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.

20Coxiella burnetii is a Gram-negative, facultative intracellular microorganism that can 21 cause acute or chronic Q fever in human. It was recognized as an obligate intracellular 22 organism until the revolutionary design of an axenic cystine culture medium (ACCM). 23Present axenic culture of C. burnetii strictly requires a hypoxic condition (<10% 24 oxygen). Here we investigated the normoxic growth of C. burnetii strains in ACCM-2 25 with or without tryptophan supplementation. Three C. burnetii strains -Henzerling 26 phase I, Nine Mile phase II and a Nine Mile phase II transformant, were included. The 27 transformant contains a pMMGK plasmid that is composed of a RSF1010 ori, a 28 repABC operon, an eGFP gene and a kanamycin resistance cassette. We found that, 29 under normoxia if staring from an appropriate concentration of fresh age inocula, 30 Nine Mile phase II can grow significantly in ACCM-2 with tryptophan, while the 31 transformant can grow robustly in ACCM-2 with or without tryptophan. In contrast, 32 long-term frozen stocks of phase II and its transformant, and Henzerling phase I of 33 different ages had no growth capability under normoxia under any circumstances. 34 Furthermore, frozen stocks of the transformant consistently caused large 35 splenomegaly in SCID mice, while wild type Nine Mile phase II induced a lesser 36 extent of splenomegaly. Taken together, our data show that normoxic cultivation of 37 phase II C. burnetii can be achieved under certain conditions. Our data suggests that 38 tryptophan and an unknown temperature sensitive signal are involved in the 39 expression of genes for normoxic growth regulated by quorum sensing in C. burnetii. 40 129 Mile phase II and NMIIpMMGK were used. In the second experiment, 9 five weeks 130 old mice were averaged into three infection groups: one month old Nine Mile phase II, 131 one-month old NMIIpMMGK and 19 months old NMIIpMMGK. At indicated days 132 post-infection, mice were weighted and sacrificed to harvest spleens to determine 133 splenomegaly (spleen weight/body weight). Each spleen was homogenized in 2 mL 134 PBS. Total DNAs from 20 μL of each tissue homogenate were purified with DNeasy 135

Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii and mainly transmitted by aerosols. This study aims at establishing a systematic and efficient mouse model of acute Q fever via intratracheal (IT) inoculation of aerosolized C. burnetii. Methods: BALB/c mice were infected with C. burnetii via IT route using a non-invasive aerosol pulmonary delivery device to directly place the living C. burnetii organisms into their tracheas. The bacterial loads, pathological lesions, and serological responses were analyzed in mice, and compared with those of mice infected via intraperitoneal (IP) route. Results: As early as at day three post-infection (pi) with a low dose of C. burnetii (1×10⁴ per mouse), a large amount of C. burnetii organisms were determined in blood, lungs, hearts, livers, and spleens of the mice. The inflammatory infiltration was observed in hearts and lungs of mice. Compared with mice infected via IP route, the mice infected via IT route exhibited a higher level of bacterial loads and more severe pathological lesions in hearts and lungs at day 3 and day 7 pi. Conclusions: These data indicated that IT route is more efficient than IP route to cause acute C. burnetii infection in mice. Overall, we successfully established a mouse model of C. burnetii infection via IT route, which is useful for investigations of pathogenesis and immunity of acute C. burnetii infection as well as evaluation of therapeutic drugs and preventive vaccines of Q fever.