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Acanthamoeba castellanii life habits and mechanisms of self preservation Deborah Moore, Wendy Trzyna, David Neff, Michael Norton, Hongwei D. Yu, and John Barry Departments of Biochemistry, Chemistry, and Biology at Marshall University and MU School of Medicine (JCESOM) Huntington, WV Spring 2016 Introduction: Ongoing work in the Trzyna lab at Marshall U. is focused on characterizing phenotypic properties and stress response mechanisms of the protozoa Acanthamoeba castellanii. These eukaryotes of clade Amoebozoa are a species of gymnamoeba (naked amoeba) that fills niches in diverse aquatic and soil environments. These organisms are also opportunistic pathogens of humans and are associated with certain pathogenic and non-pathogenic bacterial species such as Pseudomonas and E. coli. Of particular interest here are strains of Pseudomonas aeuroginosa that have been selectively cultured in the Yu lab to be more or less pathogenic and to express green fluorescent protein (GFP) to allow fluorescence imaging of the bacteria as they interact with amoebae. Specifically, we studied the feeding behaviours of A. castellanii, their relationship with bacteria both as food source and endosymbiont, and the process of encystment which protects the organism from death during times of food shortage or environmental stress. These cysts are essentially encapsulated in a double walled capsule containing cellulose. During the spring semester of 2016, we used scanning electron microscopy (SEM) and confocal laser scanning microscopy (CSLM) to study both the free living metabolically active trophozoite form and dormant cyst form. We imaged fixed and dehydrated trophozoites and cysts with SEM. Trophozoites were imaged with CSLM in transmitted light and using fluorescent emissions from GFP. Cysts were imaged with CSLM after being stained with cellulose specific fluorescent dye calcofluor white. e- source (–) relative to anode Culturing A. castellanii: To maintain axenic (i.e. without bacteria) cultures, Acanthamoeba are kept in logarithmic growth to a mid-log phase of ~2x10^6 cells/mL in PGY (Proteose Glucose Yeast) liquid media in 125mL Erlenmeyer flasks shaking at 200rpm and set to 30⁰C. These cultures were sub-cultured every 3 days to maintain healthy logarithmic cultures. Green fluorescent protein expressing bacteria were also fed to amoeba in liquid culture to observe feeding behavior and the morphological details of the amoeba/bacteria symbiotic relationship. Alternatively, motile ameobae in trophic form were cultured by feeding them bacteria immobilized on a nutrient-poor agar gel plate (as seen in figure 4 at right). To induce encystment, logarithmic cultures of amoebae were washed and transferred in NEM (Neff Encystment Media) media, which is a special media used for the sole purpose of inducing encystment. Once set in the media, cultures were placed in a shaking incubator at 200rpm and which was set at 30⁰C. Figure 2. SEM image (right) of a mature fixed and dehydrated cyst (glutaraldehyde followed by ethanol dehydration series). Image of the cell was captured using the SEM at 20kV and 5000x. Scale bar = 5 micrometers. Figure 4. Migration assay on agar plate with bacterial lawn. Amoebae migrate up bacterial end of migration assay smear (marked with black vertical lines) on plate seen at right with advancing front of proliferation seen as a dense band of amoebae feeing while moving upwards with less dense populations left behind where food start sources are depleted. This front of proliferation can be seen at higher magnification in the 3 images directly below. This assay is being used to determine virulence of bacterial species of interest based on relative migration rate of amoebae. The particular strains studied Arrow represents 24 hours of migration, here are ~0.8cm important in the pathology of cystic fibrosis. 4.5cm direction of movement Figure 5. SEM image (left) of a fixed and dehydrated trophozoite (glutaraldehyde followed by ethanol series). Imaged with SEM at 20kV and 5000x magnification. Panels below A-C show the interaction of Acanthamoeba castellanii trophozoites with Pseudomonas aeruginosa strain PAO1V Δaroa, while bottom panels D-F show interaction with a different less virulent strain PAO1V. Micrographs were taken approx. 45 minutes after first contact of bacterial cells with amoeba. Imaging was done with a confocal scanning laser microscope with blue Argon laser excitation and green narrow band emissions collected. Scale bar =10um for all images. Greyscale transmitted light images shown for context. Contractile vacuole is outlined in dashed red lines, and cell nucleus in white. 24h anode relatively (+) here 20,000V 48h Figure 1. Top photos show two of the scanning microscopes housed at Marshall University, left is the Leica SP5 CSLM and right is JEOL5310-LV SEM. Bottom row shows diagrams of each indicating that the CSLM scans a focused laser beam to elicit fluorescence which can be selected for imaging based on wavelength while the SEM scans an electron beam across the sample to generate electron signal. MU College of Science http://www.marshall.edu/cos/ MU Molecular and Biological Imaging Center http://www.marshall.edu/mbic/ Joan C. Edwards Scool of Medicine-MU https://jcesom.marshall.edu/ Figure 3. Top pictures show a maturing cyst in brightfield (left) and brightfield with calcofluor white fluorescent emission overlay (right) after 24 hours post NEM. Bottom pictures show a near mature cyst after 48 hours NEM. Long UV (405nm) laser excitation and blue narrow band emissions collected with Leica SP5 CSLM. Scale bar = 5 micrometers. At left is a time lapse image series of a single A. castellanii trophozoite feeding on GFP expressing Peudomonas bacteria in liquid media over the course of about 1 hour. The frame was shifted 2-3 times during this hour to keep the moving amoeba in field. References: Cordingley, J. S., & Trzyna, W. C. (2008). Multiple factors affecting growth and encystment of Acanthamoeba castellanii in axenic culture. Acta Protozoologica, 47(4), 307-316. Cometa, I., Schatz, S., Trzyna, W., Rogerson, A. (2011) Tolerance of naked amoebae to low oxygen levels with an emphasis on the genus Acanthamoeba. Acta Protozoologica, 50: 33-40.