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

Abstract

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

The Coxiella burnetii QpH1 Plasmid Is a Virulence Factor for Colonizing Bone Marrow-Derived Murine Macrophages

“…This enhanced virulence of NMIIpMMGK was likely associated with this strain's ability of normoxic growth. Characterization of C. burnetii normoxic growth has been described in a separate manuscript (46). Furthermore, the sizes of splenomegaly were in accordance with the C. burnetii genome equivalents in all infected mice (Fig.…”

“…Though similar to other RSF1010-ori-based shuttle vectors, the pMMGK vector conferred an unexpected capability of normoxic growth on C. burnetii, which is likely to be related to the transformant's enhanced virulence. The normoxic growth of C. burnetii was characterized in a separate manuscript (46).…”

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

“…This enhanced virulence of NMII pMMGK is likely associated with this strain’s ability of normoxic growth. Characterization of C. burnetii normoxic growth was described in a separate manuscript [42]. Moreover, the sizes of splenomegaly are in accordance with C. burnetii genome equivalents in all infected mice (Figure 8C).…”

“…Though similar with other RSF1010-ori based shuttle vectors, the pMMGK vector confers an unexpected capability of normoxic growth on C. burnetii , which likely relates to the transformant’s enhanced virulence. The normoxic growth of C. burnetii was characterized in a separate manuscript [42].…”