Lipid composition of multilamellar bodies secreted by Dictyostelium discoideum reveals their amoebal origin

Abstract

When they are fed with bacteria, Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs), which are composed of membranous material. It has been proposed that MLBs are a waste disposal system that allows D. discoideum to eliminate undigested bacterial remains. However, the real function of MLBs remains unknown. Determination of the biochemical composition of MLBs, especially lipids, represents a way to gain information about the role of these structures. To allow these analyses, a protocol involving various centrifugation procedures has been developed to purify secreted MLBs from amoeba-bacterium cocultures. The purity of the MLB preparation was confirmed by transmission electron microscopy and by immunofluorescence using H36, an antibody that binds to MLBs. The lipid and fatty acid compositions of pure MLBs were then analyzed by high-performance thin-layer chromatography (HPTLC) and gas chromatography (GC), respectively, and compared to those of amoebae as well as bacteria used as a food source. While the bacteria were devoid of phosphatidylcholine (PC) and phosphatidylinositol (PI), these two polar lipid species were major classes of lipids in MLBs and amoebae. Similarly, the fatty acid composition of MLBs and amoebae was characterized by the presence of polyunsaturated fatty acids, while cyclic fatty acids were found only in bacteria. These results strongly suggest that the lipids constituting the MLBs originate from the amoebal metabolism rather than from undigested bacterial membranes. This opens the possibility that MLBs, instead of being a waste disposal system, have unsuspected roles in D. discoideum physiology.

Fig 1

D. discoideum cells grown on K. aerogenes produce and secrete MLBs. (A) Image of a phagocytic plaque. In the center (ce) of the plaque, amoebae have consumed virtually all of the bacteria and have begun multicellular development. At the periphery (pe) of the plaque, amoebae are still in the vegetative form and are crawling and feeding on bacteria. (B) TEM image of D. discoideum cells grown in liquid medium (HL5). (C to F) TEM images of material from the periphery of a phagocytic plaque of D. discoideum cells grown on K. aerogenes. In panel C, amoebae (*), bacteria (pinheads), and secreted MLBs (arrows) are highlighted. An MLB in a lysosomal compartment is shown in panel D, while secreted MLBs are shown in panels E and F. Scale bars: A, 2 mm; B and C, 2 μm; D and E, 0.2 μm; F, 0.5 μm.

Fig 2

D. discoideum cells grown in liquid medium with K. aerogenes produce and secrete MLBs. Shown are TEM images of an MLB in an lysosomal compartment in a D. discoideum cell grown in liquid medium (HL5) in the presence of K. aerogenes (A) and a secreted MLB (B) under the same culture conditions. Scale bars, 0.2 μm.

Fig 3

H36 labeling of partially purified secreted MLBs. Secreted MLBs were purified using the protocol proposed by Barondes et al. (2). The purified material was processed for TEM (A and B) or deposited on a glass slide and processed for DAPI staining and immunofluorescence using the H36 (C), H72 (D), or H161 (E) antibody or for colabeling with H36 and H72-Zenon (F) or with H36 and H161-Zenon (G). (A and B) TEM images. Bacteria (pinheads), secreted MLBs (arrows), and small, unidentified particles (*) are highlighted in panel B. (C) Epifluorescence microscopic images. DIC, differential interference contrast. Some MLBs (DAPI-negative structures) with H36 labeling are indicated with arrows in panel C. Some MLBs with sparse, irregular, or no H36 labeling are indicated with pinheads. Some MLBs are also indicated with arrows in panels D and E. Scale bars: A, 2 μm; B, 0.5 μm; C to G, 5 μm.

Fig 4

Protocol for purification of MLBs secreted by D. discoideum. (A) Schematic representation of the purification protocol of secreted MLBs. When the bacterial lawn was almost entirely eaten by amoebae, the biomass found on the petri dish was harvested and resuspended in starvation buffer (step 1). Amoebae were removed by centrifugation. In step 2, the supernatant was put back into a coculture with fresh amoebae for 24 h. This step allowed the elimination of residual bacteria. Amoebae were removed again by centrifugation (step 3). In step 4, part of the supernatant was deposited on the top of a sodium bromide density gradient and then centrifuged to concentrate MLBs in a small aggregate separated from other contaminants. Finally, MLBs were recovered from the gradient and washed twice in PBS (step 5). (B and C) Images of a sodium bromide density gradient after centrifugation. MLBs were found in many area of the gradient but were in a pure state only in the aggregate (layer 2). The aggregate from the gradient in panel B is highlighted (white circle) in the magnified image shown in panel C.

Fig 5

Purity of MLB preparations confirmed by TEM and immunofluorescence. After the passage throughout the new purification method, the purity of MLBs from layer 2 was confirmed by TEM (A to C) and immunofluorescence (D and E). (A) Field of purified MLBs. (B) Single MLB. The purified MLBs have the typical multilayered morphology already described. (C) K. aerogenes in pure culture. (D and E) Two series of images from immunofluorescence analyses of two different fields are shown. By differential interference contrast (DIC), a lot of small, round structures corresponding to MLBs were visible (D) as well as small (D) or large (E) MLB aggregates. The H36 antibody stains these structures. The two fields shown here are DAPI negative, suggesting the absence of DNA (i.e., bacteria) after the purification procedure was performed. Scale bars for D and E, 4 μm.

Fig 6

Total lipid profile of purified MLBs, amoebae, and K. aerogenes. The lipids were extracted, separated by HPTLC, and quantified. The results are expressed as the mean values ± standard deviations of 3 independent analyses for the MLBs, 6 for amoebae, and 3 for K. aerogenes (Ka).

Fig 7

Fatty acid compositions of MLBs, amoebae, and K. aerogenes. The fatty acids were analyzed and quantified by GC. The results are expressed as the mean values ± SD of 5 independent analyses for the MLBs, 5 for amoebae, and 3 for K. aerogenes (Ka). Names of fatty acids: C_12:0, lauric acid; C_14:0, myristic acid; C_15:0, pentadecanoic acid; C_16:0, palmitic acid; C_16:1, palmitoleic acid; C₁₇ cyc, margaric acid; C_18:0, stearic acid; C_18:1, oleic acid (trans or cis); C_18:2, linoleic acid; C_18:3, linolenic acid; C₁₉ cyc, nonadecylic acid; C_20:0, arachidic acid.