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Coxiella burnetti, the etiologic agent of Q fever, is an oligate intracellular parasite of eukaryotes. Unlike the majority of successful bacterial parasites, which escape the bactericidal environment of the phagolysosome by various means, C. burnetii multiplies only in the phagolysosome. In view of the relatively harsh environment inhabited by C. burnetii, we have examined (i) the in vitro metabolism of glucose and glutamate by whole cells of C. burnetii under conditions designed to approximate the pH within the phagolysosome and (ii) the effect of manipulation of the phagolysosomal pH by lysosomotropic amines on the replication of C. burnetii in chicken embryo fibroblasts. The transport, catabolism, and incorporation of both glucose and glutamate were found to be highly stimulated by acidic conditions, whereas at pH 7.0 metabolism of these substrates was minimal. The transport processes were shown to be energy dependent and highly sensitive to inhibition by uncouplers of oxidative phosphorylation. Increasing the phagolysosomal pH ofinfected chicken embryo fibroblasts by use of the lysosomotropic agents chloroquine, methylamine, or ammonium chloride inhibited the multiplication of C. burnetii, thus demonstrating the in vivo requirement for the acidic conditions ofthe phagolysosome. This apparent dependence upon phagosome-lysosome fusion to generate pH conditions favorable to C. burnetii replication suggests a unique biochemical mechanism of parasite activation. A pathogenic mechanism based on regulation of microbial metabolism by H+-dependent stimulation of cell function is proposed.

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After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G؉C contents were found within the deletions.Coxiella burnetii is an obligately parasitic bacterium that replicates within phagolysosomes of eukaryotic hosts (1,16,27). This organism is the causative agent of Q fever in humans. It is endemic in many species of domestic animals, especially sheep, goats, and cattle. C. burnetii is extremely infectious; inhalation of one organism is enough to initiate an acute illness in a guinea pig (32). In humans, the disease usually is a moderate to severe flu-like illness with headache, fever and chills, malaise, myalgia, and anorexia or an atypical pneumonia with a dry cough (26). Recrudescence and chronic illness, usually in the form of hepatitis or intractable endocarditis, are the most serious manifestations of the disease (24,25,28,55). Humans usually contract Q fever by inhaling contaminated dust, often in barnyard, stockyard, or abattoir settings (34, 45).Some strains of C. burnetii have been observed to undergo variation in surface antigens (18,40). Continual passage of the Nine Mile strain (the tick-derived United States prototype strain) through immunocompetent hosts (306 successive passages in guinea pigs) apparently maintained the organism in its original, native (wild-type) antigenic form. However, eight subsequent passages through embryonated eggs resulted in the emergence of organisms that were easily distinguished serologically from the original isolate (40). The new phenotype reacted strongly with complement-fixing antibody found in acute-phase serum, compared with the lack of a reaction demonstrated by the guinea pig-passaged antigenic form (40). The two antigenic types differed ...

Coxiella burnetii is a gram-variable obligate intracellular bacterium which carries out its development cycle in the phagolysosome of eucaryotic cells. Ultrastructural analysis of C. burnetii, in situ and after Renografin purification, by transmission electron microscopy of lead-stained thin sections has revealed extreme pleomorphism as demonstrated by two morphological cell types, a large cell variant (LCV) and a small cell variant (SCV). Potassium permanganate staining of purified rickettsiae revealed a number of differences in the internal structures of the cell variants. (i) The outer membrane of the SCV and LCV were comparable; however, the underlying dense layer of the SCV was much wider and more prominent than that of the LCV. The periplasmic space of the SCV was not readily visualized, whereas the periplasmic space of the LCV was apparent and resembled that of other gram-negative bacteria. (ii) Complex internal membranous intrusions which appeared to originate from the cytoplasmic membrane were observed in the SCV. The LCV did not harbor an extensive membranous system. (iii) Some LCVs contained a dense body in the periplasmic space. This endogenous structure appeared to arise in one pole of the LCV as an electrondense "cap" formation with the progressive development of a dense body approximately 130 to 170 nm in diameter which was eventually surrounded by a coat of at least four layers. Our observations suggest that the morphogenesis of C. burnetii is comparable, although not identical, to cellular differentiation of endospore formation. A developmental cycle consisting of vegetative and sporogenic differentiation is proposed.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Over a 34-mo period we studied 51 patients with Q fever and 102 control subjects (with various lower-respiratory-tract infections) who were matched for age, sex, and time of onset of infection. By univariate analysis (not adjusted for multiple comparisons), cases differed significantly from controls in the following activities: working on a farm; slaughtering or dressing animals; and contact with cats, cattle, and sheep. The strongest association was with exposure to stillborn kittens--11 of 51 cases vs. none of 102 controls (P less than .00000)--and with exposure to parturient cats (odds ratio, 10.3; 95% confidence interval, 3.5-31.8). Exposures to newborn animals (chiefly kittens) and stillborn kittens were significant risk factors by multivariate analysis, as were rural residence and slaughtering or dressing animals. In 13 Q fever incidents following exposure to parturient cats, 80 people became ill, 52 of whom had serological evidence of recent Coxiella burnetii infection (most of the others were not tested).

Serological parameters were compared in 15 cases of Coxiella burnetii infection comprising 5 cases each of primary Q fever, chronic granulomatous hepatitis, and endocarditis. The diagnosis was made on the basis of clinical history and serology and on the isolation of C. burnetii phase I from biopsy specimens of liver and bone marrow from two patients with granulomatous hepatitis and from the aortic valve vegetations of five patients with endocarditis. The temporal sequences of immunoglobulin levels, rheumatoid factor, and specific antibody responses to phase II and phase I antigens of C. burnetii were evaluated as predictive correlates of the three Q fever entities. Serum levels of immunoglobulin classes G, M, and A were variable in all the entities of Q fever. Increased mean levels (in milligrams per deciliter) of immunoglobulin G (IgG) and IgA were noted with chronic disease in the sera of some patients, whereas IgM levels were not significantly different from normal values. Rheumatoid factor was significantly elevated in chronic disease but not in primary Q fever. The temporal sequence of C. burnetii phase II and phase I antibodies were compared by microagglutination, complement fixation, and indirect microimmunofluorescence tests. All of these serological tests were useful in distinguishing primary from chronic disease. Thus, the ratio of anti-phase II to anti-phase I antibodies was greater than 1, greater than or equal to 1, and less than or equal to 1 for primary Q fever, granulomatous hepatitis, and Q fever endocarditis, respectively. Moreover, the high phase-specific IgA antibody titers in the indirect microimmunofluorescence test were diagnostic for endocarditis.