title: Sustained axenic metabolic activity by the obligate intracellular bacterium Coxiella burnetii.
abstract: Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [(35)S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840, USA.
authors.forename: Anders
authors.initials: A
authors.lastname: Omsland
authors.forename: Diane C
authors.initials: DC
authors.lastname: Cockrell
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Fischer
authors.forename: Robert A
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002021:Buffers; D002244:Carbon / Q000032:analysis; D018929:Cell Culture Techniques; D016997:Coxiella burnetii / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D014176:Protein Biosynthesis / Q000187:drug effects
chemical_list: D002021:Buffers; D003470:Culture Media; D002244:Carbon; D000255:Adenosine Triphosphate