Herbert A. Thompson

Secretion of proteins by Coxiella burnetii

Viable Coxiella burnetii organisms were isolated from the culture medium of persistently infected Baby Hamster Kidney (BHK-21) fibroblasts. When these organisms were incubated in host-cell-free medium at low pH, some of the de novo-synthesized protein made by the bacteria was translocated to the exterior of the cell. The exported protein was detectable after 2-7 h incubation at 37 degrees C. No evidence was found to suggest that protein accumulation in the medium was due to leakiness caused by cell damage. Both DCCD (dicyclohexylcarbodiimide) and CCCP (carbonyl cyanide m-chlorophenylhydrazone) inhibited the process to some extent. Exported protein was represented largely by three polypeptides with molecular masses of 34, 24 and 12 kDa. De novo-synthesized proteins corresponding to these molecular masses were not detected in cytoplasmic fractions, but a membrane fraction might possess a similar form. It was concluded that a physiological process of protein translocation occurred in C. burnetii during acid activation in a defined medium. Organisms that were extracted directly from the cytoplasm of infected fibroblasts by a mechanical disruption procedure were also active in de novo protein synthesis; however they exported much less of the protein.

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

Two populations of Coxiella 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 H3 32PO4 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 microM) and hydroxyurea (10 mM), but it was not affected by chloramphenicol (310 microM) until 4 h after addition of the drug. Incorporation of H3 32PO4 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 QpH1 plasmid DNA, during acid activation of metabolism.

Permeability of Coxiella burnetii to ribonucleosides

Knowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete. The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes. Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities. Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides. Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine. Both phases were poorly active in cytidine uptake. Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil. Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism. Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process. It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.

Protein Synthesis in Cell-free Extracts of Coxiella burnetii

Some aspects of protein biosynthesis were investigated in extracts of the obligate intracellular bacterium Coxiella burnetti. Sucrose gradient analysis revealed small quantities of 30S and 50S ribosomal subunits, few 70S ribosomes and no polysomes. Functional endogenous mRNA was not detected. In translation of exogenously added poly(U), extracts required Mg2+ (17 mM) and NH4+ (60 mM) for optimal polyphenylalanine synthesis; the optimum MG2+ requirement differed from that of Escherichia coli. The translation of coliphage Q beta RNA by C. burnetti extracts required Mg2+ (13 mM), NH4+ (60 mM) and an energy source for polypeptide synthesis, and was sensitive to chloramphenicol but not to cycloheximide. Under optimal conditions, the translation of Q beta RNA proceeded at a rate and to an extent equal to that obtained in a conventional E. coli system. Electrophoretic analysis of translation products made during incubation of C. burnetti extracts with polycistronic Q beta RNA revealed a major product with a molecular weight of about 14 000; this product co-electrophoresed with the coat protein extracted from Q beta phage propagated in E. coli. The results suggested that the extracellular form of the rickettsia-like organism, C. burnetti, possessed the full array of components necessary for the initiation, elongation and termination of polypeptides.