Listeria Monocytogenes Transiently Alters Mitochondrial Dynamics During Infection

Listeria monocytogenes transiently alters mitochondrial dynamics during infection

Fabrizia Stavrua,b,1, Frédéric Bouillaudc,d,e, Anna Sartorif, Daniel Ricquierc,d,e, and Pascale Cossarta,b,1

aUnité des Interactions Bactéries-Cellules, Institut Pasteur; U604, Institut National de la Santé et de la Recherche Médicale and bUSC2020, Institut National de la Recherche Agronomique, 75015 Paris, France; cInstitut Cochin, dInstitut National de la Santé et de la Recherche Médicale U1016, and eCentre National de la Recherche Scientiﬁque Unité Mixte de Recherche 8104, Université Paris Descartes, 75014 Paris, France; and fPlate-Forme de Microscopie Ultrastructurale, Imagopole, Institut Pasteur, Unité de Recherche Associée 2185; 75015 Paris, France

Contributed by Pascale Cossart, January 20, 2011 (sent for review August 30, 2010)

Mitochondria are essential and highly dynamic organelles, con- food-borne disease listeriosis. While listeriosis is a public health stantly undergoing fusion and fission. We analyzed mitochondrial issue, L. monocytogenes has been instrumental in elucidating dynamics during infection with the human bacterial pathogen fundamental cell biological questions, e.g., actin polymerization Listeria monocytogenes and show that this infection profoundly principles (reviewed in refs. 28 and 29). alters mitochondrial dynamics by causing transient mitochondrial After cell invasion, L. monocytogenes uses the pore-forming network fragmentation. Mitochondrial fragmentation is specificto toxin listeriolysin O (LLO) to escape from the phagosome. Al- fi pathogenic Listeria monocytogenes, and it is not observed with though LLO function had been characterized rst in Listeria es- the nonpathogenic Listeria innocua species or several other intra- cape from the phagosome under acidic conditions (30), several cellular pathogens. Strikingly, the efficiency of Listeria infection is studies now indicate that this crucial virulence factor also displays affected in cells where either mitochondrial fusion or fission has activity at neutral pH and acts on cells before bacterial entry (31, 32). Indeed, low levels of LLO secreted before bacterial entry are been altered by siRNA treatment, highlighting the relevance of fi fi suf cient to activate prosurvival signaling cascades such as the mitochondrial dynamics for Listeria infection. We identi ed the NFκB and MAPK pathways (33, 34), transcriptionally reprogram secreted pore-forming toxin listeriolysin O as the bacterial factor host cells (35), and trigger global deSUMOylation (36). mainly responsible for mitochondrial network disruption and mi- Here we report that L. monocytogenes causes dramatic alter- tochondrial function modulation. Together, our results suggest ations of mitochondrial dynamics via LLO. Strikingly, mitochon- that the transient shutdown of mitochondrial function and dy- drial fragmentation induced by Listeria infection is a transient namics represents a strategy used by Listeria at the onset of in- phenomenon, indicating that mitochondria are not terminally fection to interfere with cellular physiology. damaged. We propose that modulation of mitochondrial dynamics and function is a strategy used by pathogenic Listeria at bacterial infection | calcium influx | bioenergetics the onset of infection to slow down mitochondrial activity and mitochondria-dependent processes. itochondria are essential organelles providing most cellu- Mlar ATP as well as biosynthetic intermediates. They have Results emerged as important integrators of several signaling cascades Infection with Pathogenic Listeria Induces Mitochondrial Fragmentation. (1). Fusion and fission of mitochondria occurs constantly, regu- We first investigated the effects of L. monocytogenes infection on lating their size and subcellular distribution and reflecting their mitochondria of epithelial cells by using confocal laser scanning functional state (1, 2). Disturbance of mitochondrial dynamics microscopy. Fig. 1A shows L. monocytogenes infection of HeLa leads to pathological conditions exacerbated in tissues with high cells [1 h, multiplicity of infection (MOI) of 50] inducing strong metabolic demand such as neuronal or muscle tissues (3). Al- and rapid mitochondrial network fragmentation. Importantly, this though it often is unclear whether defects in mitochondrial dy- network fragmentation is specific to pathogenic L. monocytogenes, namics are the cause or effect of the disease, mitochondria have because it is not observed with the closely related nonpathogenic now emerged as drug targets for several pathologies (4, 5). species Listeria innocua, even when cells are infected with L. At the single-cell level, long-term impairment of either fusion innocua overexpressing the L. monocytogenes invasin Internalin B or fission can cause respiratory defects (6, 7). Key players in the (InlB) to enter cells [L. innocua(InlB), Fig. 1A]. This finding fusion process include the outer mitochondrial membrane indicates that mitochondrial fragmentation is not a consequence GTPases mitofusin 1 and 2 (Mfn1/2) (8, 9), and the inner mem- of stress imposed by the engulfment of bacteria. Furthermore, L. brane GTPase optic atrophy 1 (Opa1) (10, 11), but the molecular monocytogenes-induced mitochondrial fragmentation is not re- details of the fusion mechanism remain obscure (12, 13). Mito- stricted to HeLa cells, because it also occurs in human placental chondrial fission critically depends on the GTPase dynamin- (Jeg3) and green monkey kidney (Vero) cells (Fig. S1A). related protein 1 (Drp1) (14), which oligomerizes on the outer To assess whether other invasive pathogens also cause mito- mitochondrial membrane to constrict mitochondria at division chondrial fragmentation, we infected cells with Salmonella enter- sites (15). Mitochondrial fission also occurs during apoptosis and ica serovar typhimurium, Escherichia coli(Inv) as a model for may be required for apoptosis progression under specific circum- Yersinia pseudotubercolosis (37), enteropathogenic E. coli (EPEC), stances (16), although it does not induce apoptosis per se (17–19). and Shigella flexneri. Importantly, infection with either the extra- Several pathogens including both viruses and bacteria directly cellular pathogen EPEC or with invasive pathogens that remain or indirectly target mitochondria to interfere with the host apoptotic machinery (20–24). Depending on the pathogen and host-cell type, this interference can inhibit cell death to preserve Author contributions: F.S. and P.C. designed research; F.S. and A.S. performed research; the pathogen’s replication niche or induce cell death to promote F.B. and D.R. contributed new reagents/analytic tools; F.S., F.B., and P.C. analyzed data; infection spreading (25). Mitochondria also function as signaling and F.S., F.B., and P.C. wrote the paper. platforms in the innate immune response, and this function has The authors declare no conflict of interest. been linked recently to mitochondrial dynamics during viral in- Freely available online through the PNAS open access option. fection (26, 27). 1To whom correspondence may be addressed. E-mail: [email protected] or fstavru@ To investigate the interrelation between host-cell mitochon- pasteur.fr. drial dynamics and bacterial infection, we focused on Listeria This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. monocytogenes, a facultative intracellular bacterium causing the 1073/pnas.1100126108/-/DCSupplemental.

3612–3617 | PNAS | March 1, 2011 | vol. 108 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1100126108 Downloaded by guest on October 1, 2021 Fig. 1. Mitochondrial fragmentation upon infection with Listeria monocytogenes. (A) HeLa cells infected with L. monocytogenes (1 h; MOI of 50) display strongly fragmented mitochondria, whereas cells infected with L. innocua expressing InlB to enter cells do not. Mitochondria were stained with MitoTracker Deep Red (red). L. innocua or L. monocytogenes were Fig. 2. Early Listeria infection is affected by impaired mitochondrial dy- stained with polyclonal antibodies R6 and R11 (green). Arrows indicate in- namics. (A) HeLa cells treated with two different siRNAs (#A and #B) targeting fected cells, and insets show 2× enlargements. (B) Infection (1 h, MOI of 50) the mitochondrial fusion protein Mfn1 were infected with L. monocytogenes with E.coli(Inv) as a model for Yersinia infection, Shigella flexneri (M09T), (MOI of 50) in a gentamicin survival assay. The relative number of intracellular Salmonella enterica serovar typhimurium, and enteropathogenic E. coli (EPEC) bacteria was determined by cfu count at 3 h postinfection. L. monocytogenes do not cause mitochondrial fragmentation. Cell nuclei and bacteria were infection is significantly impaired in Mfn1-knockdown cells (P < 0.001, one- stained with DAPI (blue). Salmonella additionally expresses GFP (green). Mi- tailed Student’s t test). Each experiment was performed in triplicate, and tochondria were stained as in A. Arrows indicate infected cells, and insets a representative experiment with SDs is shown. At least three independent show 2× enlargements. experiments were performed for each condition. (B) Infection of Mfn2- knockdown cells was performed as in A. Mfn2 knockdown also impairs Lis- teria infection, although a lesser extent. (C) Down-regulation of both Mfn1 confined to a phagocytic vacuole [i.e., E. coli(Inv) and Salmonella and Mfn2 has a cumulative effect in impairing Listeria infection, suggesting enterica serovar typhimurium] did not appear to affect mitochon- that these proteins play nonredundant roles in infection. (D) L. monocytogenes infection is promoted in Drp1-knockdown cells. Experiment and dria (Fig. 1B), even at an MOI of 100 and up to 3 h of infection, statistical analysis were performed as in A–C.**P < 0.005, ***P < 0.001 by supporting the notion that L. monocytogenes-induced mitochon- one-tailed Student’s t test. drial fragmentation is not a general stress response to bacterial infection. Moreover, mitochondrial fragmentation is not caused by the presence of bacteria in the cytosol, because infection with Listeriolysin O Is Sufficient to Cause Mitochondrial Fragmentation. Shigella flexneri,which,likeListeria, escapes from the vacuole and Because mitochondria appeared to fragment at early stages of polymerizes actin to move intra- and intercellularly, did not cause infection, we tested whether a secreted effector of L. mono- mitochondrial fragmentation (Fig. 1B). cytogenes could cause mitochondrial fragmentation. We first used Correlative light/transmission electron microscopy (TEM) a noninvasive mutant of L. monocytogenes lacking InlB and found showed that fragmented mitochondria in Listeria-infected cells that this mutant was still able to induce mitochondrial fragmen- had a disorganized ultrastructure with remodeled cristae com- tation (Fig. 3A). We then asked whether the best-characterized pared with mitochondria in noninfected cells (Fig. S1B). secreted effector of L. monocytogenes, the pore-forming toxin LLO, could induce mitochondrial fragmentation. Strikingly, mi- Impairment of Mitochondrial Dynamics Affects Infection by Listeria CELL BIOLOGY tochondrial fragmentation was abolished when an LLO-deletion monocytogenes. Having established that L. monocytogenes infec- mutant (L. monocytogenes Δhly) was used (Fig. 3A), indicating that tion induces mitochondrial fragmentation, we asked whether inhibiting fusion or fission by siRNA would affect early Listeria LLO is required for fragmentation of host-cell mitochondria. infection stages. Cells depleted of the fusion proteins Mfn1 and Bacteria whose hemolysin (hly) gene carries a point mutation Mfn2 (resulting in fissioned mitochondria) or of the fission pro- disrupting pore formation (38) did not affect mitochondrial mor- tein Drp1 (resulting in hyperfused mitochondria) were infected phology (Fig. 3B), revealing that fragmentation depends on the with L. monocytogenes. Infection was strongly impaired in cells pore-forming ability of LLO. fi with fissioned mitochondria, and the strongest inhibition was LLO appears necessary and suf cient to induce mitochondrial fi seen with codepletion of both mitofusins (Fig. 2 A–C and Fig. S2 fragmentation, because addition of the puri ed toxin at nano- – A and B). Infection was more efficient in cells with hyperfused molar concentrations [3 6 nM, considered noncytotoxic and not mitochondria (Fig. 2D and Fig. S2C). This result suggests that, for causing lactate dehydrogenase (LDH) release (35)] recapitulated efficient infection, Listeria requires the ability to induce mito- the mitochondrial phenotype observed upon infection (Fig. 3C). chondrial fission, because cells with mitochondria that already are Mitochondrial fragmentation was found to occur in a fast, all-or- fissioned at the onset of infection provide an unfavorable envi- nothing manner (i.e., within less than 10 min) upon addition of ronment for infection. Silencing of proteins regulating mitochon- recombinant LLO (Movie S1). Increasing incubation time or LLO drial dynamics may also affect later stages of infection or have concentration resulted in an increased number of cells displaying indirect effects on mitochondrial function and infection. fragmented mitochondria (Fig. 3D).

Stavru et al. PNAS | March 1, 2011 | vol. 108 | no. 9 | 3613 Downloaded by guest on October 1, 2021 Fig. 3. LLO induces mitochondrial fragmentation without affecting total levels of key mitochondrial dynamics proteins. (A) HeLa cells infected (1 h, MOI of 50) with a noninvasive L. monocytogenes mutant (ΔinlB) display fragmented mitochondria, indicating bacterial entry is not required. In contrast, no fragmentation occurs with infection by an LLO-deﬁcient mutant (Δhly). Bacteria were stained with anti L. monocytogenes (R11, green), mitochondria were stained with anti-cytochrome c (red), and DNA was stained with DAPI (blue). Insets show 2× enlargements of mitochondria. Arrows point to infected cells. (B) L. monocytogenes carrying a point mutation in the hly gene inactivating its pore-forming ability (W492A) do not cause mitochondrial fragmentation. Mitochondria (red) and bacteria (green) were stained as in A.(C) HeLa cell treatment with recombinant LLO (10 min, 6 nM) was sufﬁcient to induce mitochondrial fragmentation. Mito- chondria (red) were stained as in A.(D) HeLa cells were treated with different concentrations of LLO for 10 min or with 6 nM LLO for different periods of time. Morpho- metric analysis indicated that LLO-induced mitochondrial fragmentation is concentration and time dependent at the cell-population level. The percentage of fragmentation was determined by counting at least 100 cells per data point; data from at least two independent experiments were pooled, and P values were calculated using one-tailed Student’s t test (***P < 0.005, *P < 0.25). (E) Cells treated for 10 min with the indicated amounts of LLO were analyzed by Western blot for Drp1, Mfn1, or Mfn2, showing that LLO treatment does not affect their total levels.

Mitochondrial fragmentation could result from enhanced fis- demonstrates that LLO does not form pores in the mitochondrial sion or decreased fusion (39). We thus analyzed whether LLO inner membrane, a finding that is supported further by immuno- treatment would affect total levels of key mitochondrial dynamics fluorescence analysis, showing plasma membrane rather than mediators, i.e., Mfn1, Mfn2, and Drp1. Total levels of these pro- mitochondrial localization of LLO (Fig. S4). In contrast, LLO teins did not decrease in cells treated with 3–6 nM recombinant appears to permeabilize the otherwise succinate-impermeable LLO (Fig. 3E). Because LLO forms ion-permeable pores in plasma membrane, contributing to mitochondrial depolarization membranes (30), we hypothesized that LLO might modulate mi- by allowing leakage of bioenergetic substrates out of the cell. tochondrial dynamics by inducing ion flux. We tested whether Infection also affected cellular ATP levels in an LLO- blocking K+ efflux or Ca2+ influx would prevent LLO-induced dependent manner: Infection with wild-type L. monocytogenes mitochondrial fragmentation, given that changes in these ions are (1 h, MOI of 50) induced a 50% decrease in intracellular ATP known to affect mitochondrial morphology (40, 41). Blocking K+ levels, but this decrease was not observed with L. innocua(InlB) efflux from LLO-treated cells by incubation in high extracellular or the L. monocytogenes Δhly mutant (Fig. 5C). Recombinant LLO K+ concentrations (135 mM) had no effect (Fig. 4). In contrast, (6 nM, 10 min) was sufficient to cause an even more pronounced interfering with LLO-induced Ca2+ influx by performing LLO decrease in intracellular ATP levels (Fig. 5C). A similar decrease treatment in Ca2+-free medium strongly prevented mitochondrial was obtained by a combination of chemically induced mitochon- fragmentation (Fig. 4). This result suggested that Ca2+ influx drial uncoupling with carbonyl cyanide m-chlorophenylhydrazone (rather than K+ efflux) through LLO pores is the signal inducing (CCCP) and Ca2+ influx (A23187), although uncoupling or Ca2+ mitochondrial fragmentation. influx separately did not lead to a significant difference (Fig. 5D). These data strongly suggest that L. monocytogenes infection not LLO Treatment Causes Mitochondrial Membrane Potential Loss and only affects mitochondrial dynamics but also interferes with cel- a Drop in Respiration and Cellular ATP. To investigate the func- lular bioenergetics and mitochondrial function. tionality of mitochondria in LLO-treated cells, we measured the mitochondrial membrane potential ΔΨ by assessing release of LLO-Induced Mitochondrial Fragmentation Does Not Correlate with the ΔΨ-dependent mitochondrial dye tetramethylrhodamine ethyl Classical Apoptosis. Mitochondrial fragmentation has been de- ester (TMRE) and found that LLO treatment caused a significant scribed in apoptotic cells, prompting us to analyze apoptosis drop in ΔΨ compared with untreated cells (Fig. 5A). At the single- markers such as cytochrome c release. Cytochrome c was not re- cell level, time-lapse microscopy of mitochondria loaded with the leased from fragmented mitochondria upon infection or LLO TMRE derivative tetramethylrhodamine methyl ester (TMRM) treatment (Fig. 3A and Fig. S5A). In contrast, cytochrome c re- indicated that ΔΨ loss was concomitant with or immediately pre- lease was observed in the positive control, i.e., staurosporine- ceded mitochondrial fragmentation (Fig. 5B). treated cells (Fig. S5A). Interestingly, the observed mitochondrial LLO treatment caused a drop in respiratory activity as mea- fragmentation did not depend on mitochondrial transition pore sured with an oxygen consumption chamber (Fig. S3). Respiration (mTP) opening, because it was not blocked by the mTP inhibitor resumed when the mitochondrial respiratory substrate succinate cyclosporin A. To test further for apoptosis in cells displaying was added, indicating that no major damage occurred to mito- infection-induced mitochondrial fragmentation, we analyzed ac- chondria, because the respiratory chain (complex II–complex IV) tivated B-cell lymphoma 2 (Bcl2)-associated X (Bax) protein by appeared functional. Furthermore, the resumption of respiration immunostaining. The proapoptotic Bcl2 protein family member

3614 | www.pnas.org/cgi/doi/10.1073/pnas.1100126108 Stavru et al. Downloaded by guest on October 1, 2021 Fig. 4. LLO-induced mitochondrial fragmentation is mediated by calcium influx, not by potassium efflux. Cells were treated for 10 min with 6 nM LLO in the presence or absence of extracellular calcium. Mitochondria (stained with anti-cytochrome c) showed strongly inhibited fragmentation in the absence of calcium, whereas blocking potassium efflux by incubation in 135 mM KCl did not prevent LLO-induced mitochondrial fragmentation.

Bax is activated by several apoptotic signals and translocates to reproduced partially by the synergistic action of an uncoupler and a mitochondria, where it forms pores and participates in fission (42, Ca2+ ionophore but these drugs do not cause the dramatic mito- 43). We could not detect mitochondrial recruitment of activated chondrial fragmentation observed upon LLO treatment (Fig. S8). Bax upon infection or LLO treatment (Fig. S5B). In the case of Listeria infection, the bioenergetic crisis is probably Together, these experiments suggest that neither L. mono- aggravated by leakage of small molecules, including respiratory cytogenes infection nor treatment with sublytic LLO concen- substrates or glycolysis intermediates through LLO pores. trations causes classical apoptosis in HeLa cells. LLO-induced ΔΨ decrease also may reflect a host-cell response to prevent mitochondrial Ca2+ accumulation to cytotoxic levels, Infection-Induced Mitochondrial Fragmentation Is Transient. An im- because mitochondrial Ca2+ uptake is ΔΨ dependent (46). In- portant question was whether mitochondria that fragment be- deed, several markers of apoptosis were absent in cells with LLO- cause of LLO produced at initial stages of Listeria infection or infection-induced mitochondrial fragmentation. Our data are recover their original shape and reform an interconnected net- consistent with the notion that epithelial cells recover from the work. To this end, we followed recovery by infecting cells for 1 h attack of pore-forming toxins (including LLO) at sublytic con- and then observing mitochondrial morphology at different time centrations, regain membrane integrity, and resume the cell cycle points after bacteria removal. The proportion of cells with frag- (32, 47, 48). The molecular mechanisms underlying LLO recovery mented mitochondria decreased steadily (Fig. 6A). Time-lapse are currently unclear: LLO does not colocalize with endocytic imaging indicated that in most cases tubular mitochondrial mor- markers at early time points (Fig. S4). Infected cells appear to phology is recovered within the first few hours (Fig. 6B). In line restore their mitochondrial network both morphologically (Fig. 6) with these results, we found that intracellular ATP levels re- and functionally, because intracellular ATP levels recover 4 h covered by 4 h postinfection (Fig. S6), suggesting that infected postinfection (Fig. S6). Consistent with the view that cytosolic cells are not terminally damaged. LLO is inactivated rapidly (30), mitochondrial fragmentation is induced only by extracellular LLO, i.e., early during Listeria in- Discussion fection; at late time points of infection intracellular bacteria do not We show here that infection with pathogenic L. monocytogenes affect mitochondrial morphology, even though they produce LLO causes transient mitochondrial network fragmentation and iden- to escape from the vacuole (Fig. S9). Together, these data indicate tify the secreted bacterial toxin LLO as the main factor affecting that Listeria affects host-cell mitochondria only transiently. Per- mitochondrial morphology and function at early time points of manent impairment of mitochondrial function and dynamics infection. LLO induces Ca2+-dependent mitochondrial frag- would harm host cells and therefore would be counterselected for, mentation, accompanied by a decrease in the mitochondrial mem- because it would eliminate the bacterial replication niche. brane potential ΔΨ and in respiration. As ΔΨ and respiration Our data suggest that active induction of mitochondrial frag- concomitantly decrease upon LLO treatment, ATP regeneration mentation early during infection is critical for infection. Indeed,

proceeds inefficiently, contributing to a decrease in intracellular infection is impaired in cells with previously fissioned mito- CELL BIOLOGY ATP levels. This decrease suggests a transient metabolic “slow- chondria and is enhanced in cells with hyperfused mitochondria. down” of host cells, favoring early stages of infection by interfering Treatments that impair mitochondrial respiration cause fragmen- with the capacity of the host cell to respond to this event. Such tation (49), and, conversely, mitochondrial fragmentation has been strategy could be common to several bacteria secreting pore- shown to limit the spread of incoming Ca2+ across the mitochon- forming toxins, because we found that different recombinant drial network (50). Accordingly, fragmentation appears to be an pore-forming toxins had effects comparable to LLO (Fig. S7). appropriate response to avoid propagation of the consequences of LLO pore formation has been studied at the biophysical and Ca2+ influx to the entire mitochondrial network. A prefissioned physiological level and induces Ca2+ influx (32, 44). Interestingly, state would limit the extent of damage caused by LLO action and several pathogens manipulate host-cell physiology by inducing allow the mitochondrial network to restore cellular bioenergetics Ca2+fluxes (45). Importantly, this Ca2+ influx enhances Listeria more efficiently. Consequently, invading Listeria would have less entry into cells (31), suggesting that early LLO action is a crucial time to take advantage from the transient bioenergetic “slow- step in epithelial cell infection. Ca2+ influx probably represents down” it induces. The opposite effect would occur in Drp1- a first bioenergetic insult to the cell, inducing mitochondrial knockdown cells with a hyperfused mitochondrial network. fragmentation and depolarization as well as blocking mitochon- In conclusion, we propose a scenario in which the normal drial movement (Movie S1), although such damage is reversible. bioenergetic state of the cell represents a barrier to Listeria in- While neither uncoupling nor Ca2+ ionophore treatment signifi- vasion. Consequently, an LLO-induced transient metabolic re- cantly reduce intracellular ATP levels, such an ATP decrease is programming of the cell would promote efficient infection. In

Stavru et al. PNAS | March 1, 2011 | vol. 108 | no. 9 | 3615 Downloaded by guest on October 1, 2021 Fig. 6. Mitochondria fragmented upon L. monocytogenes infection can recover tubular morphology. (A) HeLa cells infected with L. monocytogenes (1 h, MOI of 50) were ﬁxed directly or were washed and further incubated for 1.5 h, 3 h, or overnight (i.e., 2.5 h, 4 h, and overnight after inoculation) in the presence of 80 μg/mL gentamicin to kill extracellular bacteria rapidly and to slow intracellular bacterial replication. The number of cells displaying Fig. 5. LLO causes a decrease in the mitochondrial membrane potential fragmented mitochondria decreases steadily with time, indicating mito- ΔΨ concomitant with fragmentation and loss of intracellular ATP. (A)The chondrial network recovery. The percentage of cells with fragmented mi- ΔΨ mitochondrial membrane potential of HeLa cells was measured in tochondria was determined by counting >100 cells per time point in two a plate-based TMRE-release assay. Cells were treated with the indicated independent experiments. (B) HeLa cells were loaded with 100 nM Mito- ΔΨ drugs and loaded with TMRE in a -dependent manner. Mitochondrial Tracker 488, treated as in A, and imaged at the indicated time points. Arrows TMRE then was released by overnight incubation at 4 °C and measured at indicate cells with fragmented mitochondria that recover tubular morphol- ﬂ ΔΨ 585 nm, re ecting drug-induced changes in . Mitochondria were ogy starting at 2.5 h postinfection. uncoupled with 10 μM CCCP/0.5 μg/mL oligomycin (control for ΔΨ de-

crease), whereas oligomycin alone induced higher ΔΨ by blocking the F1FO ATPase (hyperpolarization control). LLO treatment caused a decrease in ΔΨ. Normalized average values from two independent experiments are agreement with this notion, mitochondrial dysfunction has been shown, and P values were calculated (**P < 0.01, *P < 0.25, one-tailed linked to increased susceptibility to bacterial infection (51, 52). Student’s t test). (B) Time-lapse analysis of HeLa cells stained with Mito- Our work shows that mitochondrial dynamics plays a role in in- Tracker 488 (green) and the potential-sensitive dye TMRM (red) shows that fection with the human pathogen L. monocytogenes. Whether in ΔΨ decreases shortly before mitochondrial fragmentation induced by ad- this case specific signaling cascades are activated downstream of dition of 6 nM LLO becomes apparent. Fragmented mitochondria lose the induced mitochondrial fragmentation and dysfunction is cur- TMRM and retain only the potential-insensitive MitoTracker 488. (C)In- rently unknown, but mitochondrial dynamics and infection do tracellular ATP levels were measured 1 h after infection with L. innocua appear to influence each other mutually, because Listeria in- expressing InlB, L. monocytogenes Δhly, or wild-type L. monocytogenes. fi Wild-type L. monocytogenes causes the strongest drop in intracellular ATP fection speci cally leads to transient disruption of mitochondrial levels; the effect is more pronounced when cells are incubated with morphology and function, and prior disruption of mitochondrial recombinant LLO (10 min, 6 nM). Cell permeabilization with detergent (0.1% dynamics by siRNA affects Listeria infection efficiency. Inter- Triton X-100, 10 min) causes complete intracellular ATP release. Experiments estingly, the cytomegalovirus protein vMia induces mitochondrial were performed three times in duplicate, and mean values (normalized to fragmentation and thereby prevents innate immune signaling untreated cells) are shown. ***P < 0.0005, **P < 0.005, *P < 0.05, one-tailed downstream of mitochondrial antiviral signaling protein (MAVS) Student’s t test. (D) HeLa cells were treated for 10 min with 6 nM LLO or 0.1% (26). Listeria may act similarly, explaining MAVS-independent Triton X-100, for 30 min with the mitochondrial uncouplers CCCP (100 μM), fl μ activation of the innate immune response (53). Given that bio- carbonyl cyanide-p-tri uoromethoxyphenylhydrazone (FCCP) (1 M), and energetics affect mitochondrial shape, and vice-versa (2, 49), it is valinomycin (100 nM), or with the Ca2+ ionophore A23187 (1 μM). In- tracellular ATP levels do not decrease significantly upon Ca2+ influx or likely that mitochondrial localization of innate immunity com- uncoupling but do decrease significantly with the combination of both ponents serves to coordinate innate immune responses with cel- treatments. Statistical analysis was performed as in C. n.s., nonsignificant lular energy levels via sensing of mitochondrial morphology and values. function. How innate immunity components and the fission/

3616 | www.pnas.org/cgi/doi/10.1073/pnas.1100126108 Stavru et al. Downloaded by guest on October 1, 2021 fusion machinery sense changes in the bioenergetic status of mi- ACKNOWLEDGMENTS. We acknowledge the expert help of C. Schmitt and tochondria is still unknown. The strong and rapid action of LLO G. Pehau-Arnaudet of the Plate-Forme de Microscopie Ultrastructurale and the continuous support of the Plate-Forme d’Imagerie Dynamique staff at described in our work provides an additional model system to the Institut Pasteur. We thank E. Veiga and L. Dortet for pioneering experi- address this question. ments, K. Rogers and M. Lecuit for stimulating discussions and laboratory members for critical reading of the manuscript. This work received support Materials and Methods from the European Research Council (Advanced Grant 233348), the Fonda- tion Le Roch Les Mousquetaires, and the Fondation Jeantet. P.C. is a Howard Reagents, cell lines, and bacterial strains used in this study, detailed exper- Hughes Medical Institute International Fellow. F.S. was funded by a Roux imental protocols, and nonstandard abbreviations are provided in SI Mate- Fellowship (Institut Pasteur) and currently is a European Molecular Biology rials and Methods. Organization Long-Term Fellow.

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