Arf GAP 1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton
2017; Springer Nature; Volume: 19; Issue: 1 Linguagem: Inglês
10.15252/embr.201744371
ISSN1469-3178
AutoresOk‐Ryul Song, Christophe J. Queval, Raffaella Iantomasi, Vincent Delorme, Sabrina Marion, Romain Veyron‐Churlet, Elisabeth Werkmeister, Michka Popoff, Isabelle Ricard, Samuel Jouny, Nathalie Deboosère, Frank Lafont, Alain R. Baulard, Edouard Yeramian, Laurent Marsollier, Eik Hoffmann, Priscille Brodin,
Tópico(s)Bacteriophages and microbial interactions
ResumoScientific Report15 November 2017free access Source DataTransparent process ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton Ok-Ryul Song CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Equipe ATIP AVENIR, CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Christophe J Queval CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Raffaella Iantomasi CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Vincent Delorme orcid.org/0000-0001-5235-7069 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Sabrina Marion CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Romain Veyron-Churlet CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Elisabeth Werkmeister CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Michka Popoff CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France CNRS, UMR8520, Institut d'électronique, de microélectronique et de nanotechnologie, Villeneuve d'Ascq, France Search for more papers by this author Isabelle Ricard CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Samuel Jouny CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Nathalie Deboosere CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Frank Lafont CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Alain Baulard orcid.org/0000-0002-0150-5241 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Edouard Yeramian Unité de Microbiologie Structurale, CNRS UMR3528, Institut Pasteur, Paris, France Search for more papers by this author Laurent Marsollier Corresponding Author [email protected] orcid.org/0000-0001-5497-5995 Equipe ATIP AVENIR, CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France Search for more papers by this author Eik Hoffmann Corresponding Author [email protected] orcid.org/0000-0003-4224-1091 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Priscille Brodin Corresponding Author [email protected] orcid.org/0000-0003-0991-7344 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Ok-Ryul Song CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Equipe ATIP AVENIR, CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Christophe J Queval CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Raffaella Iantomasi CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Vincent Delorme orcid.org/0000-0001-5235-7069 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Sabrina Marion CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Romain Veyron-Churlet CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Elisabeth Werkmeister CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Michka Popoff CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France CNRS, UMR8520, Institut d'électronique, de microélectronique et de nanotechnologie, Villeneuve d'Ascq, France Search for more papers by this author Isabelle Ricard CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Samuel Jouny CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Nathalie Deboosere CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Frank Lafont CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Alain Baulard orcid.org/0000-0002-0150-5241 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Edouard Yeramian Unité de Microbiologie Structurale, CNRS UMR3528, Institut Pasteur, Paris, France Search for more papers by this author Laurent Marsollier Corresponding Author [email protected] orcid.org/0000-0001-5497-5995 Equipe ATIP AVENIR, CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France Search for more papers by this author Eik Hoffmann Corresponding Author [email protected] orcid.org/0000-0003-4224-1091 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Search for more papers by this author Priscille Brodin Corresponding Author [email protected] orcid.org/0000-0003-0991-7344 CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea Search for more papers by this author Author Information Ok-Ryul Song1,2,3,4, Christophe J Queval1, Raffaella Iantomasi1, Vincent Delorme1,4, Sabrina Marion1, Romain Veyron-Churlet1, Elisabeth Werkmeister1, Michka Popoff1,5, Isabelle Ricard1, Samuel Jouny1, Nathalie Deboosere1, Frank Lafont1, Alain Baulard1, Edouard Yeramian6,‡, Laurent Marsollier *,2,3,‡, Eik Hoffmann *,1,‡ and Priscille Brodin *,1,4,‡ 1CNRS, Inserm, CHU Lille, U1019 - UMR8204 - CIIL - Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Univ. Lille, Lille, France 2Equipe ATIP AVENIR, CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France 3CRCINA, INSERM, Université de Nantes, Université d'Angers, Angers, France 4Institute Pasteur Korea, Seongnam-si, Gyeonggi-do, South Korea 5CNRS, UMR8520, Institut d'électronique, de microélectronique et de nanotechnologie, Villeneuve d'Ascq, France 6Unité de Microbiologie Structurale, CNRS UMR3528, Institut Pasteur, Paris, France ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 244 688314; E-mail: [email protected] *Corresponding author. Tel: +33 320 871035; E-mail: [email protected] *Corresponding author. Tel: +33 320 871184; E-mail: [email protected] EMBO Rep (2018)19:29-42https://doi.org/10.15252/embr.201744371 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase-activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions. Synopsis Mycobacterial uptake into epithelial cell is dependent on the receptor M3R and requires actin rearrangements regulated by Arf1. This pathway is controlled by ArfGAP1, demonstrating its role in the regulation of host-pathogen interactions. Colonization by M. tuberculosis depends on the M3 muscarinic receptor (M3R) and the activation of Arf1 and PLD1. M3R-mediated entry of M. tuberculosis into epithelial cells is restricted by the Arf GTPase-activating protein ArfGAP1. Mycobacterial entry involves rearrangements of the actin cytoskeleton controlled by ArfGAP1. Introduction Many pathogenic bacteria are able to interfere with host signaling to enter and replicate within cells during infection 123. Phagocytic cells, such as macrophages and dendritic cells, engulf bacteria upon engagement of phagocytic receptors, while many bacterial virulence factors interfere with the host at later stages of the phagocytic process, for example, by subverting phagosome maturation or to achieve phagosomal escape. In contrast, many pathogens are also able to enter non-phagocytic cells, such as epithelial cells and fibroblasts, by actively interfering with the actin cytoskeleton to trigger pathogen uptake. For example, Listeria monocytogenes and Yersinia pestis use bacterial surface proteins to induce Rac1/Cdc42-mediated actin rearrangement to enter the host 45, while Salmonella typhimurium and Shigella flexneri apply a type III secretion system to trigger Cdc42/Rac1/RhoA-dependent actin reorganization 67. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which remains a leading infectious disease around the world with 1.8 million deaths and more than 10 million new cases each year 8. The pathology of tuberculosis is directly linked to the tight interplay between the host immune response and the persistence of Mtb infection 9. Although mostly alveolar macrophages and dendritic cells are colonized by Mtb, several findings indicate that non-phagocytic cells are an essential niche for this pathogen to promote its replication and dissemination from the lung to other organs 101112. In pulmonary epithelial cells, the redistribution of actin filaments was shown to be altered upon Mtb infection and is important for pathogen entry 13. Although a participation of macropinocytosis in Mtb uptake has been demonstrated 14, further examination showed that Mtb entry relies also on other, yet to be characterized, cellular components to enter into non-phagocytic cells 15. Furthermore, it has been suggested that the trafficking pattern of Mtb in alveolar epithelial cells differs from the one observed in macrophages, since Mtb-containing vacuoles show features of mature, late endosomes and were labeled with Rab5 and Rab7 16. In addition, it was reported that mycobacterial heparin-binding hemagglutinin (HBHA) reduces G-actin polymerization into actin filaments and blocks autophagy pathways in epithelial cells 171819. Recently, it has been shown that HBHA interacts with syndecan 4, a mycobacterial attachment receptor in epithelial cells that promotes mycobacterial entry 20. Furthermore, it also has been shown that the Mtb vaccine strain, Mycobacterium bovis BCG, is able to manipulate G protein-coupled receptors (GPCRs) in epithelial cells, such as CXCR1 and CXCR2, resulting in Rac1 upregulation and altered actin cytoskeleton signaling 21. However, despite these recent findings, the precise mechanisms regulating Mtb entry and persistence in epithelial cells remain to be elucidated. Here, we aimed to identify the main players influencing Mtb infection of epithelial cells using a genomewide RNAi screen, similar to the one reported previously in macrophages 22, and investigated the underlying mechanisms that lead to the colonization of this cell type. We identified Arf GTPase-activating protein 1 (ArfGAP1) as a key host restriction factor of Mtb entry. By dissecting the molecular mechanisms that ArfGAP1 engages to modulate mycobacterial colonization, we characterized a novel signaling pathway involved in regulating the uptake of mycobacteria into epithelial cells. We found that Mtb infection induced ADP-ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1) activation downstream of the muscarinic receptor 3 (M3R). M3R is belonging to the GPCR family and was originally identified as the major receptor for acetylcholine neurotransmitter for regulating physiological activities in various organs 23. Knock-down (KD) of ArfGAP1 resulted in actin stress fiber formation activating massive colonization of epithelial cells by Mtb. Importantly, we demonstrated that these actin cytoskeleton rearrangements favor specifically mycobacterial entry but not uptake of other bacteria, such as Shigella flexneri and Yersinia pseudotuberculosis, and that this pathway is specific to epithelial cells and not to other cell types. Therefore, our findings demonstrate a novel role for ArfGAP1 and M3R in the regulation of host–pathogen interactions in epithelial cells by modulating and controlling the actin cytoskeleton. Results and Discussion To assess the interactions between epithelial cells and Mtb, we decided to identify host effector proteins that restrict the colonization of the pathogen during infection. We applied a phenotypic assay based on genomewide RNAi screening, which we developed previously 2425, to human lung epithelial A549 cells. This system relies on automated confocal microscopy and allowed us to quantify the intracellular localization of the EGFP-expressing Mtb H37Rv strain (H37Rv-GFP) inside infected A549 cells. Briefly, 3 days before the infection epithelial cells were transfected with smart pool siRNAs from a genomewide library (targeting 16,532 human genes; Fig 1A). Cells were then infected with H37Rv-GFP for 5 h, and extracellular bacilli were removed by extensive washing and amikacin treatment. The infected cells were incubated for 5 days, labeled by DAPI followed by image acquisition using an automated confocal microscope 2425. Customized image analysis was used for the quantification of relevant parameters, such as percentage of infected cells compared to the total number of cells 26, and non-targeting siRNA (scramble) was used as control. Classification of hits was based on Z scores taking into account the percentage of infected cells compared to the cell number (Fig 1B). Among the genes that had an impact on Mtb colonization, silencing of 109 genes resulted in increased Mtb infection rates of A549 cells, including the genes encoding the anti-inflammatory cytokine IL10 and the transcription factor XBP1, which is involved in the cellular stress response. Both molecules were shown previously to be affected by Mtb infection in epithelial cells 2728 confirming our findings. Interestingly, among the most significant siRNA hits, we identified ArfGAP1 that dramatically increased mycobacterial infection of epithelial cells upon its silencing (Fig 1B) without affecting the intracellular distribution of ArfGAP1 (Appendix Fig S1). In order to validate our finding of the siRNA screen, we investigated Mtb uptake in epithelial cells 3 days after transfecting them with ArfGAP1 siRNA. Compared to control cells, we found a threefold increase in the percentage of infected ArfGAP1 KD cells already 1 h post-infection (Fig 1C, left panel). In addition to the overall Mtb infection rate, we also found that ArfGAP1 KD cells were infected by more mycobacteria per cell at all time points (Fig 1C, right panel). These findings are not restricted to the investigated virulent Mtb strain, because a similar increase in mycobacterial colonization was found in ArfGAP1 KD cells upon infection with an attenuated, EGFP-expressing Mtb strain (H37Ra-GFP) (Fig EV1A–E). In agreement with this, Mtb colonization was also enhanced in control epithelial cells by adding QS11, a chemical inhibitor of ArfGAP1 function (Fig EV1F). Since ArfGAP1 is well known for its function in the formation of COPI vesicles at the Golgi 29, we addressed the possibility that ArfGAP1 silencing interferes with Golgi-dependent transport to the cell surface, for example, of an inhibitory factor, which might account for the observed effects on Mtb uptake. Therefore, we analyzed Golgi structure and integrity as well as secretory trafficking upon ArfGAP1 KD by confocal and spinning disk microscopy, respectively. While ArfGAP1 KD did not alter Golgi integrity compared to scramble cells, as shown by the two Golgi markers TGN46 and golgin97, Arf1 KD induced a fragmentation of the trans-Golgi network (Appendix Fig S2A–D), similar than brefeldin A (BFA) treatment 30. ArfGAP1 silencing also did not interfere with the intracellular distribution and Golgi-associated accumulation of Rab6, a GTPase that is regulating secretory trafficking from the Golgi to the cell surface 31 (Appendix Fig S2E and F). Moreover, we also did not observe differences between scramble and ArfGAP1 KD cells in the formation and exit of neuropeptide Y, a secretory cargo known to be transported from the Golgi in a Rab6-dependent manner 3132 (Movies EV1 and EV2). These findings exclude the possibility that ArfGAP1 silencing alters Golgi-dependent transport and suggest that ArfGAP1 function on the cytoskeleton is restricting Mtb uptake into epithelial cells. Altogether, our data demonstrate that ArfGAP1 works as a critical host restriction factor regulating the intracellular colonization of epithelial cells by Mtb. Figure 1. Genomewide siRNA screening identified ArfGAP1 as a host factor restricting Mtb colonization of epithelial cells A, B. A549 epithelial cells were transfected with pooled siRNA in 384-well plates and infected with Mtb H37Rv-GFP at an MOI of 5. Five days post-infection, images were acquired by automated confocal microscopy followed by image analysis. Shown are the applied workflow (A) and a Z score scatter plot of the obtained data (B, upper panel). Among the most significant siRNA hits, ArfGAP1 was identified in Mtb-infected cells. A representative confocal image of scramble and siArfGAP1-transfected cells is shown below (B, lower panel). Cell nuclei were stained by DAPI (blue), while GFP-expressing Mtb are shown in green. Scale bars: 50 μm. C, D. The impact of ArfGAP1 silencing on Mtb uptake (C) and intracellular replication of Mtb (D) were analyzed in infected A549 cells and by automated confocal microscopy. Shown are the applied workflow (upper panel) and results of three independent experiments (lower panel). Both overall infection rates (left histogram in C and D) and bacterial load per cell (right histogram in C and D) are indicated. Data analysis was carried out to obtain percentages of infected cells (n ≥ 700) and quantified areas of intracellular bacteria (px: pixels). Data are presented as mean ± SEM. ***P < 0.0005, ns: not significant (Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of ArfGAP1 silencing on mycobacterial colonization using an attenuated Mtb strain A, B. Scramble and ArfGAP1 KD cells were infected with Mtb H37Ra-GFP at MOI 10 and analyzed 4 days post-infection by automated confocal microscopy. Shown are representative images (A) and Mtb colonization of cells (B). Cell nuclei were stained by DAPI (blue), while GFP-expressing Mtb are shown in green. Scale bars: 50 μm. C, D. Similarly, these cells were also lysed and plated at different dilutions (C) to determine colony-forming units (CFU) (D). E. The impact of ArfGAP1 silencing on Mtb H37Ra uptake at MOI 5 was analyzed by automated confocal microscopy at the indicated time points post-infection. F. A549 cells were treated with the ArfGAP1 inhibitor QS11 and analyzed for Mtb H37Ra colonization at MOI 5 after 4 h post-infection. Data information: Data are presented as mean ± SEM. ***P < 0.0005, **P < 0.005, *P < 0.05, ns: not significant (Student's t-test). Histograms display at least 1,400 (B), 700 (E), 400 (F) analyzed cells in three replicates (B), in four replicates (E), and in three replicates (F). Shown are representative examples of two (B, D, F) or three (E) independent experiments. Download figure Download PowerPoint In addition to the findings of Mtb uptake, we also infected cells with Mtb at a higher MOI followed by ArfGAP1 silencing 2 h after the infection to analyze a possible influence on the intracellular replication of Mtb in A549 epithelial cells. While there was no difference 1 day post-infection, we observed an increased bacterial load in ArfGAP1 KD cells compared to scramble cells 3 and 5 days post-infection (Fig 1D), though it was less pronounced compared to the impact of ArfGAP1 silencing on Mtb uptake. These results suggest that the absence of ArfGAP1 also has an impact on mycobacterial replication. It is possible that ArfGAP1 is able to regulate processes that have an impact on phagosomal membrane integrity and rupture, which would allow Mtb to get access to the cytosol and to nutrients, which could influence Mtb replication rates. However, this would need to be further investigated in future studies. Here, we decided to focus on the role of ArfGAP1 on Mtb uptake by epithelial cells. ArfGAP1 is known to control Arf1-dependent membrane trafficking by facilitating the hydrolysis of the active, GTP-bound form of Arf1 to its inactive, GDP-bound conformation 3334. Therefore, we investigated next whether mycobacterial entry into epithelial cells involves a signaling complex comprising Arf1 and other associated proteins. We analyzed the amount of activated Arf1-GTP by pull-down and Western blotting and found, as expected, that ArfGAP1 KD of cells increased the levels of Arf1-GTP compared to control cells (Fig 2A). While levels of Arf1-GTP increase rapidly upon Mtb infection and reach a peak after 1 h post-infection (Fig 2B), ArfGAP1 expression only starts to increase significantly 1–3 h after Mtb infection (Fig 2C). Furthermore, the KD of Arf1 or of GRP1/ARNO3, the guanine nucleotide exchange factor (GEF) of Arf1 35, both reduced dramatically mycobacterial colonization (Fig 2D). Similarly, also the presence of BFA, an inhibitor of ArfGEF function 3036, induced a decrease in mycobacterial colonization (Fig 2E). Of note, already sub-nanomolar concentrations of BFA were able to impair bacterial uptake, suggesting that this effect is due to the absence of activated Arf1. These findings demonstrate that the initial activation of Arf1 is essential to promote mycobacterial entry, while ArfGAP1 expression increases downstream of these events, suggesting a negative feedback loop that downregulates Arf1 activity. The Arf GTPase activation state depends strongly on the activity of phospholipases (PLD), as it has been demonstrated previously in other cell types 37. In particular, ArfGAP1 expression is known to be induced by PLD1-mediated biosynthesis of phosphatidylinositol 38. Therefore, we decided to investigate PLD activity in Mtb-infected, epithelial cells. Similar to Arf1-GTP function, PLD activity was found to increase during Mtb infection (Fig 2F) and when Arf1GAP1 was silenced (Fig 2G). In contrast, absence of Arf1 in epithelial cells decreased PLD activity significantly. In order to verify which specific phospholipase is interfering with Mtb infection, we knocked down either PLD1 or PLD2 in epithelial cells and found that only the absence of PLD1 increased Mtb infection rate of A549 cells (Fig 2H). Moreover, we also confirmed by Western blotting that PLD1 KD is reducing the cellular expression of ArfGAP1 (Appendix Fig S3). Taken together, these results demonstrate that Mtb entry into A549 cells relies on a pathway that includes Arf1 and PLD1 activity, which is negatively controlled by ArfGAP1. Figure 2. ArfGAP1, Arf1, and PLD1 signaling modulate Mtb entry into epithelial cells A–C. A549 cells were transfected with scramble and ArfGAP1 siRNA and analyzed for active Arf1-GTP by pull-down and Western blotting (A). Tubulin was used as loading control. Similarly, Arf1-GTP was analyzed in cells infected with Mtb H37Rv at an MOI of 5 at the indicated times post-infection (B). Mtb-infected cells were also analyzed for the expression of ArfGAP1 by Western blotting (C). Shown are representative blots of two independent experiments. D. A549 cells were transfected with scramble, Arf1, and GRP1 siRNA, and Mtb colonization at MOI 20 was analyzed after 4 h. E. A549 cells were treated with the indicated concentrations of BFA for 18 h prior infection at MOI 20, and Mtb colonization was analyzed after 4 h by automated confocal microscopy. F, G. PLD activity was measured enzymatically using the Amplex Red reagent in Mtb-infected A549 cells at MOI 5 (F) and in cells that were transfected with scramble, Arf1, and ArfGAP1 siRNA (G) (n = 3). H. Mtb colonization at MOI 20 was also analyzed after 4 h by automated confocal microscopy in cells transfected with scramble, PLD1-specific siRNA, and PLD2-specific siRNA. Data information: Data are presented as mean ± SEM. ***P < 0.0005, **P < 0.005, *P < 0.05, ns: not significant (Student's t-test). Histograms display at least 200 analyzed cells in seven replicates (D), in three replicates (E), and six replicates (H). Shown are representative examples of two (D, E, H) or three (F, G) independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embr201744371-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint The correlation between Mtb colonization and Arf1 activity suggests a regulation of mycobacterial uptake by cytoskeletal organization, which is controlled by PLD1-dependent signaling. Many GPCRs at the cell surface were shown to induce PLD activity and to facilitate Arf-associated signaling pathways 39. Interestingly, the muscarinic receptor M3R has been shown to induce the BFA-sensitive activation of PLD 40 and in particular the Arf1-dependent route of PLD1 activation 41. Furthermore, we tested the impact of a selective KD of different receptors, which are known to interact with Arf1 (Fig EV2A), on Mtb colonization of epithelial cells during additional ArfGAP1 KD. The increased Mtb infection rate induced upon ArfGAP1 silencing (Fig 1C) was found for all tested receptors except for M3R (Fig EV2B), which suggests that M3R is a putative candidate receptor that is involved in Mtb uptake. Interestingly, silencing of the alpha-IIb/beta-3 integrin ITGA2B in addition to the simultaneous KD of ArfGAP1 increased Mtb uptake compared to co
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