Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells

Abstract

Two populations of CoxieIlu burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H332P04 into DNA. Incorporation occurred at pH 4.5 but not at pH 7-0, and proceeded for 12-15 h. When incorporation of [ 3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 p~) and hydroxyurea (10 mM), but it was not affected by chloramphenicol(310 p~) until 4 h after addition of the drug. Incorporation of H332P04 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpHl plasmid DNA, during acid activation of metabolism .

Coxiella burnetiiSurvival in THP-1 Monocytes Involves the Impairment of Phagosome Maturation: IFN-γ Mediates its Restoration and Bacterial Killing

“…This finding partly questions the C. burnetii paradigm in which C. burnetii replicates in the cellular compartment displaying phagolysosomal features (5,24). The first characteristic reported for C. burnetii was that acidic pH is required for bacterial metabolism (6,7). In agreement with previous reports on avirulent organisms (8, 9), we found C. burnetii in acidic vacuoles independently of bacterial virulence.…”

“…The selective use of phagocytic receptors is an active process based on interference with complement receptor type 3-mediated phagocytosis (3). Once internalized, C. burnetii survives and replicates in an acidic environment (4,5), which is needed for bacterial metabolism (6,7). This low pH also accounts for the relative inefficiency of antibiotics toward C. burnetii (1).…”

Transformation of Coxiella burnetii to ampicillin resistance

“…L929 cells were grown in spinner-modified minimum essential medium (SMEM; purchased from Biowhittaker) with 5% heat-inactivated newborn calf serum and L-glutamine. Purified C. burnetii organisms, those transformed or not transformed, were acid activated as described previously (7,15,43). Briefly, C. burnetii cells were resuspended in medium B (49 mM KH 2 PO 4 or 49 mM K 2 HPO 4 , 169 mM KCl, 16 mM NaCl, 120 mM glycine, 250 mM sucrose; pH 4.5 or pH 7.0) (15,43).…”

“…To provide evidence for the expression of ␤-lactamase from pSKO(ϩ)1000 plasmid sequences within transformants, a biological assay to detect ␤-lactamase within acid-activated C7T2-10 Ϫ6 transformants was employed (23,26). C. burnetii was acid activated in vitro for 17.5 h in the presence of the oxidizable substrates glucose and glutamate (7,15,43). Cell lysates were then examined for the capacity to neutralize ampicillin (Fig.…”

“…It is an obligate intracellular bacterium replicating within the acidic phagolysosomes of eukaryotic cells (1,4,15,24). Although C. burnetii becomes metabolically active and synthesizes protein and DNA during in vitro acid activation assays, axenic growth has not been observed (7,15,43). This obligate intracellular parasitism poses difficulties in studying virulence factors associated with this organism.…”