<i>Coxiella burnetii</i> Nine Mile II Δ <i>cbu0533</i> as a suitable laboratory replacement strain

<i>Coxiella burnetii</i> 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 <i>C. burnetii</i> virulence. The <i>C. burnetii</i> 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 <i>in vivo</i> and <i>in vitro</i>. 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 (<i>C. burnetii</i> NMII Δ<i>cbu0533</i>) as an alternative strain for BSL-2 containment studies. Growth of wild-type NMII and NMII Δ<i>cbu0533</i> in axenic media and macrophages was evaluated. Neither growth kinetics nor replication vacuole morphology significantly differed between strains. NMII Δ<i>cbu0533</i> also prevented staurosporine-induced cell death, indicating that the mutant strain maintains the capacity to alter cell signaling events similar to wild-type <i>C. burnetii</i>. Cumulatively, these data reveal that <i>C. burnetii</i> NMII Δ<i>cbu0533</i> is a suitable alternative to wild-type NMII in BSL-2 laboratory experiments that can be distributed to the field for use.IMPORTANCEAttenuated <i>C. burnetii</i> 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 <i>C. burnetii</i> NMII LPS can elongate due to a mutation reversion in <i>cbu0533</i>, resulting in increased virulence in guinea pigs. As a result, the previously exempted NMII strain is subject to regulation if the <i>cbu0533</i> mutation reversion is present, posing concerns regarding laboratory use. Experiments described here provide a suitable alternative strain to address these concerns.