Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 181

dotA-Probe (5´-CCGGAGATACCGGCGGTGGG-3´ plus FAM/TAMRA) (App-
lied Biosystems).
4. Control template DNA consisting of C. burnetii dotA amplified by PCR using the
forward primer 5´-ATGAATAAATTAGTGTCATCGCTG-3´ and reverse primer
5´-AGGACCCAAGTGTAGTTGTTTCCC-3´ and cloned into the TA-cloning
vector pCR2.1-TOPO (Invitrogen).
5. Applied Biosystems Prism 7000 Sequence Detection System (Applied
Biosystems).
3. Methods
Vero cells, a widely used cell line for animal virus propagation (17) , have also
been routinely employed as a host cell for amplification of C. burnetii (18) and
other obligate intracellular bacteria (19) . Vero cells are highly permissive for
C. burnetii infection and support robust replication of the pathogen. Moreover,
they are a hardy, fast growing cell line that does not require special growth
conditions or media. We have recently established that the stationary phase
of the C. burnetii growth cycle begins approximately 6 days post-infection in
Vero cells following an approximately 100-fold increase in C. burnetii genome
equivalents (20) . Therefore, optimal yields of the organism are obtained when
purification is conducted after this time point.
All protocols for purifying C. burnetii from host cells include a lysis step to
release intracellular organisms. Lysis methods include subjecting infected cells
to osmotic stress with distilled water (21) , passing cells through a syringe needle
(22) , and disrupting cells by sonication (23) , tissue homogenization (24) ,orby
vortexing in the presence of glass beads (25) . We find that gentle sonication
effectively breaks host cells without damaging the more fragile large cell
morphological form of C. burnetii (20) . C. burnetii preparations reasonably free
of host cell material can be obtained simply by pelleting unbroken nuclei and
intact/partially lysed cells, followed by high-speed centrifugation of the post-
nuclear supernatant to pellet released bacteria. An additional step of pelleting
bacteria through a 30%-RenoCal-76 pad will lessen the amount of host cell
carryover, as most insoluble and buoyant host cell membrane material will
remain at the pad/buffer interface.
Coxiella burnetii infection is acquired through the aerosol route, and alveolar
macrophages are considered the initial target cell of the organism (16) . While
macrophage or monocyte-like cell lines have been widely employed to study
Coxiella -host interactions (1,10,11) , the biological response of these cells to
infection may not accurately reflect the in vivo situation. Indeed, included
among the deficiencies of cell lines is attenuated oxidative killing (11,26) and
cytokine production (26) . To more closely mimic the in vivo growth conditions
of C. burnetii , we developed primary human MDMs as an in vitro model