Chemotactic responses of Acanthamoeba castellanii to bacteria, bacterial components, and chemotactic peptides

Abstract

Chemotactic responses of Acanthamoeba castellanii to bacteria, several representative bacterial products, and chemotactic peptides were studied by following migration of amebas under agar. Amebas showed a positive chemotactic response to all bacterial species tested, even to those which were not ingested by amebas because of toxic pigments. Lipopolysaccharide and lipoteichoic acid, components of the outer membrane and cell wall, respectively, of Gram-negative and Gram-positive bacteria, evoked a neutral response from the amebas indicating that they are not attractants. Cyclic adenosine monophosphate, either as a bacterial product or as a pure compound was not an attractant. The chemotactic peptide formyl-methionyl-leucyl-phenylalanine served as an attractant, but the antagonist peptides N-t-boc-norleucyl-leucyl-phenylalanine and N-t-boc-methionylleucyl-phenylalanine did not. Amebas respond to these chemical stimuli, probably by means of membrane receptors. Chemotaxis is an oriented response to a stimulus (Wilkinson, 1982). It may be an important factor in the location of bacteria by soil amebas, as it has been shown to be for mammalian phagocytes locating invading microbes (Hugli, 1989). Amebas such as Acanthamoeba, a free-living protozoon found in soil and water, share a number of similarities with phagocytes in that both types of cells ingest bacteria and probably possess some mechanism that enables them to locate these microbes. A large body of information is available about the chemotactic responses of mammalian phagocytes (Devreotes & Zigmond, 1988; Wilkinson, 1982), but less is known about mechanisms used by amebas in locating bacteria. Studies of Acanthamoeba and Hartmannella (see McIntyre & Jenkin, 1969; Tharavanij, 1965; Urquhart, 1984) have indicated that a chemotactic factor present in, or released from, bacteria serves as the signal in attracting amebas, and that both chemotaxis and chemokinesis are exhibited by Naegleria fowleri toward bacteria and chemotactic peptide (MarcianoCabral & Cline, 1987). Entamoeba histolytica was shown to migrate toward filtrates of Escherichia coli, as well as toward complement component C5a, and lysed human erythrocytes (Urban et al., 1983). This study was undertaken to examine chemotaxis in Acanthamoeba, using migration under agar toward bacteria, bacterial components, and chemotactic peptides as a means of evaluating the ability of soil amebas to locate microbial I The authors thank Dr. Savanat Tharavanij, Mahidol University, Bangkok, Thailand, for making available sections of his doctoral thesis dealing with chemotaxis of amebas. We thank Professor David Raab, Brooklyn College, Psychology Department, for advice on statistical treatment of data. This research was supported in part by a City University of New York research award 669171 and by NIH-NIGMS Grant 5T34 GM08078 (NIH-MARC Program). Portions of this research were presented at meetings of the Society of Protozoologists. TRANS. AM. MICROSC. Soc., 112(1): 43-61. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.57 on Sat, 10 Sep 2016 05:40:07 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. food sources. Our results support a chemotactic response of amebas to bacteria, as well as to the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, a chemoattractant for mammalian phagocytes (Schiffman et al., 1975). MATERIALS AND METHODS Acanthamoeba castellanii (Neff strain) was grown axenically in Oxoid proteose-peptone (2% w/v)/yeast extract (0.5% w/v)/glucose (0.5% w/v), pH 7.2 at room temperature (ca. 25?C), with a generation time of about 13 h. Cultures were maintained in Corning tissue culture flasks (75 cm2), and amebas for use in experiments were harvested while in logarithmic growth phase (2-3 days old). At time of harvest, flasks were chilled on ice and amebas dislodged from the growth surface by several sharp raps to the side of the growth vessel. Amebas were washed three times in dilute saline (Page, 1967) at 4?C in a clinical centrifugation (200 x g), and kept chilled until ready to use. The ameba suspension was counted in a Coulter Counter (model ZF) and cell numbers were adjusted for use in experiments. Preparation of chemotaxis plates. Five ml of sterile melted Difco Noble agar (1.5% w/v) prepared in dilute saline was pipetted into sterile 60 x 15 mm Nunclon? tissue culture Petri dishes (Nunc, Denmark) having a grid pattern of 2-mm squares. The agar was allowed to harden, at which time wells (6-mm diameter) were punched in the agar with a no. 3 cork borer, using a template. The agar plug was removed with a Pasteur pipette attached to a vacuum line. Agar plates were freshly prepared at the time of each experiment to minimize drying of the agar and, for the same reason, holes were punched in the agar at the time they were to be filled. The cork borer used for making wells was sterilized by immersion in 95% (v/v) ethanol, followed by flaming to burn off the alcohol. Agarose (Litex, type HSA; Accurate Chemical & Scientific Corp., Westbury, New York) was used instead of agar in several experiments at 1 and 1.5% w/v. Three wells were made in a line in the agar (Nelson & Herron, 1988). One well was located in the center of the plate and the other two wells along an axis with the center well (one well at 1200 hours and the other at 1800 hours), using the grid pattern for orientation. The center-to-center spacing between wells was 20 mm (or 10 squares). Amebas were added to the center well; the test substance (bacteria, bacterial products, etc.) in agar was added to the second well (the 1200 hours well). The third well (the 1800 hours well) served as the control well, receiving saline or solvent used in preparing the test material added to agar. Each well could hold 50 tl of fluid. Figure 1 is a schematic representation illustrating the experimental design as seen on the plate surface, with stippling to indicate the distribution of amebas around the center well. Preparation of test substance. When live bacteria were used as test substance, they were grown either in nutrient broth or brain heart-infusion media at 37?C. Cultures were transferred daily over several days. For experiments, overnight cultures grown with shaking were harvested and washed three times with dilute saline using a Sorvall RC-2 centrifuge (12,000 x g), and suspended in saline to 44 This content downloaded from 207.46.13.57 on Sat, 10 Sep 2016 05:40:07 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 1, JANUARY 1993