Coxiella burnetii Nine Mile II Δcbu0533 as a suitable laboratory replacement strain

Coxiella burnetii is an intracellular bacterial pathogen that causes the disease Q fever. Both secretion of effector proteins by a type IVB secretion system and expression of lipopolysaccharide (LPS) are critical for C. burnetii virulence. The C. burnetii Nine Mile strain lineage is routinely used in laboratories, with Nine Mile I RSA493 (fully virulent) expressing phase I LPS, Nine Mile Crazy RSA514 (lightly attenuated) expressing intermediate LPS, and NMII RSA439 clone 4 (NMII, attenuated) expressing phase II LPS. NMII was exempted from the U.S. Centers for Disease Control and Prevention Federal Select Agent Program due to a genetic deletion predicted to prevent phase I LPS synthesis; however, recent findings demonstrated that NMII LPS elongation occurs in vivo and in vitro. These findings raise concerns regarding the suitability of NMII for manipulation at biosafety level-2 (BSL-2) containment. Here, we evaluated a mutant strain lacking the gene responsible for LPS elongation (C. burnetii NMII Δcbu0533) as an alternative strain for BSL-2 containment studies. Growth of wild-type NMII and NMII Δcbu0533 in axenic media and macrophages was evaluated. Neither growth kinetics nor replication vacuole morphology significantly differed between strains. NMII Δcbu0533 also prevented staurosporine-induced cell death, indicating that the mutant strain maintains the capacity to alter cell signaling events similar to wild-type C. burnetii. Cumulatively, these data reveal that C. burnetii NMII Δcbu0533 is a suitable alternative to wild-type NMII in BSL-2 laboratory experiments that can be distributed to the field for use.IMPORTANCEAttenuated C. burnetii NMII RSA439 clone 4 (NMII) expresses truncated lipopolysaccharide (LPS) and was long predicted to be unable to produce elongated LPS due to a large chromosomal deletion. As a result, this strain was exempted from the CDC Federal Select Agent Program's regulation and is commonly used under biosafety level-2 laboratory conditions. Recently, it was shown that C. burnetii NMII LPS can elongate due to a mutation reversion in cbu0533, resulting in increased virulence in guinea pigs. As a result, the previously exempted NMII strain is subject to regulation if the cbu0533 mutation reversion is present, posing concerns regarding laboratory use. Experiments described here provide a suitable alternative strain to address these concerns.