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 -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- 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 . 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 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 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 (-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. The appearance of this polar vacuole (Fig. 1E and Fig. 2. The autophagic machinery is specifically recruited to the distal pole of ejecting M. marinum.(A–D) Fluorescence microscopy of infected cells Movie S2) is reminiscent of an expanding autophagic membrane. fixed and stained with anti-Atg8 showing polar accumulation of Atg8 at We therefore looked for the presence of autophagosomal different stages of ejection. Early in ejection, Atg8 (green) is found as a large markers around bacteria in the course of ejection. Consistent pocket around the bacterium (outlined with a broken white line) (B), which with the TEM images, immunofluorescence imaging showed that gets more focused as the bacterium becomes more extracellular (C and D). Atg8, a protein that specifically incorporates into expanding The outline of the cell is depicted with a red line. (E–G) Fluorescence mi- autophagosomal membranes (19), strongly localized around the crograph of an infected cell expressing GFP (E), GFP-SQSTM1 (F), or stained distal pole of ejecting M. marinum (Fig. 2A and Movie S3). This with anti-ubiquitin (UB; G). (A and E–G) Bacteria are shown in blue, actin is Atg8-positive cup was associated with M. marinum at all stages of shown in red, and the respective marker is shown in green. Ejectosomes are indicated by a white arrow, the distal poles of bacteria are highlighted ejection, from the very early stages when the bacterium is mainly with a white arrowhead. (H) Quantification of association of ubiquitin and inside the cell, until the bacterium is completely ejected, where Atg8 with cytoplasmic and ejecting bacteria. n = 3. (I and J) Cytoplasmic an Atg8-positive cup still caps the extremity of the almost fully M. marinum associated with ubiquitin (UB, I) and Atg8 (J). The bacteria are extracellular bacterium (Fig. 2 B–D). shown in blue, the respective marker is shown in green. (Scale bars: 2 μm.)

E688 | www.pnas.org/cgi/doi/10.1073/pnas.1423318112 Gerstenmaier et al. Downloaded by guest on September 28, 2021 PNAS PLUS at 24 h postinfection (hpi), all ejecting bacteria were labeled at 100 100 ABn.s. their intracellular pole (Fig. 2H). Furthermore, although the 80 80 on ti

nonejecting cytosolic bacteria recruited ubiquitin, atg8, and 60 ia 60 c GFP-SQSTM1 to patches along their entire surface (Fig. 2 I and 40 *** 40 J and Fig. S3), this was restricted to the distal pole during ejec- 20 20 tion. These observations indicate that the recruitment of the *** *** % asso % Atg8 association

autophagy machinery during ejection is by a specialized pathway T - VE W tg6 tg18 with a higher level of regulation and organization. atg5- a atg7- A 2xFY SQSTM1- The Canonical Autophagy Machinery Is Required for Cup Formation. C D To determine whether the recruitment of Atg8-positive mem- brane to ejecting bacteria required the canonical autophagy pathway, we infected Dictyostelium mutants with a defective atg1 gene (atg1−). Atg1 (Ulk1/2) is a key component of the canon- ical autophagy initiation complex and the Dictyostelium atg1− M. marinum RD1 M. smegmatis mutant is strictly impaired in autophagy (21). atg1− null cells Atg8 Atg8 were however still able to form ejectosomes closely resembling ** ** E 0.8 * ** the actin-rich structures generated in wild-type cells, with the WT same frequency (Fig. 3 A and B). In atg1− cells, none of the racH- 0.6 ejecting bacteria were associated with Atg8 (Fig. 3C), although atg1- rcells half of them (46.4% ± 3.6) retained the association with ubiq- o 0.4 uitin at their distal pole (Fig. S4). Consistent with this, TEM analysis in atg1− cells showed a total loss of the vacuolar cup 0.2 structures around the distal pole of ejecting bacteria (Fig. 3 D infected don

Infected acceptor per 0 and E). To test the requirement for other components of the 6242632 canonical autophagic pathway, we also tested for the recruitment hours post infection

of Atg8 to ejecting bacteria in mutants of the ubiquitin-like Atg8 n.s. n.s. F H 5x104 *** I 30 *** conjugation machinery (Atg5 and Atg7) and the phosphatidyl inositol 3-kinase (PI3K) complex (Atg6/Beclin1−) (Fig. 4A). 4x104 20 We found that atg5 and atg7 were strictly required for asso- 3x104 ciation of Atg8 with the ejectosome, whereas disruption of

per cell 2x104 one of the two Dictyostelium atg6 orthologs gave a significant, Atg8 10

G %deadcells although less penetrant defect, consistent with the partial block 1x104

in autophagy reported by others (22) (Fig. 4A). We therefore Mean PI-fluorescence 0 conclude that the core autophagic machinery is required for the non- inf. non- inf. non- inf. non- inf. formation of the ejectosome-associated vacuole. inf. inf. inf. inf. WT atg1- WT atg1-

Fig. 4. Elimination of autophagy reduces cell-to-cell transmission and cau- ses cell leakage. (A) Dictyostelium knockout mutants were infected with M. marinum 4 M. marinum A Actin B C Actin M. marinum and the recruitment of Atg8 to the distal poles of ejectosomes 3 Atg8 was scored in three independent experiments. Statistical significance was cal- culated using the one way ANOVA. (***P ≤ 0.001; n.s., not significant). (B) 2 Wild-type cells expressing GFP-Atg18 and GFP-2xFYVE were infected with 1 M. marinum and the accumulation of the respective marker at the distal pole % ejectosomes of ejecting bacteria was scored (n = 3). (C and D) Dictyostelium wild-type cells 0 were coinfected (Fig. S5) with M. marinum wild-type and M. marinum ΔRD1 WT atg1- (C)andM. smegmatis (D), respectively. Ejectosome structures (white arrow- D atg1- E atg1- F WT heads) are formed by both, M. marinum ΔRD1 (C, blue) and M. smegmatis (D, blue). Distinct Atg8 (green) localization at the distal pole (white arrows) is observed for both, M. marinum ΔRD1 (C)andM. smegmatis (D)alike M. marinum wild-type. (E) Cell-to-cell transmission was measured by flow

cytometry. Acceptor cells (wild-type) were mixed with infected donor cells CELL BIOLOGY (wild-type, racH−,andatg1−) at 6 hpi and transmission measured at 6, 24, 28 and 32 hpi. Both racH− and atg1− cells are significantly impaired in trans- mitting bacteria to acceptor cells (n = 3, mean ± SEM; *P ≤ 0.1; **P ≤ 0.01). Two-way ANOVA analysis). (F and G) Ejectosome structures (white arrowhead) formed by atg1− cells showed spilling of cytosolic Atg8 (green) on the extra- cellular side of the ejecting bacterium (indicated by white arrow). (Scale bar: Fig. 3. The atg1− mutant forms nonfunctional ejectosomes. (A)Ejectosomes 2 μm.) (H) In contrast to wild-type (WT) Dictyostelium cells, atg1− cells show are present in atg1− cells (white arrowheads). Actin is shown in red, bacterium a significant increase in mean PI accumulation upon M. marinum infection in blue. (B) Ejectosome frequency is unaffected in atg1− cells compared with (24 hpi), n = 3. Similarly, cell death was significantly increased (I)whenatg1− wild-type cells (WT), n = 3. Infected atg1− cells stained for Atg8 (C) illustrate cells were infected with M. marinum, whereas wild-type cells were unaffected the absence of Atg8 recruitment. Actin is in red, Atg8 antibody staining in (24 hpi). Statistical analysis was performed using one-way ANOVA analysis (n = green. (D and E) Transmission electron micrograph of an ejectosome in atg1− 3–4; mean ± SEM; ***P ≤ 0.001). (Scale bars: 2 μm.) cells confirms absence of the distal vacuole. (D) The ejectosome region is in- dicated with white arrowheads. (Scale bar: 1 μm.) (E) The distal pole of the ejecting bacterium is cytosolic and not capped by a vacuole. (Scale bar: To characterize this compartment further, we also examined the 500 nm.) Material spilling from the ejectosome region is indicated with black arrows (D and E). (F) A transmission electron micrograph of the distal pole of localization of both GFP-Atg18 (ortholog of the mammalian an ejecting bacterium from wild-type cells (WT). The vacuole engulfing the proteins WIPI1/2), and the PI(3)P reporter GFP-2xFYVE. Ca- bacterium is indicated with black arrows. (Scale bar: 500 nm.) nonically, both proteins are only recruited to the early, expanding

Gerstenmaier et al. PNAS | Published online February 2, 2015 | E689 Downloaded by guest on September 28, 2021 isolation membrane, in contrast to Atg8, which remains associated Although atg1− cells still form a tightly localized actin ring with the autophagosome throughout (23, 24). We observed that around ejecting bacteria, we frequently observed by light-mi- Atg18 localized to 38 ± 2.9% and 2xFYVE was present at 8.9 ± croscopy cytoplasmic material spilling out on the extracellular 4.4% of ejecting bacteria (Fig. 4B). This indicates that the pocket side of the ejectosome (Fig. 4 F and G). Extracellular material is formed at the site of ejection, by the canonical autophagic around the ejecting bacterium was also observed by EM (Fig. 3 D pathway. Furthermore, as Atg8 could be observed on >90% of and E, black arrows). This was never observed in wild-type cells, ejecting bacteria (Fig. 2H), this pocket must also be undergoing suggesting that the ejectosomes in atg1− cells may not be closing maturation to some extent. However, when we labeled the ly- as tightly around the ejecting bacteria. We therefore used the sosomal system by feeding cells fluorescent dextran, we never nuclear accumulation of the membrane impermeable dye pro- observed an accumulation at ejecting bacteria, indicating that pidium iodide (PI) to determine the membrane integrity of fusion with endolysosomes does not occur (Fig. S6). infected autophagy-deficient cells. After a 10 min exposure of To examine how the autophagic machinery is recruited, we infected cells (24 hpi) to PI, 40 ± 3.2% of live atg1− cells showed also disrupted the recently identified Dictyostelium ortholog of a significantly higher mean fluorescence than noninfected atg1− SQSTM1, the sole known autophagic adaptor in this organism cells indicating that the cells become partially permeable to the (22). Surprisingly however, loss of SQSTM1 had no affect on dye (Fig. 4H and Fig. S8). In contrast, the mean fluorescence of ± Atg8 recruitment. The alternative autophagy adaptors identified wild-type cells increased only moderately (10 6%) upon in- in mammals are poorly conserved through evolution and no clear fection. Cell leakiness will also eventually result in cell death. orthologs can be identified in Dictyostelium. Therefore, either Although other ejectosome-independent roles of autophagy may additional unknown adaptors must exist, or an alternative also contribute to this, when we quantified the number of dead mechanism of recruitment is responsible for the recruitment of cells at 24 hpi using the PI-staining and the characteristic for- the autophagy machinery. ward/side scatter of dead cells (Fig. S8) we found significantly more dead cells when autophagy is blocked (Fig. 4I). Recruitment of the Autophagic Machinery Is ESX-1 Independent. The Discussion specific recruitment of the autophagy machinery to the distal pole of ejecting bacteria raises the question whether polar mycobac- In this paper, we describe a previously unidentified interaction terial virulence factors are involved in this recruitment and between pathogenic M. marinum and the autophagic machinery. whether this is specific for pathogenic M. marinum. The virulence In contrast to previously described host-pathogen interactions, secretion system ESX-1 is enriched at the poles of M. marinum this is specific to ejecting bacteria and is required for the trans- (25) and is encoded in the RD1-region. Involvement of this se- mission of bacteria to a naïve host cell. Although this mechanism cretion system in ejection has been demonstrated before (17). We of transmission clearly benefits the bacteria, it might be con- therefore asked whether local secretion activity of ESX-1 at the sidered to also benefit the host, as the polar autophagic vac- uole apparently prevents plasma membrane leakage and host pole might be involved in the observed activation of autophagy. As cell death. ESX-1 is required for the bacteria to escape from Recently, a number of studies have demonstrated autophagy- into the cytosol before ejection, we established a coinfection independent roles for several autophagy-related proteins. We protocol that used wild-type M. marinum to break phagosomes show that the recruitment of the double-membrane cup to ejecting that contained M. marinum mutants lacking the RD1-locus bacteria requires proteins from the canonical autophagy pathway. M. marinum Δ ( RD1) at the same time (assay depicted in Fig. S5). Importantly, this includes the Atg1 initiation complex, which Immunofluorescence microscopy showed that both wild-type and is dispensible for the autophagy-independent recruitment of ΔRD1 M. marinum, were able to form ejectosomes and recruit – Atg8/LC3 to the single membranes of phagosomes and macro- Atg8 to their distal poles (Fig. 4C). Therefore, ESX-1 mediated pinosomes in mammalian cells (26). In addition, the requirement ESAT-6 secretion is required for ejectosome formation (17), the for the PI3K complex subunit Atg6 and the ubiquitin-like con- polarization of this secretion system and its local activity does not jugation machinery components Atg5 and Atg7, as well as the mediate recruitment of the autophagy machinery. To monitor presence of PI(3)P and Atg18 at the bacterial pole indicate that whether the observed ejection is restricted to pathogenic the ejectosome-associated vesicle is formed at the site of ejection mycobacteria we performed coinfection with nonpathogenic by the canonical autophagy machinery. However, although the M. smegmatis. Immunofluorescence micrographs showed that presence of GFP-Atg18 on only a subset of these vesicles indi- M. smegmatis was indeed able to form an ejectosome and the cates that they undergo some maturation, the lack of dextran autophagy machinery was recruited to its distal pole (Fig. 4D). delivery to this compartment implies that it does not undergo fusion with the endolysosomal system and therefore is unlikely to Autophagy Is Required for Efficient Nonlytic, Cell-to-Cell Transmission. become degradative. Although not required for their formation, we next asked whether The autophagic membrane is tightly restricted to the distal autophagy was required for ejectosome function. Ejection is the pole of ejectosome-associated bacteria, however, it is unclear major mechanism of cell-to-cell transmission in Dictyostelium (17). how this polar recruitment is orchestrated. It has previously been Therefore, to determine whether ejectosomes generated in atg1- reported that the autophagy machinery can be recruited to deficient cells are functional, we used a flow cytometry-based assay damaged membranes via galectins (27) and the protrusion of to monitor the transmission of GFP-expressing M. marinum from a bacterium through the plasma membrane may well be inter- mutant donor cells to mRFP-expressing wild-type acceptor cells preted by the cell as plasma membrane wounding. However, at (Fig. S7). The acceptor cells were added to infected donor cells early stages of ejection, the distal pole of these rod-shaped (wild-type, atg1−,orracH−) at 6 hpi and transmission was mea- bacteria is several microns away from the plasma membrane sured starting from 24 hpi onwards. Whereas wild-type cells effi- (Fig. S1C), and it is therefore possible that the recruitment of ciently transmitted the bacteria to the acceptor cells, transmission this vacuole is directed by the bacteria themselves in some way. was reduced by 50% in atg1− mutants (Fig. 4E). This decrease in The virulence secretion system ESX-1 localizes to the poles of transmission is comparable to the defect in racH− cells that we M. marinum (25) and is encoded in the RD1-region. Involvement previously showed to be deficient in nonlytic cell-to-cell trans- of this secretion system in ejection has been demonstrated before mission due to their complete inability to form ejectosomes (17). (17) in particular the secreted factor ESAT-6. However, the Therefore, although not required for ejectosome biogenesis, results of our coinfection assay show that neither the RD1 region autophagy is essential for efficient cell-to-cell transmission. nor M. marinum-specific virulence factors are responsible for

E690 | www.pnas.org/cgi/doi/10.1073/pnas.1423318112 Gerstenmaier et al. Downloaded by guest on September 28, 2021 distal vacuole generation, which would rather favor a host-driven Mycobacteria Cultivation, Strains, and Plasmids. Mycobacteria were cultivated PNAS PLUS process. At a molecular level, the accumulation of ubiquitin and with agitation at 32 °C in Middlebrook 7H9 (Difco), supplemented with SQSTM1 suggest recruitment of the autophagy machinery by 10% (vol/vol) OADC (oleic acid, albumin, dextrose, catalase) (Becton Dickinson), 5% (vol/vol) glycerol and 0.2% Tween80 (Sigma Aldrich). M. marinum wild factors which are directly present on the bacterial surface. The type (M-strain) and the msp12::GFP plasmid were kindly provided by observation that bacteria ubiquitination is partly reduced when L. Ramakrishnan (Washington University, Seattle) and the pCHERRY3 plas- atg1 is disrupted (Fig. S4) also implies that the autophagic mid (No. 24659; ref. 35; Tanya Parish, Infectious Disease Research Institute, membrane somehow helps stabilize the ubiquitination. The re- Queen Mary’s School of Medicine, University of Washington, Seattle) was cruitment of Atg8 even in the absence of SQSTM1, suggests the supplied by Addgene. Transformed bacteria were selected either with μ involvement of additional autophagy adaptors. In mammalian hygromycin (pCHERRY) or kanamycin (msp12::GFP) at 50 g/mL. cells, multiple adaptors have been shown to mediate xenophagy Phalloidin and Antibodies. Rabbit polyclonal antibody was raised against full- including NDP52 and optineurin (28, 29). Although orthologs of length recombinant Atg8, FK2H (directed against ubiquitinylated conjugates) these proteins have yet to be found in lower , given was bought from Enzo (BML-PW0150), and anti-RFP from Chromotek (5F8). the universal challenge of containing intracellular pathogens Secondary antibodies were goat anti-rabbit or goat anti-mouse/chicken IgG (particularly if you are a professional phagocyte) it seems highly coupled to Alexa 488, Alexa 568, or Alexa 647 (Invitrogen). Phalloidin staining likely that additional adaptors will exist. was performed with Alexa Fluor488-, Alexa Fluor568-, or Alexa Fluor647- Previously identified roles for autophagy during infection are phalloidin (Molecular Probes).

aimed at capturing the bacteria within a vacuole, either to kill it, or 8 Infection Assay. Mycobacteria were grown to an OD600 =1(∼5 × 10 cells per allow it to replicate. In contrast, during ejection the autophagic mL) and clumps disrupted by passing through 50 μm CellTrics-filters (Partec) membrane is required both to maintain host cell plasma mem- and blunt 25-gauge needles (Dispomed, Neoject). Adherent Dictyostelium brane integrity and promote cell-to-cell transmission. Plasma wild type or mutant cells were grown overnight without antibiotics to membrane damage can be sealed by lysosomal fusion in a calcium- a density of 80–100%. Bacteria were added at a multiplicity of infection dependent fashion (30). Recently, a new calcium-dependent (MOI) of 50–100 and centrifuged on the Dictyostelium cells (500 × g, four wound healing mechanism has been discovered. The endosomal times for 5 min each). The cells were left to phagocytose for additional 10–30 min before free bacteria were washed off with HL5c. To inhibit ex- sorting complexes required for transport (ESCRT) machinery tracellular proliferation of bacteria the cells were subsequently resuspended has been demonstrated to catalyze the shedding of small vesicles in HL5c supplemented with 5 μg/mL streptomycin and maintained at 25 °C. carrying membrane wounds (31). Even though no molecular The moment when the bacteria were added to the attached Dictyostelium mechanism has been proposed, autophagosome interactions with cells was defined as 0 hpi. the plasma membrane have been suggested to allow the release of virus particles (32) and the cell-to-cell transmission of Brucella Immunofluorescence and Phalloidin Staining. Dictyostelium cells were infected (2). Recently, using a fluorescent timer approach, it was shown as above with GFP or mCHERRY expressing M. marinum. At 24 hpi, cells were centrifuged on poly-lysine coated coverslips (500 × g, 5 min) and fixed either that the autophagic pathway contributes to the shedding of in methanol (−80 °C, 1 h) or with Soerensen buffer (14.7 mM KH2PO4, microvesicles that harbor infectious coxsackievirusB and con- 2.5 mM NaHPO4, pH 6.3) containing 4% paraformaldehyde and stained as tribute to the spreading of the virus from cell to cell (33). described (9, 36). The fluorescence images were documented using an Autophagy has also been shown to promote the cell-to-cell Olympus IX81 confocal microscope with a 60× 1.35 NA or 100× oil immersion transmission of other pathogens, although the molecular mech- objective and Fluoview software v1.7b. Recording parameters for fields of anisms are unknown (2, 3). In contrast to our observations, in 1024 × 1024 pixels with appropriate electronic zoom (6–12×) were 3× line which the ejecting bacteria are cytosolic, Starr and colleagues averaging (Kalman). To adjust the brightness and contrast of complete images ImageJ (imagej.nih.gov/ij/) was used. showed that the Brucella replicating vacuole converts into a vac- uole with autophagic features, and that this is independent of Correlative Light and Electron Microscopy (CLEM). The combination of light Atg5 and Atg7. Interestingly, they also show that this conversion and electron microscopy was used to analyze the ultrastructure of ejecto- is necessary for efficient bacterial transmission. Furthermore, somes. Adherent Dictyostelium cells expressing Lifeact-GFP were infected they also report no increase in cell death upon Brucella release, as described above with mCHERRY expressing M. marinum. Infected cells suggesting a nonlytic form of egress, reminiscent of ejectosome- were plated in a sterile 35 mm μ-dish with an imprinted grid (Ibidi). After mediated transmission. 24 h post infection (hpi), the cells were fixed for two hours with 2% para- formaldehyde (Electron Microscopy Sciences) and 0.25% glutaraldehyde Here, we show that the autophagic machinery is necessary for (Electron Microscopy Sciences) in HL5c medium. Fixed cells were washed and nonlytic cell-to-cell transmission of M. marinum. We propose stained for 30 min with Alexa Fluor488-phalloidin. Localization and fluo- that the membrane generated by the autophagic machinery at rescence images of ejectosome structures were documented using the the distal pole of ejecting bacteria helps seal the membrane Olympus IX81 confocal microscope as described above. Subsequently, the cells were fixed with 2.5% glutaraldehyde in 1× PBS

wound generated by the ejection (Fig. S9). The loss of the polar CELL BIOLOGY autophagic membrane leads to increased permeability at the (137 mM NaCl, 2.7 mM KCl, pH 7.4) overnight. After washing the samples twice, they were incubated with OsO4 (1% in PBS, 30 min), washed again ejectosome and death of the host cell. and finally incubated with gallic acid (1% in 0.5× PBS, 30 min). After sample The range of interactions between intracellular pathogens and dehydration in a rising ethanol series they were embedded in 50% Epoxy the autophagy machinery continues to expand. The previously resin (Roth, 8619.2) in 100% Ethanol overnight at room temperature and unidentified and unexpected role for autophagy highlighted in polymerized at 60 °C with fresh 100% Epoxy resin for 6 h. Consecutive ul- our study indicates the complexity of host-pathogen interactions trathin sectioning was performed with a Leica EM UC7 μL tramicrotome and that have evolved over millions of years, and how pathogen the samples were examined with a Tecnai Spirit transmission electron mi- virulence and host defense pathways have reached a form of croscope at 80 kV (FEI). For 3D reconstruction the image stack was aligned with ImageJ (stackreg; imagej.nih.gov/ij/) and reconstructed in Imaris (6.2). equilibrium that benefits both organisms. Movies were exported from Imaris and rendered in AdobeAfterEffects and AdobeMediaEncoder. Materials and Methods Dictyostelium Cell Culture. Wild-type Dictyostelium cells (Ax2) were axenically Transmission Assay. All Dictyostelium strains (Ax2, racH−, and atg1−, donor cultivated in HL5c medium (Formedium) at 22 °C. The Dictyostelium atg1− cells) were infected as described above with GFP-expressing M. marinum. cells were described (34) and racH− cells from F. Rivero (University of Hull, Infection rates were measured by flow cytometry (Accuri C6, BD Biosciences), Hull, United Kingdom). Dictyostelium mutants atg5−, atg6−, and atg7− and normalized to each other by dilution with the appropriate strain. At 6 hpi were received from dictyBase (www.dictybase.org). Dictyostelium mutant the infected donor cells were mixed with RFP-expressing acceptor Dictyos- SQSTM1- was generated using the pKOSG-IBA-Dicty1 system from Stargate. telium cells at a 1:5 ratio. At each timepoint (6, 24, 28, and 32 hpi), cells were

Gerstenmaier et al. PNAS | Published online February 2, 2015 | E691 Downloaded by guest on September 28, 2021 fixed with 4% paraformaldehyde in Soerensen buffer and immunostained with added (ratio 10:1). The live cells were imaged at 25 °C using a Zeiss 510 Meta an anti-RFP antibody to enhance the red signal. Subsequently, 250,000 cells confocal microscope equipped with a 63× Europlan apochromat oil immer- were measured by flow cytometry. The results were analyzed and plotted sion objective (N.A. 1.4) and the Zeiss confocal microscope software release with Flowjo 7.6.3 (TreeStar). To quantify transmission of M. marinum from 3.2. Two different laser-lines were used (Argon-Laser 488 nm and HeNe- donor to acceptor cells the ratio of infected acceptor versus infected donor Laser 543 nm). The imaging was performed in a multitrack-mode with a cells was calculated. pinhole of 2 Airy units. Cells were imaged for 10 min with time intervals of 6 s between each scan. Brightness and contrast adjustments to images, as Permeability Test with Propidium Iodide (PI). Dictyostelium cells were infected well as annotations, were performed using ImageJ. with GFP-expressing bacteria as described above. At 24 hpi 2 × 106 cells were centrifuged (5 min, 500 × g) and resuspended in 500 μL Soerensen sorbitol Coinfection Assay. ABD-GFP expressing Dictyostelium cells were simulta- buffer (120 mM sorbitol). PI (Sigma Aldrich, stock solution: 0.5 mg/mL) was neously infected with both green fluorescent (GFP-expressing) M. marinum added at a dilution of 1:10 and fluorescence (FL3) of 250,000 cells was and red fluorescent (mCHERRY-expressing) M. smegmatis or M. marinum measured (530 nm excitation, 610 nm emission) by flow cytometry (Accuri ΔRD1 as described under Infection assay. The bacteria were grown sepa- C6, BD Biosciences). As a positive control for dead and leaky cells, Dictyos- telium cells were heat killed (60 °C, 10 min), stained, and measured as rately in shaking culture and added to the adherent Dictyostelium cells at described above. the same time (defined as 0 hpi) with MOIs of 100 for M. marinum-GFP and Δ To obtain the percentages of permeable cells which are right-shifted the 30 for mCHERRY-expressing M. smegmatis or M. marinum RD1. Phagocy- shown, data were normalized between compared data sets and areas were tosis and washing was performed as described (infection assay) and cells computed using the density estimate of channel FL3 (PI). Area estimates for prepared for immunofluorescence (as described) at 24 phi. the overlap region between two curves (noninfected and infected) was computed and used to obtain the fraction of the total area shifted right of ACKNOWLEDGMENTS. We gratefully acknowledge Francisco Rivero for pro- the overlap. All computations were preformed in R (R Core Team (2014). R: viding Dictyostelium mutant racH− and Lalita Ramakrishnan for providing A language and environment for statistical computing. R Foundation for M. marinum and plasmids; and Ulrike Fröhlke and Carola Schneider for tech- Statistical Computing, Vienna, Austria). nical help. This work was supported by The Leibniz Society, Deutsche For- schungsgemeinschaft (DFG; HA3474/3-1, M.H.), Ciencia sem fronteiras (CsF, BEX2607-13-1, postdoctoral fellowship to R.P.), Cancer Research-UK and The – Live-Cell Imaging. Lifeact-RFP/Atg8-GFP expressing Dictyostelium cells were Royal Society Research Grant RG130655 (to J.S.K.), and a grant from the Swiss infected with mCHERRY-expressing M. marinum and at 4 hpi transferred to National Science Foundation (to the T.S. laboratory). This work would not have a35mmμ-dish (Ibidi). At 6 hpi, nonfluorescent Dictyostelium cells were been possible without dictyBase (37).

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E692 | www.pnas.org/cgi/doi/10.1073/pnas.1423318112 Gerstenmaier et al. Downloaded by guest on September 28, 2021