A new genus and species of obligate intracellular bacterial parasite of small free-living amoebae is described. This bacterium causes fatal infections in amoebae belonging to the Acanthamoeba-NaegZeria group. It does not grow on any artificial substrate deprived of living amoeba cells. The entry of the bacterium into a host occurs by phagocytosis, but growth occurs in the cytoplasm, not in phagosomes. This parasite is readily distinguished from other kinds of previously recognized bacteria that live within amoeba cells on the basis of its host cell lytic activity. The bacterium is a gram-negative, short rod with tapered ends. It multiplies intracellularly by a transverse central pinching-off process. Cells are motile by means of a polar tuft of flagella. The bacterium is surrounded by a distinct multilayered cell envelope with a chemtype A,y peptidoglycan. The peptidoglycan is unique in its high content of glucosamine residues with free amino groups. The DNA base composition of this organism is 43 mol% guanine plus cytosine. The name Sarcobium Zyticum gen. nov., sp. nov. is proposed for this bacterium. The type strain of S. Zyticum is strain L2 (= PCM 2298).More than 30 years ago a new obligate intracellular bacterial parasite (OIBP) which causes fatal infections of hosts was found in a raw culture of amoebae that were freshly isolated from soil (7). This OIBP was isolated from soil from the Lublin area, Poland. In previous reports I have described the infection processes for Acanthamoeba castellanii, Hartmannella rhysodes, Hartmannella astronyxis, Mayorella palestinensis, Didasculus thorntoni, Schizopyrenus ruselli, and Naegleria gruberii, as well as 41 other unclassified amoebae isolated from soil and water reservoirs (8, 10). The OIBP did not grow on any medium when living amoebae were not present. Even a thick suspension of disrupted amoeba cells was ineffective. It has been shown that the OIBP cannot multiply if after infection the mixed culture is heated at 43°C for 10 min; this treatment is sufficient to kill trophic forms of the amoeba, but the bacterium survives. Microscopy has shown (8,11). that the parasites are readily phagocytized by amoebae and that their immediate place of residence is within phagosomes. The neighboring phagosomes fuse extensively with parasite-containing vacuoles; however, even dead bacteria evade lysosoma1 attack because the cell wall is resistant to the host's bacteriolytic enzymes. Later, living bacteria escape from phagosomes into the cytoplasm, where multiplication occurs. The final effect of the infection is total lysis of the host cells. Although the morphology of the OIBP and the presence of peptidoglycan in its cell wall suggest that this organism is a bacterium, it has not been possible to classify it previously because of a lack of nutritional tests; the OIBP does not correspond to any bacterium described in Bergey's Manual of Determinative Bacteriology (3). Most of the many kinds of previously recognized bacteria that live within amoeba cells behave as temperate endos...
The composition of phospholipids from the cellular envelope of Legionella lytica grown on artificial medium was determined by two-dimensional thin-layer chromatography. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidyl-N-monomethylethanolamine were the predominant phospholipids, while diphosphatidylglycerol, phosphatidylglycerol, and phosphatidyl-N,N-dimethylethanolamine were present at low concentrations. A trace amount of lipids carrying glycosyl residues was also observed. The fatty acids and their distribution in individual phospholipids were characterized using liquid chromatography/mass spectrometry (LC/MS), matrix-assisted laser desorption ionization-time of flight, and gas chromatography/MS methods. The characteristic feature of L. lytica phospholipids was the presence of an unbranched chain (which differentiates this bacterium from Legionella pneumophila) and branched iso and anteiso fatty acids as well as cis-9,10-methylenehexadecanoic acid. According to spectroscopic LC/MS data, the localization of saturated and unsaturated fatty acid residues on phosphorylglycerol was determined. Some aspects of the significance of phosphatidylcholine, one of the main phospholipids in L. lytica, are addressed and taxonomic implications of the data are discussed.
Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3-hydroxymyristic acid. The cellfree preparation also exhibited amidase activity cleaving about 50% of the amide-bound 3-hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester-bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl-8 1,6-glucosamine disaccharide was not degraded and remained bound to the oligosacchdride part (heptose/3-deoxyoctulosonic acid) of the lipopolysaccharide.Lipopolysaccharides are major constituents of the cell wall of gram-negative bacteria. Their general structural principle has been evaluated and their biological significance as endotoxins studied. The lipid component, lipid A, has been recognized as representing the endotoxically active component of lipopolysaccharides [l].It has been shown previously that the slime mold Dictyostelium discoideum, when fed on Salmonella bacteria, would degrade the corresponding lipopolysaccharide by removing and metabolizing the fatty acyl residues of the lipid A component, and to excrete the water-soluble non-toxic residual polysaccharide molecule into the medium [2]. Later, Khorana and coworkers isolated and purified two amidases from D. discoideum [3 -51. These enzymes act in a very specific way in vitro on lipopolysacchdrides by sequentially removing the two amide-bound 3-hydroxyacyl residues, however, only after the ester-bound fatty acids have previously been removed from the molecule by alkaline hydrolysis.It has long been known that the endotoxic properties of lipopolysaccharides (and lipid A) are dependent on the presence of ester-linked fatty acids [I]. Their removal by alkali treatment results in a substantial decrease in endotoxic activity of lipopolysaccharides and lipid A. However, the possibility could not be excluded that such chemical treatment of lipopolysaccharides might cause unrecognized degradation reactions of the molecule in addition of cleaving 0-acyl residues. We therefore were interested in obtaining esterases which would specifically remove 0-acyl groups enzymatically. This paper describes the presence of esterases and other hydrolytic enzymes in cell-free crude extracts of the protozoa Correspondence to W . Drozanski, Instytut Mikrobiologii, Uniwersytet M. Curic-Sklodowskiej,
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