Lipid A Has Significance for Optimal Growth of Coxiella burnetii in Macrophage-Like THP-1 Cells and to a Lesser Extent in Axenic Media and Non-phagocytic Cells

Abstract

Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A in Coxiella burnetii growth in axenic media, monkey kidney cells (BGMK and Vero), and macrophage-like THP-1 cells by using a potent LpxC inhibitor -LPC-011. We first determined the susceptibility of C. burnetii LpxC to LPC-011 in a surrogate E. coli model. In E. coli, the minimum inhibitory concentration (MIC) of LPC-011 against C. burnetii LpxC is < 0.05 μg/mL, a value lower than the inhibitor's MIC against E. coli LpxC. Considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to 7 days, the stability of LPC-011 in cell cultures was assessed. We found that regularly changing inhibitor-containing media was required for sustained inhibition of C. burnetii LpxC in cells. Under inhibitor treatment, Coxiella has reduced growth yields in axenic media and during replication in non-phagocytic cells, and has a reduced number of productive vacuoles in such cells. Inhibiting lipid A biosynthesis in C. burnetii by the inhibitor was shown in a phase II strain transformed with chlamydial kdtA. This exogenous KdtA enzyme modifies Coxiella lipid A with an α-Kdo-(2 → 8)-α-Kdo epitope that can be detected by anti-chlamydia genus antibodies. In inhibitor-treated THP-1 cells, Coxiella shows severe growth defects characterized by poor vacuole formation and low growth yields. Coxiella progenies prepared from inhibitor-treated cells retain the capability of normally infecting all tested cells in the absence of the inhibitor, which suggests a dispensable role of lipid A for infection and early vacuole development. In conclusion, our data suggest that lipid A has significance for optimal development of Coxiella-containing vacuoles, and for robust multiplication of C. burnetii in macrophage-like THP-1 cells. Unlike many bacteria, C. burnetii replication in axenic media and non-phagocytic cells was less dependent on normal lipid A biosynthesis.

Culture under normoxic conditions and enhanced virulence of phase IICoxiella burnetiitransformed with a RSF1010-based shuttle vector

“…In our attempts to culture C. burnetii in regular CO 2 incubators (∼20% oxygen), we found that a C. burnetii Nine Mile phase II transformant-NMII pMMGK can grow robustly (∼3 logs increase) if starting from a reasonably high inoculum concentration in the regular ACCM-2 medium (without tryptophan supplementation) (Figure 1A). The pMMGK plasmid is composed of the pMMB207 backbone (a RSF1010 ori and a repABC operon), an eGFP gene and a kanamycin resistance cassette (10). This initial finding of C. burnetii normoxic growth was made in T-flask cultures.…”

“…The pMMGK plasmid is composed of a RSF1010 ori, a repABC operon, an eGFP gene and a kanamycin resistance cassette. Construction of pMMGK was described previously (10).…”

“…LPS from phase II C. burnetii is severely truncated and only contains lipid A and partial core oligosaccharide. Lipid A, the basal component of LPS, is essential for C. burnetii growth in macrophage-like THP-1 cells but nonessential in non-phagocytic cells (10).…”

TheCoxiella burnetiiQpH1 plasmid is a virulence factor for colonizing bone marrow-derived murine macrophages

“…C. burnetii transformants were expanded in ACCM-2, suspended in SPG (0.25M sucrose, 10mM sodium phosphate, 5mM L-glutamic acid) and kept frozen at −80°C. Genome copy numbers were determined by Taqman probe qPCR specific to dotA [53].…”

“…Total DNAs were extracted with DNeasy Blood Tissue Kit (Qiagen). Genome copy numbers of C. burnetii in both axenic and cell cultures were determined by Taqman probe qPCR as previously described [53]. PCR conditions were as follows: initial denaturation at 94°C for 10min, followed by 40 cycles of amplification at 94°C for 15 s, 60°C for 1min.…”