The Autophagic Machinery Ensures Nonlytic Transmission of Mycobacteria
Total Page：16
File Type：pdf, Size：1020Kb
The autophagic machinery ensures nonlytic PNAS PLUS transmission of mycobacteria Lilli Gerstenmaiera,1, Rachel Pillaa,1, Lydia Herrmanna, Hendrik Herrmanna,2, Monica Pradoa, Geno J. Villafanoa, Margot Kolonkoa, Rudolph Reimerb, Thierry Soldatic, Jason S. Kingd, and Monica Hagedorna,3 aSection Parasitology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; bElectronmicroscopy, Heinrich-Pette-Institute, 20251 Hamburg, Germany; cDepartment of Biochemistry, University of Geneva, 1211-Geneva, Switzerland; and dDepartment of Biomedical Sciences, University of Sheffield, Sheffield S10 2TN, United Kingdom Edited by Ralph R. Isberg, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA, and approved January 7, 2015 (received for review December 9, 2014) In contrast to mechanisms mediating uptake of intracellular bacterial M. tuberculosis and M. marinum in the amoeba Dictyostelium,is pathogens, bacterial egress and cell-to-cell transmission are poorly nonlytic for the host cell, even though its plasma membrane is understood. Previously, we showed that the transmission of path- perforated at the site of ejection. Previously, we showed that ogenic mycobacteria between phagocytic cells also depends on ejectosome formation is dependent on ESAT-6 (Early secretory nonlytic ejection through an F-actin based structure, called the antigenic target 6), a secreted virulence factor encoded in the ejectosome. How the host cell maintains integrity of its plasma RD1-locus, and the Dictyostelium small GTPase RacH. How- membrane during the ejection process was unknown. Here, we ever, both the structure and mechanistic details of ejectosome reveal an unexpected function for the autophagic machinery in function remain unknown. nonlytic spreading of bacteria. We show that ejecting mycobacteria Using the Dictyostelium–M. marinum system (9, 17, 18) to are escorted by a distinct polar autophagocytic vacuole. If autophagy further dissect the mechanism of ejectosome formation and is impaired, cell-to-cell transmission is inhibited, the host plasma function, we demonstrate an unexpected role for autophagic membrane becomes compromised and the host cells die. These membranes in both mycobacteria egress and concomitant cell-to- findings highlight a previously unidentified, highly ordered interac- cell transmission. tion between bacteria and the autophagic pathway and might rep- resent the ancient way to ensure nonlytic egress of bacteria. Results Correlative Microscopy Reveals a Vacuolar Structure at the Distal Pole autophagy | Dictyostelium discoideum | Mycobacterium marinum | ejection of Ejecting Bacteria. To better understand the mechanism of nonlytic bacterial ejection, we examined the ultrastructure of the n recent years, our understanding of the interactions between ejectosome. Using a correlative approach, we were able to Ithe host autophagic machinery and intracellular pathogens has identify ejectosomes by fluorescence microscopy (Fig. 1A and rapidly expanded. These interactions are complex; although, in Fig. S1 A and E), before ultrastructural analysis of serial thin many cases, the engagement of autophagy protects the host by sections by transmission electron microscopy (TEM) (Fig. 1 B–D capturing and destroying the pathogen, some bacteria actively and Movie S1). Fig. 1C shows a representative section of a bac- subvert this pathway to promote their own survival (reviewed in terium at a very late stage of ejection with the proximal pole ref. 1). Autophagy has also been suggested to promote cell-to- cell transmission of Brucella (2, 3), although the molecular Significance mechanisms are unknown. Both Mycobacterium tuberculosis, which causes tuberculosis in Pathogenic mycobacteria can be transmitted by direct ejection humans, and the closely related species M. marinum have been from one host cell to another. However, the mechanism of shown to interact with the autophagy machinery of their host cell ejection, and how lysing the host cell is prevented are un- (4–7). After uptake by immune phagocytes, the bacteria arrest known. This study explains how the host cell remains intact phagosomal maturation and convert their vacuole into a replica- and alive while Mycobacterium marinum breaks through its tion-permissive compartment. Both bacteria can translocate into plasma membrane during ejection. We show that a membra- the host cell cytosol dependent on an intact Region-of-Difference- neous cup is specifically recruited to the distal pole of ejecting 1-locus (RD1) (8–11). The genomic RD1-locus encodes a secre- M. marinum. We demonstrate that these membranes are tion system, ESX-1 (Type-VII secretion system), which has been formed by the canonical autophagic pathway, though they do associated with mycobacterial virulence (ref. 12, reviewed in refs. not mature to autophagolysosomes. Disruption of autophagy CELL BIOLOGY 13 and 14). Once in the cytosol, M. marinum becomes ubiqui- causes the host cells to become leaky and die during ejection. tinated (4) likely recruiting adaptor proteins, such as members This dramatically reduces cell-to-cell transmission of the in- of the sequestosome-1 family (SQSTM1), which also bind LC3 fection, demonstrating an important and unexpected role for (microtubule-associated proteins 1A/1B light chains 3A/LC3A and autophagy in maintaining plasma membrane integrity during 3B/LC3B), here referred to as Atg8, on autophagosomal mem- mycobacterial infection. branes. In this way, bacteria are normally targeted to autophago- Author contributions: L.G., R.P., R.R., T.S., and M.H. designed research; L.G., R.P., L.H., somes and killed, but M. marinum efficiently escapes this fate, H.H., M.P., G.J.V., M.K., J.S.K., and M.H. performed research; L.G., R.P., H.H., M.P., most probably by shedding the ubiquitinated material as a decoy G.J.V., M.K., and M.H. analyzed data; R.R., T.S., J.S.K., and M.H. contributed new (4). However, infection by M. tuberculosis can be overcome by reagents/analytic tools; and J.S.K. and M.H. wrote the paper. stimulating the classic autophagic pathway (15) and autophagy can The authors declare no conflict of interest. reduce the bacterial burden in vivo (7). This article is a PNAS Direct Submission. It was previously thought that M. marinum and M. tuberculosis 1L.G. and R.P. contributed equally to this work. leave their host cell by inducing necrotic or apoptotic cell death 2Deceased July 28, 2014. (16). However, we recently showed that these bacteria also 3To whom correspondence should be addressed. Email: [email protected]. exit their host cell and spread via an F-actin structure, termed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the ejectosome (17). This form of egress, which is common to 1073/pnas.1423318112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1423318112 PNAS | Published online February 2, 2015 | E687–E692 Downloaded by guest on September 28, 2021 A B The presence of Atg8-containing membranes at bacteria dur- * ing ejection indicates the specific recruitment of the autophagic * machinery. Selective autophagy is mediated by ubiquitination of the target and recruitment of adaptor proteins such as SQSTM1 that contain both ubiquitin- and Atg8-binding domains (reviewed in ref. 20). Consistent with this pathway, both ubiq- Actin Dictyostelium M. marinum uitin and DdSQSTM1 (GFP-SQSTM1), the single ortholog of SQSTM1 accumulated in a pocket around the distal C D pole of ejecting bacteria, similarly to Atg8 (Fig. 2 E–G). Extra- cellular M. marinum was never associated with Atg8 or GFP- * SQSTM1, indicating that the autophagic membrane is retained * inside the host. Importantly, the bacterial localization of both ubiquitin and Atg8 was largely restricted to ejecting bacteria. Although less than 15% of total cytoplasmic bacteria were as- sociated with ubiquitin (Fig. 2 H and I) or Atg8 (Fig. 2 H and J) E Atg8 C’ A Fig. 1. A vacuole caps the distal pole of ejecting M. marinum.(A)ADic- tyostelium cell ejecting a M. marinum bacterium was localized by confocal fluorescence microscopy. The bacterium is shown in red, actin is shown in green. (B) Overlay of the corresponding brightfield and transmission elec- tron microscopy (TEM) image after processing. (Scale bar: 2 μm.) (C) TEM image of the ejecting bacterium. The white arrow indicates the distal pole of the ejecting bacterium, the white arrowhead points toward the proximal BCD pole. (Scale bar: 2 μm.) (C’) shows a magnification of the region where the bacterium perforates the plasma membrane. Typical for ejection, the plasma membrane is protruding and tightly apposed to the ejecting bacterium (indicated by black arrows). (D) High magnification of the distal pole of the ejecting bacterium. (Scale bar: 500 nm.) Black arrowheads indicate the vac- uolar membrane apposed to the bacterium, black arrows point to the membrane exposed to the host cell cytosol. The actin-rich ejectosome is in- Atg8 Atg8 Atg8 dicated with an asterisk in all images. (E) 3D-model of the vacuolar pocket around the pole of an ejecting bacterium. Serial thin sections were imaged E F G by TEM and surface rendered. Bacteria are depicted in red, polar vacuole is depicted in yellow, and plasma membrane is depicted in green. (white arrowhead) being extracellular and the distal pole (white arrow) lagging behind within the cell. The plasma membrane, GFP GFP-SQSTM1 UB which is ruptured at the site of ejection, is tightly apposed to the bacterium (Fig. 1C’, arrows). Strikingly, in every electron mi- H intracellular ejecting I crograph, the distal pole of the bacterium in the course of 100 ejection was tightly enclosed by a vacuolar structure. Both spa- 80 cious (with an electron lucent lumen, Fig. 1D and Fig. S1 F and UB G), and flat cisternal structures were observed (Fig. S1 B–D), but 60 J 3D-reconstruction using serial thin sections revealed these cis- 40 ternal structure always formed a vacuole around the distal bac- % association 20 terial pole (Fig. 1E, Fig. S2, and Movie S2). 0 Atg8 UB Atg8 The Autophagic Machinery Is Present at the Distal Pole of Ejecting Bacteria.