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.

Fig. 1.

Supplemented CCM supports enhanced C. burnetii metabolic activity. Effects of casamino acids and L-cysteine on C. burnetii catabolic capacity were determined by incubating organisms in CCM or CCM supplemented with 2.5 mg/ml casamino acids, 1.5 mM L-cysteine or both (ACCM). Bacteria were preincubated in the respective media for 24 h, then labeled with [³⁵S]Cys/Met in labeling buffer (pH 4.5) for 3 h. (A) De novo protein synthesis by C. burnetii was measured by quantification of radiolabel incorporation by scintillation counting. Results are expressed as fold increase in incorporation when compared with incorporation of bacteria preincubated in CCM, then labeled in labeling buffer (pH 7.0) (negative control). Casamino acids and L-cysteine significantly improved C. burnetii metabolic activity. (B) SDS/PAGE and autoradiography confirmed incorporation of radiolabel into bacterial proteins. Values are mean ± SEM (n = 3). The level of radiolabel incorporation in CCM (pH 7.0) is normalized to 1.

Fig. 2.

The number of substrates oxidized by C. burnetii increases with decreasing oxygen availability. The ability of C. burnetii to oxidize substrates in different oxygen environments was assessed using PM. (A) C. burnetii genes encoding terminal oxidases associated with aerobic (cytochrome o) and microaerobic (cytochrome bd) metabolism suggested C. burnetii can respire under microaerophilic conditions. (B) Purified C. burnetii was added to PM-1 plates and incubated for 24 h in 20%, 5%, and 2.5% oxygen. The number of metabolites oxidized increased with decreasing oxygen tension, consistent with microaerophilic metabolism. Representative PM plate images are shown. (C) Seventeen substrates were efficiently oxidized by C. burnetii in 2.5% oxygen. Signal intensities were measured using an OmniLog detection system and expressed as relative OmniLog units (OLU). Quantitative analysis is representative of at least 3 independent experiments. Substrate key (rows A-H, columns 1–12): A1, no substrate control; A5, succinate; A8, L-proline; A11, D-mannose; B12, L-glutamate; C2, D-galactonic acid-γ-lactone; C9, α-D-glucose; D6, α-ketoglutarate; E1, L-glutamine; E12, adenosine; F5, fumarate; F6, bromo succinate; G4, L-threonine; G5, L-alanine; G9, mono methyl succinate; H8, pyruvate; H9, L-galactonic acid-γ-lactone; H11, phenylethylamine.

Fig. 3.

ACCM supports axenic cell division of infectious C. burnetii under microaerobic conditions. (A) C. burnetii GE were assessed by QPCR daily for 6 days. Incubation in 20% (■) oxygen did not support C. burnetii replication while incubation in 2.5% (•) oxygen resulted in considerable C. burnetii replication. (B) Increases in C. burnetii GE during incubation in ACCM correlated with production of infectious bacteria as determined by a quantitative FFU assay. Values are mean ± SEM (n = 3). (C) Representative staining of FFUs contained in equal aliquots of ACCM harvested at 2, 4, and 6 days post inoculation. A magnified view of the inset in the 6 days post inoculation panel in also shown. (Scale bars, 30 μm.)

Fig. 4.

C. burnetii SCV to LCV development occurs in ACCM. To determine whether C. burnetii transitions between nonreplicative SCV and replicative LCV developmental forms during incubation in ACCM, medium was inoculated with purified SCVs and TEM used to assess developmental transitions. (A and B) TEM showed the inoculum had ultrastructural characteristics of the SCV including small cell size (average diameter: 0.188 ± 0.0044 μm) and condensed chromatin. Organisms incubated in ACCM for 3 days exhibited ultrastructural characteristics of the LCV including increased cell size (average diameter: 0.456 ± 0.0078 μm) and dispersed chromatin. Following 6 days of incubation, a mixed population of SCVs and LCVs was observed, resulting in an overall reduction in cell size (average diameter: 0.290 ± 0.0087 μm). Values are mean ± SEM (n = 60 cells). (Scale bar, 0.5 μm.) (C) Immunoblot demonstrating the presence of ScvA (3.5 kDa), a SCV-specific DNA-binding protein, only in the SCV inoculum and the LCV/SCV mixture present in stationary phase.