitable solution to alleviate the stresses from increasing potable water demand and climate/demographic change (1), when used (following adequate treatment) in largeand small-scale irrigation, toilet flushing, and car washing (2-4). Defined as household wastewater that is not contaminated with toilet waste, GW is further categorized as light (derived from bathroom showers, tubs, and washbasins) and dark (derived from laundry machines, dishwashers, and kitchen sinks) gray water (5). In households surveyed internationally, light GW constitutes about 47% of total GW, with the remaining 27 and 26% originating from kitchen sinks/dishwashers and laundry washing machines, respectively (reviewed in reference 5). The composition of GW varies greatly with light GW containing mostly personal care products and dark GW composed mostly of dish and laundry detergents, food residue, and fats, oils, and grease.Few studies report on the densities of microbial contaminants within treated GW, and of these, most present fecal indicator data, with less information on pathogens (6). When reusing GW there is concern for fecally borne pathogens (such as species in the bacterial genera Campylobacter, Salmonella, and Vibrio [7-10]), as well as an emerg...

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Encystment of Vermamoeba (Hartmannella) vermiformis: Effects of environmental conditions and cell concentration

Cited by 21 publications

References 30 publications

Shedding light on microbial dark matter: a TM6 bacterium as natural endosymbiont of a free‐living amoeba

Shedding light on microbial dark matter: a TM6 bacterium as natural endosymbiont of a free‐living amoeba

Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii

Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

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