A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis

Abstract

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.

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

“…Consistent with previous studies, this multi-vacuole phenotype was also observed when expression of STX17 (syntaxin 17) was silenced to block autophagy progression [13,24] (Figure 1(c)). STX17 is responsible for facilitating fusion of autophagosomes with lysosomes [25].…”

“…In addition to having only one large CCV per cell, a normal Coxiella infection of HeLa cells results in recruitment of LC3B to the lumen of the CCV, representing significant fusion of the CCV with autophagosomes [13,15]. The multi-vacuole phenotype observed above is indicative of a CCV fusion defect in the absence of CLTC.…”

“…This is a multi-protein apparatus which spans both the bacterial and vacuolar membranes, to enable the translocation of bacterial proteins, termed effectors, into the host cell [12]. The secretion system itself, and a subset of the effectors translocated by this system are essential for intracellular replication [10,11,13]. The cohort of approximately 150 effector proteins are collectively responsible for manipulating host cellular functions to facilitate the intracellular replication of Coxiella [10].…”

“…Lysosomal markers, including LAMP1 (lysosomal associated membrane protein 1), the vacuolar-type H + -translocating ATPase, and CTSD (cathepsin D), surround the CCV [14]. Additionally, the autophagy marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) is located inside the CCV, and is recruited there during autophagosome fusion with the CCV [5,13,15]. These fusion events benefit the establishment of the CCV, since induction of autophagy by starvation allows for more infected cells and larger CCVs [16].…”

“…In the absence of CLTC, CCVs remain small and support minimal Coxiella replication [17,18]. More recently, the Coxiella effector Cig57 was shown to interact with FCHO2 (FCH domain only 2), an accessory protein in clathrin-mediated endocytosis, and contribute to Coxiella virulence, as Cig57 is essential for intracellular replication of Coxiella and CCV biogenesis [13,18]. Both Cig57 and CvpA play vital, non-redundant roles in facilitating intracellular replication of Coxiella through their interactions with clathrin-dependent processes, however the biochemical functions of these important effectors remain to be determined.…”