[Outer membranes of Francisella tularensis and their protein composition]. / Naruzhnye membrany tuliaremiinogo mikroba i ikh belkovyi sostav.

Four strains of the species Francisella tularensis were used in the present work: a live vaccine strain 15/10 and three virulent strains (503, Schu, 543) from three different subspecies. The bacterial membranes were prepared by the 0.5% N-laurylsarcosinate (Sarcosyl) treatment. These membranes were identified as the outer membranes by morphological, immunological and biochemical analyses. The outer membrane proteins contained up to 30-35 polypeptides with three dominant fractions having the 63, 48 and 41-43 kD molecular masses. Despite the significant similarity between the membranes protein profiles there were some quantitative and qualitative differences between the three variants of Francisella tularensis in polypeptides compositions and patterns.

Identification of a heat-modifiable protein of Francisella tularensis and molecular cloning of the encoding gene.

As an initial step in defining the constituents of the outer surfaces of Francisella tularensis, membrane fractions were prepared, and the immunoreactivity of constituents examined by Western immunoblotting. One protein, thought to be an outer membrane protein, was found to be heat and beta-mercaptoethanol (2-ME)-modifiable and was named FopA. This protein migrates at an apparent molecular weight of 34 kilodaltons (kDa) when cell extracts are solubilized below 80 degrees C, but migrates as a doublet of 41- to 43-kDa when cell extracts are solubilized at 95 degrees C. A cosmid bank was constructed and two recombinants were found to express FopA. The recombinant FopA was also heat and beta-mercaptoethanol modifiable and was found to localize in the outer membrane of Escherichia coli.

A capsule-deficient mutant of Francisella tularensis LVS exhibits enhanced sensitivity to killing by serum but diminished sensitivity to killing by polymorphonuclear leukocytes.

The live vaccine strain (LVS) of Francisella tularensis is killed by human polymorphonuclear leukocytes as a result of strictly oxygen-dependent mechanisms (S. Löfgren, A. Tärnvik, M. Thore, and J. Carlsson, Infect. Immun. 43:730-734, 1984). We now report that a capsule-deficient (Cap-) mutant of LVS survives in the leukocytes. In contrast to the encapsulated parent strain, the Cap- mutant was avirulent in mice and was susceptible to the bactericidal effect of nonimmune human serum. The mutant was killed by serum as a result of activation of the classical pathway of complement by naturally occurring immunoglobulin M. This killing by serum was mitigated by the presence of human polymorphonuclear leukocytes. After opsonization in complement component C5-deficient nonimmune serum, the Cap- mutant was ingested and survived in the leukocytes. Under these conditions, the parent strain was killed. The leukocytes responded to both the parent and the Cap- strain with a very low chemiluminescent response. Only the response to the parent strain was inhibited by superoxide dismutase. When the Cap- mutant was opsonized with immunoglobulin G, it induced a higher and superoxide dismutase-inhibitable chemiluminescent response and was killed by the leukocytes. In conclusion, the capsule of F. tularensis LVS seemed to protect this organism against the bactericidal effect of serum. When deprived of the capsule, the organism failed to induce an antimicrobial response in polymorphonuclear leukocytes and survived in the leukocytes. Survival in phagocytes is a key characteristic of intracellular parasites. The Cap- mutant of F. tularensis may become a useful tool in experiments to explain the differences between pathways of ingestion of intracellular parasites, evidenced by the death or survival of the parasite.

Fatty acid distribution in the phospholipids of Francisella tularensis.

Francisella tularensis, LVS (live vaccine strain) grown in a chemically defined medium was found to have a lipid content of 21% by dry weight. The two major phospholipids were identified as phosphatidylethanolamine (PE; 76%) and phosphatidylglycerol (PG; 24%) by thin layer chromatographic analysis, staining characteristics and quantitative chemical analyses of fatty acid, phosphate and glycerol constituents. PE contained a high proportion of 24:0 fatty acid, with lesser amounts of 24:1, 22:0 and 10:0. The major fatty acids of PG were 18:1 and 22:0. Hydroxy fatty acids, which are prominent components of F. tularensis, were conspicuously lacking in these phospholipids; it is therefore concluded that hydroxy fatty acids are constituents of other structures of the organism.

Determination of monounsaturated double-bond position and geometry in the cellular fatty acids of the pathogenic bacterium Francisella tularensis.

The nonhydroxy fatty acid composition of Francisella tularensis is reported in detail. The double-bond configuration of the monounsaturated acids has been determined by capillary gas chromatography-mass spectrometry of the derivatized fatty acids. The monounsaturated fatty acids detected, in decreasing order of abundance, were 24:1 omega 15c, 18:1 omega 9c, 22:1 omega 13c, 20:1 omega 11c, 16:1 omega 7c, 26:1 omega 17c, and 14:1 omega 7c. The fatty acid profile found in F. tularensis, in particular the double-bond positions, represents a valuable taxonomic characteristic of this pathogenic bacterium.

Comparison of the effects of acid and base hydrolyses on hydroxy and cyclopropane fatty acids in bacteria.

The cellular fatty acid compositions of Legionella oakridgensis, Brucella suis, Pseudomonas aeruginosa, and Francisella tularensis were compared after base hydrolysis (saponification), acid hydrolysis, and acid methanolysis procedures were used to release the fatty acids. The branched-chain, unsaturated, saturated, and ester-linked hydroxy acids were released as effectively with saponification at 100 degrees C for 30 min as with acid hydrolysis or acid methanolysis at 85 degrees C for 16 h. Although the amide-linked hydroxy acids were released more effectively by acid hydrolysis or acid methanolysis, these methods degraded the cyclopropane fatty acids, producing a number of new peaks or artifacts in the chromatograms. Cyclopropane fatty acids were not degraded by saponification, and at least 50% of the hydroxy acids were released when the cells were saponified with 15% NaOH in 50% aqueous methanol. Thus, the results show that saponification for 30 min at 100 degrees C with 15% NaOH, followed by methylation is an excellent method for routine fatty acid analysis of bacteria and for screening cultures whose identity and fatty acid composition are unknown.

[Higher fatty acid composition of the intraspecific taxa of Francisella tularensis]. / Sostav vysshikh zhirnykh kislot u vnutrividovykh taksonov Francisella tularensis.

The higher fatty acid composition of 27 F. tularensis strains differing in their biological properties and belonging to different intraspecific taxons has been determined with the use of gas and liquid chromatography. All the strains under study have been found to possess similar fatty acid spectra, characterized by the presence of long straight-chain fatty acids (saturated and unsaturated) with the number of carbon atoms varying from 10 to 26. Quantitative differences content of individual fatty acids (C10:0, C14:0, C24:0) have been revealed in various F. tularensis subspecies, which can serve as an additional criterion for their differentiation. The peculiar features of the higher acid composition of F. Tularensis give grounds for considering this species to be taxonomically independent and characterize its intraspecific taxons by the quantitative content of individual fatty acids.

Cellular fatty acid composition of Francisella tularensis.

Several unusual fatty acids characterized strains of Francisella tularensis. Long-chain (C20-C26) acids and the hydroxy acids 2-hydroxy-decanoate, 3-hydroxy-hexadecanoate, and 3-hydroxy-octadecanoate appeared to be of special diagnostic value.

[Cytopathic effect of the tularemia microbe on a culture of peritoneal macrophages]. / Izuchenie tsitopaticheskogo deistviia tularemiinogo mikroba na kul'turu peritoneal'nykh makrofagov.

Morphological analysis of the process of interaction of tularemia microbe strains differing by virulence with macrophages demonstrated that all these strains produced a lethal effect on macrophages obtained from the animales sensitive to the infection. The macrophages obtained from the animals were but little sensitive to tularemia and were resistant to the action of the causative agent of this infection. The data obtained led to a supposition on the presence in the tularemia causative agent of a factor responsible for its lethal action on the macrophages.

Virulence factors of Francisella tularensis.

The mechanism causing viable Francisella tularensis to lose virulence in aerosols has been investigated. Fully virulent organisms were found to be encapsulated and avirulent organisms from aged aerosols, decapsulated. Capsules were also removed by suspension of F. tularensis in hypertonic sodium chloride. The resulting naked, but viable, organisms were predominantly avirulent for guinea-pigs challenged intraperitoneally. Capsular material and cell walls were found to contain large amounts of lipid, about 50 and 70% (w/w) respectively, and to differ in lipid and sugar composition. Isolated capsular material was not found to contain a lethal toxin for mice or guinea-pigs, or to induce an immunological response in these animals to fully virulent F. tularensis.

Polynucleotide homologies of Brucella deoxyribonucleic acids.

Deoxyribonucleic acids (DNA's) extracted from organisms presently placed in the genus Brucella (B. abortus, B. melitensis, B. neotomae, and B. suis) possessed very similar polynucleotide sequences. Unlabeled, single-stranded DNA fragments from B. abortus, B. melitensis, B. neotomae, and B. suis were equally effective in competing with the interaction of corresponding radiolabeled, single-stranded DNA fragments with their homologous DNA-agars. Unlabeled fragments of B. ovis, however, did not compete as effectively as the homologous, unlabeled DNA's, and this organism, therefore, had a detectably different polynucleotide composition. The mole percentages of guanine plus cytosine in Brucella DNA's (56 to 58%) were also similar. DNA's from Francisella tularensis, Escherichia coli, and the slow loris did not compete.