Coxiella burnetii Type IV Secretion-Dependent Recruitment of Macrophage Autophagosomes

Abstract

cCoxiella burnetii is an intracellular Gram-negative bacterium that causes human Q fever, a flu-like disease that can progress to chronic, life-threatening endocarditis. In humans, C. burnetii infects alveolar macrophages and promotes phagosomal fusion with autophagosomes and lysosomes, establishing a unique parasitophorous vacuole (PV) in which to replicate. The pathogen uses a Dot/Icm type IV secretion system (T4SS) to deliver effector proteins to the host cytoplasm, where they alter cellular processes to benefit the pathogen. The T4SS is required for PV expansion and prevention of apoptosis, but little else is known about the role of the system during intracellular growth. Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV to deliver nutrients to the pathogen and provide membrane for the expanding vacuole. In this study, we examined the role of the T4SS in mediating PV interactions with autophagosomes. We found that the autophagy-related proteins LC3 and p62 localized to wild-type PV but not to T4SS mutant organism-containing phagosomes in human macrophage-like cells, primary human alveolar macrophages, and Chinese hamster ovary cells. However, while lipidated LC3 levels were elevated regardless of T4SS activity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the pathogen recruits preformed autophagosomes. When the T4SS was activated 24 h after infection, autophagosome recruitment ensued, indicating that autophagosome interactions are dispensable for initial PV maturation to a phagolysosome-like compartment but are involved in vacuole expansion. Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactions by using the Dot/Icm T4SS.

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…Cell lysates were collected for SDS-PAGE analysis at 72 h post-infection. In agreement with previous studies [26], there was a lack of SQSTM1 degradation during infection with WT Coxiella . In fact, SQSTM1 levels were increased under these conditions, but could be rescued with starvation (Figure 5(e)).…”

“…Increasing amounts of lipidated LC3B were observed at 48 and 72 h post-infection. Thus, in agreement with previous literature [26], it was observed that LC3B-I to LC3B-II conversion occurs specifically in response to Coxiella infection (Figure 5(a)).…”

“…Thus, the membrane capacity of the CCV is expanded as autophagosomes deliver extra membrane to the growing CCV. In agreement with previously published work [26], the data presented here indicates there is a block in the autophagic flux during Coxiella infection due to the accumulation of SQSTM1 in cells. This has previously led to the conclusion that autophagy is not induced by the bacteria, rather that LC3B-II is not being degraded, however we can show that increasing autophagy through starvation leads to further CCV expansion.…”

“…Another report found no difference in the LC3B lipidation profile between uninfected and infected HeLa cells [13]. Within this study we observed autophagy induction in a Dot/Icm-dependent manner during infection of HeLa cells, which has previously been reported in THP1 cells [26]. This induction likely assists the CCV in expanding to its normal size, since further autophagy, induced through starvation, increased the CCV size significantly.…”

“…Whether Coxiella infection induces the initiation of autophagy remains under debate. It has been reported that LC3B lipidation is induced during Coxiella infection of macrophages, although this was not dependent upon the Dot/Icm secretion system [26]. Another report found no difference in the LC3B lipidation profile between uninfected and infected HeLa cells [13].…”