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Type: Thesis
Title: Cell biology of the interaction between Listeria monocytogenes and Colpoda spp.
Author: Raghu Nadhanan, Rethish
Issue Date: 2012
School/Discipline: School of Molecular and Biomedical Science
Abstract: Intracellular bacterial pathogens, such as Listeria monocytogenes, survive and multiply within mammalian cells. However, interaction with protozoans in the external environment may protect this pathogen from harsh conditions. In particular, if L. monocytogenes is able to survive within these protozoans, these cellular hosts may act as a vehicle that links contamination of food processing environments to contamination of foods. Though Colpoda ciliates are a very common type of protozoa, current knowledge on the interaction between intracellular bacterial pathogens and Colpoda at cellular level is limited. The interaction of L. monocytogenes and Colpoda ciliates was investigated in the present study. Co-cultures of planktonic and biofilm L. monocytogenes DRDC8 with Colpoda sp. strains RR (isolate of natural environment) and strain MLS-5 (isolate of food processing environment) at 25°C were used to examine this interaction. Bacteriological counts and microscopy (fluorescence and transmission electron microscopy (TEM)) were used to track the fate of internalized L. monocytogenes within the ciliates. TEA chloride was used to inhibit phagocytosis to determine if L. monocytogenes induce its own uptake into Colpoda. Grazing of Colpoda on L. monocytogenes biofilms and changes in biofilm structures were evaluated by crystal violet assay and scanning electron microscopy (SEM). Mechanisms utilized by Colpoda RR in killing and degradation of DRDC8 were investigated by using chemical inhibitors of phagosome-lysosome fusion (NH₄Cl), vacuolar acidification (bafilomycin A1 and monensin), proteases (protease inhibitor cocktail) and nitric oxide (L-NMMA). In addition, the ability of Colpoda to secrete faecal pellets containing bacteria following co-cultures with DRDC8 was examined. Co-culture of DRDC8 with Colpoda RR and MLS-5 provided direct evidence that these ciliates were able to actively phagocytose and kill planktonic and biofilm forms of DRDC8. L. monocytogenes was unable to initiate its own uptake into either Colpoda RR or MLS-5 and the level of expression of Listeriolysin O did not influence the outcome of coculture. The increase in viable counts of Colpoda following feeding with DRDC8 together with a concomitant reduction in viable counts of intra-ciliate DRDC8 within a 4 h period, indicated Colpoda used L. monocytogenes as a food source. This was confirmed by observations that internalized DRDC8 were confined within tight vacuoles the presence of large food vacuoles containing many electron-dense bacteria-sized particulates within Colpoda cytosol. Colpoda RR also effectively phagocytosed and degraded S. Typhimurium C5, as well as several non-pathogenic bacteria such as B. subtilis and E. coli DH5α. An important and novel outcome was the observation that induction of encystment of DRDC8-fed Colpoda RR, lead to the entrapment of bacterial cells within cyst outer walls and the cytosol. Furthermore, co-cultures of DRDC8 with Colpoda RR and MLS-5 resulted in the secretion of faecal pellets containing intact, viable and respiring DRDC8 cells. Bacteriological counts confirmed that faecal pellet-located DRDC8 were resistant to concentrations of gentamycin (up to 100 μg mL⁻ ¹) and sodium hypochlorite (up to 10%), that were well above concentrations that are otherwise lethal to suspensions of DRDC8. Fluorescence microscopy of acidotrophic stains Lysosensor(TM) Blue DND 167 and acridine orange treated Colpoda cells showed lysosomes fused with DRDC8-containing vacuoles within Colpoda RR. Following treatment of co-cultures with NH¹Cl, bafilomycin A1 and protease inhibitor cocktail, viable intra-ciliate bacterial counts and TEM showed evidence of survival without replication of DRDC8 within inhibitor-treated Colpoda RR for up to at 24 h post feeding. These outcomes indicated that Colpoda RR employs phagosome-lysosome fusion, vacuole acidification and proteases as mechanisms to kill intra-ciliate L. monocytogenes. However, nitric oxide was not involved in the killing of DRDC8 within Colpoda. Both Colpoda strains used, actively phagocytosed and killed L. monocytogenes. L. monocytogenes were unable to escape the ciliate phagocytic vacuole and establish an intracellular lifestyle within Colpoda. This conclusion is in stark contrast to observations of the fate of L. monocytogenes cells within mammalian cells in which these bacteria escape into the cytosol in a Listeriolysin O dependent manner and then spread from cell to cell. However, the release of Colpoda-derived faecal pellets containing viable L. monocytogenes, indicated that these encapsulated forms of bacteria may provide a reservoir and a mechanism for transmission of pathogens. Considering that a faecal pelletlocation endows bacteria with resistance to disinfectants and cleaning agents used in food manufacturing and preparation facilities, this may explain why L. monocytogenes is difficult to eradicate from food processing facilities.
Advisor: Thomas, Connor Jocelyn
Morona, Renato
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2012
Keywords: Listeria monocytogenes; Colpoda; phagocytosis; co-culture
Appears in Collections:Research Theses

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