Efficient method of cloning the obligate intracellular bacterium Coxiella burnetii

Abstract

Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.

FIG. 1.

MAb A6 reacts against NMII LPS. (A) LPS was extracted from C. burnetii NMI, NMC, NMII, and Australian QD (Aus) isolates by the hot phenol water method (14). Samples were separated by SDS-PAGE and stained with silver (left) or transferred to an Immobilon-P membrane for immunoblotting (right). Only LPS from NMII reacted with MAb A6. The relative sizes of molecular mass markers in kDa are shown on the left of each panel. (B) Vero cells were infected with NMI, NMC, NMII, or Aus for 5 days, fixed with methanol, and dual stained for immunofluorescence with MAb A6 (red) and serum from an infected guinea pig (green). Guinea pig serum stained all four isolates, whereas MAb A6 stained only NMII, as indicted by the yellow color produced by overlapping green and red fluorescence. Bar, 15 μm.

FIG. 2.

NMI and NMII C. burnetii bacteria coinhabit the same vacuole in Vero cells. Vero cells were infected with NMI and NMII C. burnetii bacteria at MOI of 100 and 1, respectively. Cells were fixed with methanol at 4 days postinfection and NMI (green) and NMII (red) selectively stained by indirect immunofluorescence as described in Materials and Methods. (A) Low-magnification epifluorescence micrograph showing singly (arrowheads) and coinfected (arrows) cells. Bar, 15 μm. (B) High-magnification confocal fluorescence micrograph showing the characteristic “jelly doughnut” appearance of coinhabited vacuoles with NMI forming an outer ring around an inner core of NMII. Bar, 2 μm.

FIG. 3.

Individual C. burnetii PVs can be harvested using micromanipulation. (A) A phase-translucent PV is easily visible by phase-contrast light microscopy at 4 d postinfection in panel 1 (arrow). Panels 2 and 3 depict the PV excision process. (B) PVs can reseal following extraction of vacuole contents. Vero cells on gridded coverslips were infected with NMII C. burnetii for 4 d. Panels 1, 2, and 3 are a sequential series of micrographs depicting the extraction of C. burnetii from an isolated PV (arrow). Panel 4 depicts the same PV 7 h later, which has resealed and is structurally intact. Remaining phase-dense C. burnetii bacteria are visible in the upper portion of the vacuole. (Slight movement of the PV and accompanying cell occurred during the 7-h incubation.) Bars, 15 μm.

FIG. 4.

Cloning of C. burnetii by micromanipulation. Vero cells were coinfected with NMII and NMC C. burnetii bacteria at MOI of 1 and 10, respectively. At 5 d postinfection, nine PVs were randomly excised and their contents suspended in 40 μl of K-36 buffer. DNA from 8 μl of the PV suspension was whole genome amplified. Samples were then subjected to PCR according to the strategy depicted in panel A to determine whether the sample contained NMC (PCR product of 401 bp), NMII (PCR product of 825 bp), or both (PCR products of 401 and 825 bp, respectively). PVs 1 and 2 contained NMII, PVs 3 and 4 contained NMC, and PVs 5 through 9 contained both NMC and NMII (B). The remaining 32-μl suspension of PVs 5 through 9, containing mixtures of NMC and NMII, was used to reinfect separate Vero monolayers. At 5 d postinfection, three PVs were randomly excised from each infected culture and the samples reanalyzed by PCR as described above. All PVs obtained from the second-round infection contained clonal populations of NMC or NMII.