Host cell-free growth of the Q fever bacterium Coxiella burnetii

Abstract

The inability to propagate obligate intracellular pathogens under axenic (host cell-free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen that replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism's metabolic requirements using expression microarrays, genomic reconstruction, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log 10) of C. burnetii in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic of in vivo grown organisms. Axenic cultivation of C. burnetii will facilitate studies of the organism's pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellular pathogens.axenic growth ͉ metabolism ͉ microaerophile ͉ obligate intracellular pathogen

Developmental stage-specific metabolic and transcriptional activity of Chlamydia trachomatis in an axenic medium

“…Reduced oxygen tension is critical for axenic replication of the obligate intracellular parasite Coxiella burnetii (28). Similarly, C. trachomatis encodes cytochrome bd, a terminal oxidase with increased affinity for oxygen and thus associated with metabolic activity under reduced oxygen conditions.…”

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…Coxiella burnetii Nine Mile Phase II, strain RSA 439, was cultured axenically in acidified citrate cysteine media 2 (ACCM-2; made in-house) as previously described [40]. When required, chloramphenicol (Boehringer Mannheim, 634,433) and/or kanamycin (Amresco, 0408) were added to ACCM-2 at 3 µg/ml and 300 µg/ml respectively.…”

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

“…C. burnetii Nine Mile phase II (clone 4, RSA439) was cultured axenically in ACCM-2 as previously described (12,16). E. coli TOP10 and BL21-AI (Invitrogen) were grown in Luria-Bertani medium for recombinant DNA procedures and protein purification.…”

“…C. burnetii encodes a Dot/Icm type 4B secretion system (T4BSS) homologous to the T4BSS of L. pneumophila (14). Recent advances in C. burnetii host-cell-free culture (12) and genetic manipulation (15) have enabled confirmation that type 4B secretion is essential for productive infection. Himar1 transposon mutagenesis revealed that icmL and icmD are required for translocation of effectors and colonization of host cells (16,17).…”

“…PV maturation in macrophages culminates in acquisition of the endolysosomal proteins Rab7, lysosomal-associated membrane protein 1 (LAMP1), CD63, active cathepsins, and a pH of ∼4.8 (9,10). Indeed, C. burnetii requires the acidic pH of the PV for metabolic activation and replication (11,12) and resists degradative conditions that quickly destroy Escherichia coli (10).…”