title: The association between legionellae and amoebae
Creators: name: Doust, Seyed Reza Hosseini
Publisher: name: University of Glasgow
Year: 1995
Subjects: subject: Microbiology.

description: Legionella pneumophila and Acanthamoeba castellanii were selected as the model for investigation of amoebae-legionellae interactions. The number of legionellae recovered from the co-culture, increased with time of incubation, while E. coli (serving as particulate food for amoebae in the Laboratory) numbers fell, due to ingestion and digestion by the amoebae during the same period of incubation. Amoebae appeared to restrict the multiplication of legionella at lower temperatures. However, when the temperature was increased (37°C) legionellae multiplied within amoebae and finally lysed the amoebae host cells. Cytochalasin D and methylamine were used in order to investigate the phagocytosis process in amoebae. Cytochalasin D (2 muM) (an inhibitor of actin filaments), had no significant effect on uptake and intra-amoebal growth of legionella. We concluded that uptake and intra-amoebal multiplication of legionella may occur, in part by a microfilament- independent process. The phagocytic activity of amoebae was inhibited in the presence of methylamine (100 mM) in amoeba -legionella co-culture. We concluded that receptor-mediated pinocytosis may be involve in uptake and intra-amoebal growth of legionella. The intra-amoeba conditions had abstruse effects on the legionella cell envelope composition. Unique lipopolysaccharide patterns were found in intra-amoeba grown legionella. Some of the major components of the legionella cell envelope, such as MOMP (28 kD) were expressed by either in vitro or intra-amoeba grown legionella. A striking feature of intra-amoeba grown legionella in this study was the expression of a 15 kD novel outer membrane protein. The appearance of new surface components in in vivo grown cells, such as the 15 kD protein identified in this study, may fulfil functions such as inhibition of phagosome-lysosome fusion in the amoeba host cell. We attempted to show whether or not the fusion of lysosomes with phagosomes is inhibited following phagocytosis of legionellae by amoebae. The information obtained by fluorescent microscopy, indicated that legionellae were phagocytosed by amoebae in a manner similar to E. coli and latex beads, but legionella-containing phagosomes persisted and failed to fuse with amoebae lysosomes. Further details of phagosome- lysosome fusion at ultra-structural level would be useful to confirm the present conclusion. Lucigenin-dependent chemiluminescence was used to measure photon emission in amoebae following phagocytic activity. We attempted to compare the magnitude of the oxidative burst following ingestion of E. coli, latex beads and L. pneumophila by measuring the lucigenin- dependent CL responses. CL in Acanthamoeba castellanii, an alternative professional phagocytic cell, generated a low level of lucigenin-dependent CL following phagocytosis of these stimulants. However, this CL response was barely above the background level, and two orders of magnitude lower than the responses found in mammalian phagocytic cells. The association of legionellae and amoebae was investigated in water samples obtained from different hospitals in the UK. A novel enrichment method using amoebae suitable for legionella detection in environmental water supplies was developed. Legionellae and free-living amoebae were isolated from 25% of water samples. Different genera of free-living amoeba and ciliates were observed in 46% of water samples. Totally, 49% of water samples were contaminated by potential pathogens, either legionellae or amoebae. Using the above novel enrichment method, we isolated L. pneumophila from 15% of water samples which were reported negative by the conventional culture method. We recommend this enrichment method together with PCR (where possible) for detection of legionella, especially in culture-negative water samples.