Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.     

Properties of phase I purified cellular antigen of Coxiella burnetii

An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests. Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient. The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C. burnetii in the following tests: complement fixation and microagglutination. Only fractions containing protein were serologically active. They proved to be of similar specificity and sensitivity as the antigens obtained by standard method. Moreover, it was found that after formalin treatment of C. burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.     

Nature and linkage type of fatty acids present in lipopolysaccharides of phase I and II Coxiella burnetii

The constituent fatty acids of lipopolysaccharides (LPS) of Coxiella burnetii (phase I and II) were qualitatively and quantitatively analysed by combined gas-liquid chromatography/mass spectrometry. The total fatty acid content (per mg LPS) was determined as 90.0 nmol (2.3 wt%) for LPS of phase I cells (LPS I) and 179.1 nmol (4.8 wt%) for LPS of phase II cells (LPS II). Of the 24 different acyl residues characterized (12 to 18 carbon atoms), nine were 3-hydroxy fatty acids (normal, iso- and anteiso-branched) which quantitatively predominated. All 3-hydroxylated fatty acids were found to possess the (R)-configuration, to be exclusively amide-linked and to be acylated at their 3-hydroxyl group. Ester-linked nonhydroxylated fatty acids (normal, iso- and anteiso-branched) were present but ester-bound 3-hydroxy- or 3-acyloxyacyl residues were lacking from C. burnetii LPS I and LPS II. As the major acyl group (R)-3-(12-methyl-tetradecanoyloxy)-12-methyl-tetradecanoic acid was identified. Our results show that the complex fatty acid spectrum of C. burnetii differs considerably from that of LPS of other Gram-negative bacteria. They further suggest an enormous heterogeneity of the lipid A component of C. burnetii LPS I and LPS II.     

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.     

Mouse resident peritoneal macrophages partially control in vitro infection with Coxiella burnetii phase II

Coxiella burnetii, the agent of Q fever in man and of coxiellosis in other species, is a small, dimorphic, obligate intracellular bacterium, sheltered within large, acidified, and hydrolase-rich phagosomes. Although several primary and established cell lines, macrophage-like cells, and primary macrophages from other species have been infected with C. burnetii, the infection of mouse primary macrophages has not been sufficiently characterized. In this report quantification of DAPI (4', 6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy, and transmission electron microscopy were used to compare the infection of three mouse-derived cells, L929 fibroblasts, J774 macrophage-like cells, and resident peritoneal macrophages, with a phase II clone of C. burnetii known to be non-virulent for mammals. Infected peritoneal phagocytes differed from L929 or J774 cells in that: (a) large vacuoles took longer to appear (3-5 d instead of 2), and were only found in a subset (20-30%) of macrophages, as opposed to in more than 70% of the other cells; (b) total and vacuole-associated relative bacterial loads in L929 and J774 cells were several-fold higher than in peritoneal macrophages; (c) estimated doubling times of the bacteria were about 68 h in the primary macrophages, 18 h in J774 and 22 h in L929 cells. Thus, mouse resident peritoneal macrophages control both the formation of the large vacuoles and the intracellular proliferation of C. burnetii phase II.     

A growth study of Coxiella burnetii Nine Mile Phase I and Phase II in fibroblasts

Coxiella burnetii, a slow-growing, gram-negative, obligate intracellular bacterium, is the causative agent of Q fever in humans. The avirulent Phase II C. burnetii Nine Mile strain can invade and establish persistent infections in a wide variety of laboratory cell lines, and is generally considered to be easier to grow in culture than the wild-type Phase I organism. Efforts to improve Phase I organism yield in the BHK-21 cell line demonstrated that high CO2 conditions and the use of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose supplementation resulted in higher organism yields. Phase II organisms grown in the same cell line and conditions showed lower growth rates. Analysis revealed that increased average numbers of C. burnetii Phase I organisms within fibroblasts was due to higher growth rates within the hosts rather than to increased uptake or to increased cell-to-cell spreading. Addition of the nucleoside cytidine to the growth medium stimulated growth of Phase II but not Phase I organisms.     

Endophytic bacilli--producers of type II restriction endonucleases

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.     

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

Background  

Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).     

Structure and function of type II restriction endonucleases

More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.     

3-C-branched aldoses in lipopolysaccharide of phase I Coxiella burnetii and their role as immunodominant factors

Mild acid hydrolysis with 1% acetic acid (100 degrees C, 15-60 min) of lipopolysaccharide (LPS) isolated from Coxiella burnetii phase I cells leads to a drastic decrease in its serological reactivity as shown by the passive hemolysis test. This decrease in reactivity occurs parallel or even prior to the cleavage of LPS into free lipid A and the polysaccharide moiety. During this mild hydrolysis two unusual sugars (X and Y) are released from the LPS, which were obtained in pure state by thin-layer chromatography. Analysis of their alditol acetate derivatives by gas chromatography/mass spectrometry revealed that sugar X is a 6-deoxy-3-C-methyl-hexose and sugar Y a 3-C-(hydroxymethyl)-pentose. Using a range of authentic standards and different thin-layer and gas chromatographic conditions, X could be recognized as 6-deoxy-3-C-methyl-gulose (virenose), very probably as the L form of this sugar (L-virenose). Y has been identified as 3-C-(hydroxymethyl)-lyxose (dihydrohydroxystreptose) by comparing it with newly synthesized 3-C-(hydroxymethyl)-pentoses (Dahlman, O., Garegg, P. J., Mayer, H., Schramek, S., unpublished results). Both branched sugars are (at least partially) in terminal positions since methylation analysis of LPS afforded (mainly) their permethylated derivatives. This analysis further showed virenose to be linked in C. burnetii phase I LPS as pyranose and dihydro-hydroxystreptose as furanose. The terminal linkage and the chemical nature of X and Y are in accordance with the observed acid-lability of the serological determinants.     

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.     

Genetics of Coxiella burnetii

Abstract Those organisms considered to be obligate intracellular bacteria are interesting objects for genetic studies. Little is known about their mechanisms for natural genetic exchange. Many genes from the bacterium Coxiella burnetii , an obligate intraphagolysosomal pathogen, have therefore been cloned and characterized using the heterologous host Escherichia coli . Recently, use of electroporation methodology followed by long-term selection periods have provided initial data on genetic transformation in C. burnetii .     

Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 10(7) phase I C. burnetii.     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.