Three-dimensional quantitative characterization of Coxiella burnetii infection using focused ion beam-scanning electron microscopy

Coxiella burnetii is a highly virulent intracellular pathogen that causes acute and chronic Q fever in humans. The bacterium utilizes a Dot/Icm type IV secretion system (T4SS) to translocate over 100 effectors into host cells to facilitate biogenesis of Coxiella-containing vacuoles (CCVs), which are specialized lysosome-derived organelles that support bacterial replication. During replication in CCVs, C. burnetii undergoes a unique biphasic developmental cycle defined by two distinct cellular forms: the infectious and metabolically dormant small cell variant (SCV) that converts to a replicative large cell variant (LCV). This developmental cycle was believed to be intimately coupled with biogenesis of CCVs, but the underlying mechanisms remained unclear. Here, we combine advanced focused ion beam-scanning electron microscopy (FIB-SEM) with machine learning-based data analyses to visualize host cells infected by C. burnetii. We reveal significant pleiomorphism of both the bacteria and CCVs in three dimensions. We show that this technology can be leveraged to characterize CCV biogenesis defects displayed by C. burnetii mutants that are unable to generate the large CCV displayed by wild-type C. burnetii. Analysis of HeLa cells infected with a cig2::Tn mutant confirmed that this mutant creates vacuoles that have a defect in homotypic fusion but that the proportions of SCV to LCV in vacuoles in which this mutant resides are nearly identical to CCVs containing wild-type C. burnetii, which indicates the vacuole biogenesis defect displayed by the cig2::Tn mutant did not impact the developmental cycle intracellularly. Collectively, this study provides unprecedented three-dimensional images of the complex intracellular lifestyle of C. burnetii. This imaging technology also provides unique insights into the biphasic developmental cycle and will be a powerful approach to dissect CCV biogenesis defects displayed by mutant C. burnetii.