Developmental transitions of Coxiella burnetii grown in axenic media

Coxiella burnetii undergoes a biphasic developmental cycle within its host cell that generates morphologically and physiologically distinct large cell variants (LCV) and small cell variants (SCV). During the lag phase of the C. burnetii growth cycle, non-replicating SCV differentiate into replicating LCV that in turn differentiate back into SCV during stationary phase. Nearly homogeneous SCV are observed in infected Vero cells after extended incubation (21 to 28 days). In the current study, we sought to establish whether C. burnetii developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supported developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21 days). Instead, C. burnetii exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21 days of cultivation. Transcriptional profiling of C. burnetii following 14 days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. ACCM-2 differs from ACCM-1 by the substitution of methyl-b-cyclodextrin (Mb-CD) for fetal bovine serum. Addition of Mb-CD to ACCM-1 at 7 days post-inoculation rescued C. burnetii viability and lowered expression of oxidative stress genes. Thus, Mb-CD appears to alleviate oxidative stress in ACCM-2 to result in C. burnetii developmental transitions and viability that mimic host cell-cultivated organisms. Axenic cultivation of C. burnetii in ACCM-2 and new methods Coxiella axenic media 1 vs 2