Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

Subcellular localization of rickettsial invasion protein, InvA.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

The genome of Rickettsia prowazekii and some thoughts on the origin of mitochondria and hydrogenosomes.

The sequence of an alpha-proteobacterial genome, that of Rickettsia prowazekii, is a substantial advance in microbial and evolutionary biology. The genome of this obligately aerobic intracellular parasite is small and is apparently still undergoing reduction, reflecting gene losses attributable to its intracellular parasitic lifestyle. Evolutionary analyses of proteins encoded in the genome contain the strongest phylogenetic evidence to date for the view that mitochondria descend from alpha-proteobacteria. Although both Rickettsia and mitochondrial genomes are highly reduced, it appears that genome reduction in these lineages has occurred independently. Rickettsia's genome encodes an ATP-generating machinery that is strikingly similar to that of aerobic mitochondria. But it does not encode homologues for the ATP-producing pathways of anaerobic mitochondria or hydrogenosomes, leaving an important issue regarding the origin and nature of the ancestral mitochondrial symbiont unresolved.

The concentrations of stable RNA and ribosomes in Rickettsia prowazekii.

The obligate intracellular parasite, Rickettsia prowazekii, is a slow-growing bacterium with a doubling time of about 10 h. In the present study, DNA and RNA were obtained from the rickettsiae by two independent methods, i.e. simultaneous isolation of DNA and RNA from the same sample by phenol:chloroform extraction and CsCI gradient centrifugation. In addition, ribosomal RNA was obtained by sedimentation of partially purified ribosomes from the rickettsiae. The results demonstrated that, after correction for the cell volumes, the concentrations of stable RNA and ribosomes in R. prowazekii, a slow-growing organism, were about 62 fg micron-3 and 17,000 per micron3, respectively, which were very similar (66 fg micron-3 and 21,000 per micron3) to those in Escherichia coli with a generation time of 40 min. However, on a per cell basis, R. prowazekii had 5.6 fg of RNA and 1500 ribosomes per cell, which was only about 8% of the amount of both stable RNA (71.2 fg) and ribosomes (24,000) per cell as was found in E. coli. These results indicated that R. prowazekii possesses a ribosome concentration greater than might have been predicted from its slow growth rate. This high concentration of ribosomes could be due to a large population of nonfunctioning ribosomes, a low efficiency of amino acid production, or a high rate of protein turnover. However, this study also demonstrated that the rickettsiae have very limited protein turnover. Knowledge of the kinetics and control mechanisms for protein synthesis in R. prowazekii remains to be established to determine the logic of the extra rickettsial ribosomes.

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

[The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]. / Izuchenie poverkhnostnykh antigenov Rickettsia prowazekii pri pomoshchi monoklonal'nykh antitel.

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.

Roles of the Fc receptor and respiratory burst in killing of Rickettsia prowazekii by macrophagelike cell lines.

It is known that the virulent strain of Rickettsia prowazekii grows in macrophagelike cell lines, but if the rickettsiae are treated with antirickettsial serum before infection, the intracellular rickettsiae fail to grow and are destroyed. The uptake of rickettsiae by macrophagelike cell lines was increased by treatment of the rickettsiae with immune serum and with purified immunoglobulin G (IgG) from this serum but not by treatment with the F(ab')2 fragment derived from this IgG. This suggested that the normal rickettsial pathway of entry could be augmented by the Fc receptor-mediated pathway. However, rickettsiae treated with these F(ab')2 fragments which contained no Fc region were destroyed as effectively as those treated with immune serum or IgG. Internalization of R. prowazekii (whether virulent, avirulent, treated, or untreated) did not lead to an increased release of CO2 from [1-14C]glucose, an increase that would have been indicative of a respiratory burst. Furthermore, a mutant macrophagelike cell line, incapable of a respiratory burst, was able to destroy rickettsiae treated with immune serum as effectively as did the parental cell line. Electron micrographs of macrophagelike cells which had been incubated with either antirickettsial IgG or with F(ab')2 fragments derived from this IgG both demonstrated marked deterioration of the rickettsiae, which were primarily within vacuoles but occasionally free in the cytoplasm. In contrast, untreated rickettsiae displayed morphologically normal rickettsiae which were mostly in the cytoplasm but occasionally in the intact and damaged vacuoles. These results indicated that (i) a respiratory burst was not a significant part of the mechanism used by macrophagelike cells to destroy R. prowazekii treated with immune serum, (ii) the destruction of the rickettsiae by the macrophage was not dependent on a diversion to the Fc receptor-mediated pathway of entry, and (iii) the locus of damage to the rickettsiae was most likely the phagolysosome of the macrophagelike cell line.

Methods for purification of Rickettsia prowazekii separated from the host tissue: a step-by-step comparison.

Two methods for purification of Rickettsia prowazekii strains E, E Vir, and Breinl grown in chick embryo yolk sacs are described. These methods combine either differential centrifugation or sucrose mix, centrifugation through sucrose cushion, 10 mmol/l MgCl2 treatment, filtration through a glass filter AP-20 and 2 cycles of verografin discontinuous density gradient centrifugation. The purification procedure including sucrose mix allowed to recover about 38-42% biologically active rickettsiae, a yield which was by 10% higher than that obtained by the method beginning at differential centrifugation. The rickettsiae free of host cell components preserved their infectious activity. The obtained biomass was suitable for immunological and biological characterization of Rickettsia prowazekii and for isolation of its total DNA.

[Inactivation of concentrated biomasses of Rickettsia prowazekii]. / Inaktivatsiia kontsentrirovannykh biomass Rickettsia prowazekii.

The methods used for the sparing inactivation of highly concentrated R. prowazekii biomass and for the decrease of its infectious activity are described. These methods are recommended for use in experiments in the field of molecular biology, as well as for disinfection of different materials contaminated with rickettsiae. As conditions for complete inactivation, incubation at 50 degrees C for 1 hour without chemical disinfectants, treatment with 0.5% phenol solution at 30 degrees C for 12 hours and with 0.1% formaldehyde solution at 4 degrees C for 24 hours have been selected. Treatment with 0.5% phenol solution at 36 degrees C for 1 hour or incubation at 45 degrees C without the use of disinfectants ensures an essential decrease in the infectivity of the material if the work with viable infective agents is necessary. Ultraviolet irradiation for 1.5 hours and exposure to the action of 0.1-0.5% sodium azide are less effective.

Electron microscopic analysis of in vitro interaction of Rickettsia prowazekii with guinea pig macrophages. II. Macrophages from immune animals.

Alike to macrophages from intact animals, reproduction, destruction and formation of spheroplast-like forms were observed in macrophages from immune guinea pigs 2 months post-infection (p.i.) with the virulent Breinl strain of Rickettsia prowazekii. Unlike to the former, immune macrophages revealed phagolysosomes which were larger in size and contained more rickettsiae showing morphologic signs of destruction. Spheroplast-like forms occurred more often and were more numerous than in intact animals. Structures morphologically similar to L-forms of gram-negative bacteria and that of chlamydiae were also detected. After adding immune serum, more intact rickettsiae and spheroplasts were found in phagosomes as well as more phagolysosomes contained rickettsiae and spheroplasts with morphologic signs of destruction. It is suggested that clearance of immune macrophages from rickettsiae is mediated by at least two processes: on one hand by destruction of rod-shaped rickettsiae within phagolysosomes and, on the other hand, by formation and subsequent destruction of spheroplast-like forms within vacuoles, which probably also function as phagolysosomes.

Electron microscopic analysis of in vitro interaction of Rickettsia prowazekii with guinea pig macrophages. I. Macrophages from nonimmune animals.

Monolayer cultures of peritoneal macrophages of intact guinea pigs were infected with Rickettsia prowazekii (strain Breinl) and examined by electron microscopy after 30 min, 4 and 24 hr post-infection (p.i.). Three parallel processes developed in infected macrophages: reproduction of rickettsiae in macrophage cytoplasm, destruction in phagolysosomes and production of spheroplast-like forms. Reproduction of rickettsiae yielded 2 cell types: those with dense and with light cytoplasm; they were located side by side in the microcolony and seemed to have a common capsule-like coat. Relatively small spheroplast-like forms of about 1 micron in size were regularly detected. Addition of immune serum to macrophages increased the number of rickettsiae, both of rod-shaped as well as of spheroplast-like ones located within phagosomes, but elicited no increase in the number of digested pathogen cells.

Peculiarities of Rickettsia prowazekii in the cell culture as revealed by cryoultramicrotomy.

Ultrastructure of Rickettsia prowazekii has been followed in L-929 cells 4 days post-infection (p.i.) by cryoultramicrotomy. Groups of rickettsiae were present in the cytoplasm outside of vacuoles forming microcolonies. The size of rickettsiae amounted to 400 X 700 nm, the average thickness of the cell wall was 5 nm, that of periplasmic space and cytoplasmic membrane 14 and 6 nm, respectively. Within intracytoplasmic colonies the rickettsiae were tightly packed and their cell walls were closely adjacent to each other. No halo or capsule-like coating around them was detected. No ultrastructural details were observed in the light translucent spaces between cells. Marginal rickettsiae of the microcolonies were often in close contact with the host cell mitochondria.

[Characteristics of rickettsial morphology during intracellular development]. / Osobennosti morfologii rikketsii pri ikh vnutrikletochnom razvitii.

The normal anatomy of rickettsiae has been characterized with the use of R. prowazekii, R. conorii and R. akari in continuous cell cultures L-929, Al, FL and in primary chick embryo fibroblast culture. Rickettsiae are short rod-shaped cells with the dense cytoplasm and the regular structure of the cell wall--cytoplasmic membrane complex. The study has shown the absence of polymorphism in rickettsiae growing under permissive conditions, but at the same time these organisms easily develop into pathological forms. Pathological forms can be detected alongside normal rickettsiae in the same cells. The classification of the pathological forms of rickettsiae is presented. In this classification the compensating (reversible) and destructive (irreversible) forms of alterations, as well as hypertrophic and dystrophic processes on the level of the whole rickettsial cell or its organelles, are pointed out.

Some characteristics of heavy and light bands of Rickettsia prowazekii on Renografin gradients.

Suspensions of partially purified Rickettsia prowazekii yielded two bands of organisms when centrifuged to equilibrium in Renografin density gradients. Rickettsiae from the lower, heavy band were defective in their infective and metabolic activities, as compared to organisms from the light band. The greater density in Renografin of heavy-banding organisms was due to their lack of permeability barrier to it, as evidenced by the absence of plasmolysis in hypertonic Renografin. In contrast, light-banding rickettsiae were able to exclude Renografin, since they were plasmolyzed in it. The proportion of heavy-banding organisms in a rickettsial suspension was influenced by the growth phase they were in when harvested from infected yolk sacs, as well as by the conditions and media to which they subsequently were exposed. We have concluded that these defective forms arise from the degeneration of light-banding rickettsiae. This separation of two functional classes of rickettsiae in Renografin density gradients has been exploited (i) to increase the uniformity of the suspensions by removing many noninfectious particles and (ii) to determine rapidly the integrity of certain properties of the cytoplasmic membrane of organisms exposed to a variety of conditions.

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.

Electron microscopy of surface structures of Rickettsia prowazeki stained with ruthenium red.

In studying surface structures of Rickettsia prowazeki (E and Breinl strains) by ruthenium red staining, a microcapsular layer 125-165 A thick, composed of subunits 85--100 A in diameter with a periodicity of 100--120 A as well as the inner layer of the cell wall 40--60 A thick were clearly revealed. In tangential sections of cells, subunits of the microcapsular layer were found in parallel striation arrays. These structures presumably contain acid mucopolysaccharides detectable by ruthenium red staining. Besides, hitherto unreported intracytoplasmic membrane structures were detected in ruthenium red-stained rickettsiae.