Developmental transitions coordinate assembly of the Coxiella burnetii Dot/Icm type IV secretion system

Abstract

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique biphasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV) that transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and for intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and assembly of the Dot/Icm T4SS. Importantly, assembled Dot/Icm T4SSs were not present in the infectious SCV. The appearance of an assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginated to form tightly packed stacks during the LCV to SCV transition at late stages of infection, which could enable the SCV to LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.

Structural remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography

“…On the other hand, and despite several data processing trials, we could not resolve the cytoplasmic ATPase complex. A contemporaneous study on C. burnetii T4BSS used more than 7000 particles to generate an in situ average (46), but their structure also lacked the cytoplasmic complex suggesting that the C. burnetii T4BSS ATPase complex is either flexible or rarely associated. Earlier studies have shown that acidification of the CCV inside the host triggers the differentiation of SCVs into LCVs.…”

“…While T4ASS is known to harbor extracellular pili containing VirB2 and VirB5 proteins (48), the T4BSS has no putative pilin candidates and there are no reports of this system producing a pilus. Previous studies proposed that the C. burnetii T4BSSs interact with the CCV membrane using short tethers that could facilitate translocation of T4BSS effectors (49), and a contemporaneous study also reported short filamentous densities between the bacterial OM and the CCV membrane (46). It is possible that the short tubular structures above some T4BSS particles are used to establish such an intimate contact between the bacterial OM and CCV membrane.…”