Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth

Abstract

Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.

The Coxiella burnetii T4SS Effector AnkF Is Important for Intracellular Replication

“…The functional redundancy might explain the lack of a replication-defect in the absence of vimentin ( Figure 9C) (Guignot and Servin, 2008;Kumar and Valdivia, 2008;Truchan et al, 2016). Furthermore, actin patches surrounded the CCV (Figure 9), as reported previously (Miller et al, 2018). Miller and colleagues showed that the lack of actin patches did not affect C. burnetii replication.…”

“…Indeed, cytokeratin 8 and 18 as well as actin act in concert with vimentin to ensure stability of the C. trachomatis inclusion (Kumar and Valdivia, 2008). Furthermore, actin was shown to associate with the CCV, and network disruption might result in smaller CCVs (Aguilera et al, 2009;Miller et al, 2018). It was proposed that actin stabilizes the CCV (Colonne et al, 2016) and participates in vesicular trafficking events (Aguilera et al, 2009;Miller et al, 2018).…”

“…Furthermore, actin was shown to associate with the CCV, and network disruption might result in smaller CCVs (Aguilera et al, 2009;Miller et al, 2018). It was proposed that actin stabilizes the CCV (Colonne et al, 2016) and participates in vesicular trafficking events (Aguilera et al, 2009;Miller et al, 2018). In order to elucidate the association of microfilaments, microtubules, and cytokeratins with bacterial compartment, immunofluorescence of C. burnetii-infected HeLa cells was performed.…”

“…The activity of T4SS effector proteins allows the massive expansion of the CCV, which can occupy the majority of the host cell´s volume (Lührmann et al, 2017). How C. burnetii ensures the stability of this huge compartment is not understood, but galectins (Mansilla Pareja et al, 2017) and actin (Colonne et al, 2016;Miller et al, 2018) might be involved.…”

Replication of Coxiella burnetii in a Lysosome-Like Vacuole Does Not Require Lysosomal Hydrolases

“…This behavior likely reflects defects in fusion between the CCV and endocytic vesicles that manifest due to changes on the CCV membrane that are induced by accumulated waste products. CD63 and LAMP1 are uniformly distributed on CCVs in GNPTAB Ϫ/Ϫ cells as opposed to a typical punctate localization (33). The uniform distribution of CCV membrane molecules could result from deficient lipid raft formation.…”