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

<p><i>Coxiella burnetii</i> is an intracellular bacterial pathogen which causes Q fever, a human infection with the ability to cause chronic disease with potentially life-threatening outcomes. In humans, <i>Coxiella</i> infects alveolar macrophages where it replicates to high numbers in a unique, pathogen-directed lysosome-derived vacuole. This compartment, termed the <i>Coxiella</i>-containing vacuole (CCV), has a low internal pH and contains markers both of lysosomes and autophagosomes. The CCV membrane is also enriched with CLTC (clathrin heavy chain) and this contributes to the success of the CCV. Here, we describe a role for CLTC, a scaffolding protein of clathrin-coated vesicles, in facilitating the fusion of autophagosomes with the CCV. During gene silencing of <i>CLTC</i>, CCVs are unable to fuse with each other, a phenotype also seen when silencing genes involved in macroautophagy/autophagy. MAP1LC3B/LC3B, which is normally observed inside the CCV, is excluded from CCVs in the absence of CLTC. Additionally, this study demonstrates that autophagosome fusion contributes to CCV size as cell starvation and subsequent autophagy induction leads to further CCV expansion. This is CLTC dependent, as the absence of CLTC renders autophagosomes no longer able to contribute to the expansion of the CCV. This investigation provides a functional link between CLTC and autophagy in the context of <i>Coxiella</i> infection and highlights the CCV as an important tool to explore the interactions between these vesicular trafficking pathways.</p

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of <i>Coxiella burnetii</i>

<div><p><i>Coxiella burnetii</i> is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When <i>Coxiella</i> is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate <i>Coxiella</i>-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of <i>Coxiella</i>. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in <i>cig57</i>-disrupted <i>Coxiella</i>, siRNA gene silencing of <i>FCHO2</i> or clathrin (<i>CLTC)</i> inhibits <i>Coxiella</i> growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of <i>Coxiella</i> and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic <i>Coxiella</i> replicative vacuole and enable intracellular success of this human pathogen.</p></div

Depletion of FCHO2 or clathrin inhibits <i>C</i>. <i>burnetii</i> growth.

<p>HeLa cells reverse transfected with siRNA against human clathrin heavy chain (si-<i>CLTC</i>) and siRNA against human <i>FCHO2</i> (si-<i>FCHO2</i>) were infected with wild-type <i>C</i>. <i>burnetii</i> for 2, 4 or 6 days. Non-targeting (OTP-NT) siRNA was used as a control. <b>(A)</b> Western blots depict knockdown over the timecourse from the day of infection (D0) to day 6 (D6) and include OTP-NT (–) and targeting (+) samples for each timepoint. β-actin was used as a loading control and the amount of knockdown achieved for each timepoint was graphed relative to the band intensities of β-actin. <b>(B)</b> <i>Coxiella</i> growth over the timecourse was measured by qPCR. Primers towards <i>ompA</i> of <i>Coxiella</i> were used to amplify this gene and graph the relative fold growth from the inoculum reading at day 0. Results were then normalized to OTP-NT and graphed relative to the fold change seen in this control. Dotted line corresponds to the fold change of 1.0 relative to OTP-NT and error bars represent SEM. * = P<0.05, ** = P<0.01. <b>(C)</b> Immunofluorescent images of the growth of <i>C</i>. <i>burnetii</i> at 4 days post infection. Cells were stained with anti-<i>Coxiella</i> (red) and anti-LAMP1 (green) and the nucleus in DAPI (blue). Scale bars represent 10 μm. <b>(D)</b> Images from three independent experiments were used to measure vacuole area during targeting and non-targeting conditions. A total of 50 vacuoles were measured for each condition in each experiment (n = 3). * = P<0.05, *** = P<0.001, error bars = SEM. <b>(E)</b> Results of vacuole sizes for one experiment were plotted as individual datapoints (n = 50) to reveal the spread of vacuole sizes seen in each condition. **** = P<0.0001. <b>(F)</b> The number of vacuoles per cell were counted in greater than 30 cells per experiment and condition and given a value of either 1, 2 3 or more than 4 vacuoles per cell. Results are displayed as a percentage of the total number of cells counted and represent three independent experiments.</p

Interaction between Cig57 and FCHO2.

<p><b>(A)</b> A yeast-two hybrid screen, using a HeLa cDNA library, revealed FCHO2 as a binding partner of Cig57. Growth on double dropout (DDO) plates for all transformations indicates plasmid retention and yeast viability. Growth on quadruple dropout (QDO) plates indicates the interaction between FCHO2 and Cig57 (segment 2) and the lack of growth in segment 1 and 3 of this plate indicates no interaction with the empty vectors present. <b>(B)</b> A Pull-down assay verified the interaction between Cig57 and GFP-FCHO2 during <i>Coxiella</i> infection of HEK 293T cells. Cells were infected with wild-type (WT) <i>Coxiella</i>, or <i>Coxiella cig57</i>::Tn strain expressing 3xFLAG-Cig57 from a plasmid, and transfected with GFP constructs (GFP or GFP-FCHO2). GFP proteins were pulled out using GFP-Trap beads. The presence of 3xFLAG-Cig57 in the pull down lysates indicates interaction between GFP-FCHO2 and Cig57. Results are shown for one repeat of the experiment. <b>(C)</b> Representative images showing HeLa cells transfected to express mCherry or mCherry-Cig57 (red) during infection with wild-type <i>C</i>. <i>burnetii</i> (green). Nuclei are stained in blue with DAPI. Scale bar = 10 μm. <b>(D)</b> HeLa cells stably expressing GFP-FCHO2 were transfected with pmCherry-Cig57 and nuclei stained with DAPI (blue). Scale bars represent 10 μm and the CCV is denoted by an asterisk.</p

Growth of <i>Coxiella</i> is attenuated in cell lines devoid of FCHO2.

<p><b>(A)</b> Immunoblots of HeLa cells (+) and FCHO2 knockout (KO) (–) cells using α-FCHO2 and α-β actin antibodies depicting the lack of FCHO2 expression in the KO cell line. <b>(B)</b> Genome equivalents of wild-type <i>Coxiella</i> were measured by qPCR against the <i>ompA</i> gene, in wild-type HeLa parent cells and in FCHO2 KO cells. The fold increase in genome equivalents was measured relative to the day 0 genomic equivalents, taken 4 hours post-infection. * = P<0.05. Results are representative of three independent experiments and error bars represent SEM <b>(C)</b> Micrographs of HeLa cells or FCHO2 KO cells at day 3 post-infection with wild-type <i>Coxiella</i>. <i>Coxiella</i> (red) and LAMP1 (green) were used as markers of the vacuole and host cell nuclei are stained in blue with DAPI. Scale bar = 10 μm. Vacuole areas were measured over three experiments <b>(D)</b> and for an individual experiment <b>(E).</b> At least 50 vacuoles were measured for each experiment and error bars represent SEM. ** = P<0.01, **** = P<0.0001.</p

The tyrosine endocytic sorting motif is bound by FCHO2 (1–433).

<p>Yeast two-hybrid interactions for co-transformation of pGADT7-<i>FCHO2</i> (1–433) and pGBKT7-<i>cig57</i>, or the site-directed mutations of Cig57 endocytic sorting motifs as indicated. <b>(A)</b> Growth on double dropout (DDO) plates controls for yeast viability and plasmid uptake, and growth on quadruple dropout (QDO) plates indicates an interaction between the indicated proteins. Conditions that indicate protein-protein interactions have been highlighted in bold. <b>(B)</b> Immunoblots of yeast cell lysate demonstrate the expression of the proteins. α-HA recognises expression of HA-tagged FCHO2 1–433, 65.7 kDa, from the pGADT7 plasmid, and α-c myc represents expression of tagged Cig57 derivatives (66.6 kDa) from the pGBKT7 plasmid.</p

Clathrin localization in FCHO2 KO cells.

<p><b>(A)</b> HeLa cells, or HeLa FCHO2 KO cells were infected with WT <i>Coxiella</i>, and fixed 72 hours post infection. Coverslips were stained with a clathrin antibody (green) and anti- <i>Coxiella</i> (red). Nuclei are stained in blue with DAPI. Confocal micrographs are representative of three independent experiments and the scale bar represents 10 μm. <b>(B)</b> Measurements of clathrin intensity were taken around the CCV as well as in the cytoplasm, and a ratio was formed for the intensity CCV/cytoplasm. 20 cells were measured for each of three experiments, and results in <b>(C)</b> show all data points from one experiment. Error bars represent SEM and * = P<0.05, ** = P<0.01.</p

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.Originally included in thesis in manuscript form. </p