L. monocytogenes is ubiquitous in environment and can grow and survive in a wide range of environmental conditions. It contaminates foods via raw materials or food processing environments. However, the current knowledge of its ecology and in particular, the mode of environmental survival and transmission of L. monocytogenes remains limited. One important aspect of environmental survival of L. monocytogenes may be contact with other microorganisms, including amoebae, which naturally feed on bacteria as a source of nutrients. In this context, research has shown that several intra-cellular pathogens are able to survive or replicate within free-living amoebae. In view of the potential for amoebae to act as environmental reservoirs for bacteria, the interaction of L. monocytogenes with freeliving Acanthamoeba spp. and the impact of plasmid-associated genes on their interaction were investigated. Several strains of environmental and clinical isolates of L. monocytogenes were used for co-culture with amoebae. Axenic amoebae were isolated from environmental sources (water, soil) by cultivation in PYG supplemented with antibiotics. L. monocytogenes strains were co-cultured with amoebae on plates, in trays (as monolayers of amoeba cells) and in flasks to provide qualitative and quantitative assessments of the survival of bacteria and amoebae. Bacteriological methods and microscopy (fluorescence, TEM and phase contrast) were used to track the fate of internalized bacteria. A vector that allowed GFP expression under control of the prfA promoter was used to assess the expression of Listeria virulence genes within amoeba cells. The role plasmid encoded genes in interactions of L. monocytogenes with amoebae was assessed using plasmid-cured versus wild type in the co-cultures. Finally the mechanisms of bacterial uptake and killing by A. polyphaga were assessed using chemical inhibitors that affected actin polymerization (Cytochalasin D, Wortmannin), phagosome-lysosome fusion (Suramin) and phagosomal acidification (ammonium chloride, Bafilomycin A and Monensin). Sequence analysis of a section of the large plasmid from strain DRDC8 revealed a high level of similarity of gene organization and DNA sequences with plasmid-borne genes found in other Listeria spp. and Gram-positive bacteria. While the majority of environmental isolates of L. monocytogenes contained a large plasmid, it was absent in clinical isolates. Further, plasmid of DRDC8 was lost during serial passage in HeLa cells. This data indicated that the plasmid may be readily lost during isolation procedures or during growth within host animals/cells and thus plasmid instability may explain the absence of plasmid in clinical isolates. Co-culture of L. monocytogenes with Acanthamoeba spp. showed these amoebae are able to actively phagocytose and kill bacteria within 2-5 h irrespective of temperature used. Amoebae killed both plasmid cured and LLO mutants with the same rate for the parental bacteria. Fluorescence microscopy and TEM of bacteria within trophozoites showed the bacteria become confined within tight vacuolar structures surrounded by lysosomes and mitochondria and degraded after 4 to 5 h post phagocytosis. This data indicated that although L. monocytogenes is an effective pathogen of mammalian cells, it could not escape from phagosomes and evade the killing mechanisms of amoeba trophozoites. Consequently, bacteria are killed within phagolysosome in trophozoites before they express their virulence genes to escape from phagosomes and get access to cytoplasm. This was confirmed by observing no GFP expression by bacteria carried prfA::gfp construct in co-culture with amoebae whereas it was observed during co-culture with HeLa cells. Using inhibitors, the mechanisms involved in phagocytosis, and killing of L. monocytogenes cells by A. polyphaga were assessed. The results showed that the uptake of bacterial cells is mediated by the trophozoites but not the bacteria and mannose binding protein is not involved in this process. Furthermore, pre-treatment of trophozoites with inhibitors indicated both phagosomal acidification and phagosome lysosome fusion are involved in killing of bacteria. These results suggest that compared to mammalian cells e.g. HeLa cells, amoeba trophozoites are better able to effectively inactivate and destroy internalized L. monocytogenes cells. In conclusion, Acanthamoeba spp. are able to uptake and kill L. monocytogenes cells in phagolysosome compartments. However, bacteria can saprophyticaly grow on materials released from amoeba trophozoites. Thus this group of amoebae is not able to harbour L. monocytogenes cells, or act as environmental reservoirs for this opportunistic pathogen under the laboratory conditions tested. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292868 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007
| Identifer | oai:union.ndltd.org:ADTP/264443 |
| Date | January 2007 |
| Creators | Akya, Alisha |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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