Interactions of free-living amoebae with the rice fungal pathogen, Rhizoctonia solani.

OBJECTIVE: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol. RESULTS: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.

Vermamoeba vermiformis: a Free-Living Amoeba of Interest.

Free-living amoebae are protists that are widely distributed in the environment including water, soil, and air. Although the amoebae of the genus Acanthamoeba are still the most studied, other species, such as Vermamoeba vermiformis (formerly Hartmannella vermiformis), are the subject of increased interest. Found in natural or man-made aquatic environments, V. vermiformis can support the multiplication of other microorganisms and is able to harbor and potentially protect pathogenic bacteria or viruses. This feature is to be noted because of the presence of this thermotolerant amoeba in hospital water networks. As a consequence, this protist could be implicated in health concerns and be indirectly responsible for healthcare-related infections. This review highlights, among others, the consequences of V. vermiformis relationships with other microorganisms and shows that this free-living amoeba species is therefore of interest for public health.

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

Vermamoeba vermiformis is a free-living amoeba (FLA) which is widely distributed in the environment. It is known to colonize water systems and to be a reservoir of pathogenic bacteria, such as Legionella pneumophila. For these reasons the control of V. vermiformis represents an important health issue. However, FLA may be resistant to disinfection treatments due to the process of encystment. Thereby, it is important to better understand factors influencing this process. In this aim, we investigated the effect of temperature, pH, osmotic pressure and cell concentration on the encystment of two V. vermiformis strains. Encystment was quite fast, with a 100% encystment rate being observed after 9h of incubation. For the two strains, an optimal encystment was obtained at 25 and 37°C. Concerning pH and osmotic pressure, there were different effects on the encystment according to the tested strains. For the reference strain (ATCC 50237), the patterns of encystment were similar for pH comprised between 5 and 9 and for KCl concentrations ranging from 0.05 to 0.2 mol L(-1). For the environmental strain (172A) an optimal encystment was obtained for basic pH (8 and 9) and for a concentration in KCl of 0.1 mol L(-1). The results also clearly demonstrated that the encystment rate increased with cell concentration, suggesting that there is an inter-amoebal communication. The present study establish for the first time environmental conditions favoring encystment and would lay the foundations to better control the encystment of V. vermiformis.

Growth of Acanthamoeba castellanii and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey.

The growth responses of two species of amoeba were evaluated in the presence of live, heat-killed and heat-killed/5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)-stained cells of Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Klebsiella ozaenae and Staphylococcus aureus. The specific growth rates of both species were significantly higher with live bacterial prey, the only exception being Hartmannella vermiformis feeding on S. aureus, for which growth rates were equivalent on all prey states. There was no significant difference between growth rates, yield or ingestion rates of amoebae feeding on heat-killed or heat-killed/stained bacterial cells, suggesting that it was the heat-killing process that influenced the amoeba-bacteria interaction. Pretreatment of prey cells had a greater influence on amoebic processing of Gram-negative bacteria compared with the Gram-positive bacterium, which appeared to be as a result of the former cells being more difficult to digest and/or losing their ability to deter amoebic ingestion. These antipredatory mechanisms included microcolony formation in P. aeruginosa, toxin production in K. ozaenae, and the presence of an intact capsule in K. aerogenes. E. coli and S. aureus did not appear to possess an antipredator mechanism, although intact cells of the S. aureus were observed in faecal pellets, suggesting that any antipredatory mechanism was occurring at the digestion stage.

Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis.

The potential role of inhaled protozoa as a niche for intrapulmonary replication of Legionella pneumophila was investigated in vivo with mutant strains of L. pneumophila which have reduced virulence for the amoeba Hartmannella vermiformis. L. pneumophila AA488 and AA502 were derived from wild-type strain AA100 after transposon mutagenesis. These mutants have reduced virulence for H. vermiformis but are fully virulent for mononuclear phagocytic cells. A/J mice, which are susceptible to replicative L. pneumophila lung infections, were inoculated intratracheally with L. pneumophila AA100, AA488, or AA502 (10[6] bacteria per mouse) or were coinoculated with one of the L. pneumophila strains (10[6] bacteria per mouse) and uninfected H. vermiformis (10[6] amoebae per mouse). The effect of coinoculation with H. vermiformis on intrapulmonary growth of each L. pneumophila strain was subsequently assessed. In agreement with our previous studies, coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of the parent L. pneumophila strain (AA100). In contrast, intrapulmonary growth of L. pneumophila AA488 or AA502 was not significantly enhanced by coinoculation of mice with H. vermiformis. These studies demonstrate that L. pneumophila virulence for amoebae is required for maximal intrapulmonary growth of the bacteria in mice coinoculated with H. vermiformis and support the hypothesis that inhaled amoebae may potentiate intrapulmonary growth of L. pneumophila by providing a niche for bacterial replication.

Bacteriolytic activities of the free-living soil amoebae, Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis.

Bacteriolytic activities of axenically grown free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of both Acanthamoeba species were higher as those of the three Hartmannella strains. Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus and Pseudomonas fluorescens were more easily lysed than the other bacteria tested. Agrobacterium tumefaciens, Klebsiella aerogenes and Serratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3-10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities in Acanthamoeba and Hartmannella were shown. Bacteriolytic activities of Hartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0-9.3 while for Acanthamoeba the range was pI 4.5-8.9.

[Associations of the soil amoeba Hartmannella rhysodes with the bacterial causative agents of plague and pseudotuberculosis in an experiment]. / Izuchenie assotsiatsii pochvennykh ameb Hartmannella rhysodes s bakteriiami--vozbuditeliami chumy i psevdotuberkuleza v éksperimente.

Some aspects of relationships between soil ameba and the causative agents of plague and pseudotuberculosis, capable of forming natural associations, are considered. Ameba can phagocytose bacteria causing plague and pseudotuberculosis. Cases of the preservation of individual bacterial cells at the stationary phase and in precysts of amebae have been registered.