A method for purifying obligate intracellular Coxiella burnetii that employs digitonin lysis of host cells

Abstract

Purification of the obligate intracellular bacterium Coxiella burnetii requires physical disruption of infected cells. Here we describe a gentle and safe digitonin lysis procedure to release C. burnetii from infected cells. The purity, yield, and infectivity of digitonin-prepped organisms are comparable to that of organisms purified using cell lysis by sonication.

Fig. 1

The C. burnetii PV membrane is cholesterol-rich and solubilizes following treatment with SPD, a digitonin-containing buffer (A) Vero cells were infected with C. burnetii for 6 days, then fixed with methanol. C. burnetii (red) was stained by indirect immunofluorescence and cholesterol (green) was stained with filipin as previously described (Howe and Heinzen, 2006). Epifluorescence images show filipin labeling of the C. burnetii PV membrane (solid arrow) that is comparable to that of the cholesterol-rich plasma membrane (open arrow). Cholesterol-rich host material is also evident in the PV lumen (arrowhead) (B) Phase contrast light micrographs showing a time course of dissolution of a Coxiella-infected Vero cell monolayer during a 30 min treatment with SPD buffer. Prototypic phase-translucent and spacious replication vacuoles harboring Nine Mile phase II Coxiella are evident before treatment (arrow). After a 1 min treatment, the vacuolar membrane collapses and vacuoles lose their spacious appearance. Around 15 min post-treatment, holes in the monolayer begin to appear with a corresponding release of Coxiella (inset, arrow). At 30 min post-treatment, nearly the entire monolayer has disintegrated and extracellular Coxiella have become clearly visible. (Note: each micrograph is a different field of view.)

Fig. 2

Transmission electron micrographs of sonication and SPD-prepped C. burnetii. Transmission electron micrographs of sonication and SPD-prepped organisms after centrifugation through a 30% RenoCal cushion show that both preparations are highly enriched for C. burnetii (upper panels). Contaminating host cell material is also present, mostly in the form of intact or damaged mitochondria displaying characteristic cristae (black arrow). Substantially less host cell contamination is observed with organisms following an additional 40/44/54% RenoCal-76 step gradient (lower panels). Prototypic C. burnetii SCV and LCV developmental forms are denoted with an arrow and arrowhead, respectively. Abbreviation: RC, RenoCal-76.