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Import of bacterial pathogenicity factors into mitochondria
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Import of bacterial pathogenicity factors into mitochondria Vera Kozjak-Pavlovic, Katharina Ross and Thomas Rudel Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane b-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors. Addresses Max Planck Institute for Infection Biology, Department of Molecular Biology, Research Group for Molecular Infection and Cancer Biology, Charite´platz 1, D-10117 Berlin, Germany Corresponding author: Kozjak-Pavlovic, Vera ([email protected]), Ross, Katharina ([email protected]) and Rudel, Thomas ([email protected])
Current Opinion in Microbiology 2008, 11:9–14 This review comes from a themed issue on Bacteria Edited by Thomas Meyer and Ilan Rosenshine Available online 14th February 2008 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.12.004
Introduction It is generally accepted that mitochondria have arisen from an endosymbiotic alpha-proteobacterial ancestor [1,2]. This fact is reflected in the protein content of mitochondria, many of mitochondrial proteins having counterparts in modern-day bacteria [3]. During the establishment of endosymbiosis, mitochondria have transferred the information for the majority of their proteins to the host cell nucleus. As a consequence, the import of nuclear-encoded proteins into mitochondria www.sciencedirect.com
made necessary the development of protein import machines. It is possible that one process was accelerated by the other, or that both processes happened simultaneously. Mitochondria have been identified as the target of an increasing number of bacterial proteins, which are transferred to the cell during infection, a process that often plays a crucial role in bacterial pathogenicity [4]. Consistent with a central role of mitochondria in apoptosis regulation [5], nearly all identified bacterial proteins targeting mitochondria modulate host cell death. They frequently induce the release of pro-apoptotic factors like cytochrome c and cause a breakdown of the mitochondrial membrane potential (DCm). Less is known about cell entry route and mitochondrial import, whereas mitochondrial phenotypes caused by bacterial effectors are well investigated. Multiple traits indicate parallels between bacterial and mitochondrial protein transport systems. For example, a peptidase from Rickettsia prowazekii with structural homology to mitochondrial processing peptidase (MPP) can cleave MPP substrates, the authentic signal sequences of proteins targeting mitochondria [6]. Additionally, certain bacterial signal peptides are recognized by mitochondrial import receptors and mediate protein transport into mitochondria [7]. Moreover, bacterial pathogenicity factors have been shown to contain N-terminal mitochondrial targeting signals [8,9] capable of directing to mitochondria any protein fused to it. Bacterial porins can also be targeted to host mitochondria [10–12]. They assume b-barrel topology and are therefore similar to mitochondrial porin, or voltage-dependent anion-regulated channel (VDAC). Remarkably, b-barrel proteins are found exclusively in outer membranes of bacteria, mitochondria, and chloroplasts, pointing to a common evolutionary origin of this protein class. Besides proteins with N-terminal mitochondrial presequence or b-barrel structure, a heterogeneous class of bacterial proteins including many bacterial toxins target mitochondria by so far unknown import routes [13–20]. Here, we will summarize the current view on the mitochondrial targeting and import of an emerging class of bacterial pathogenicity factors.
Protein import into mitochondria Protein import machines of mitochondria are complex and differ between organisms, though a core of each machine seems to be present in all eukaryotes [21]. Current Opinion in Microbiology 2008, 11:9–14
10 Bacteria
Figure 1
Import and sorting pathways of proteins in mammalian mitochondria. Proteins enter the mitochondrial intermembrane space via the translocase of the outer membrane (TOM) complex. The sorting and assembly machinery (SAM) complex mediates membrane insertion of b-barrel proteins. Proteins are sorted to the inner membrane by the translocase of the inner mitochondrial membrane (TIM) complexes TIM22 and TIM23. Proteins interacting with the TIM complexes are either integrated into the inner mitochondrial membrane or transported into the mitochondrial matrix.
Most of the components have been first described and functionally studied in fungal systems, to be later identified in mammals [22]. In the outer membrane, practically all proteins are first recognized by the translocase of the outer mitochondrial membrane (TOM) complex, which consists of the core component Tom40, receptors Tom20, Tom22, and Tom70, and various numbers of small Tom proteins (Figure 1). Proteins are afterwards transferred either to the TIM complexes in the inner mitochondrial membrane or to the SAM/TOB complex in the outer mitochondrial membrane. The TIM23 complex, with the PAM complex, is responsible for the import of proteins containing an N-terminal presequence, which are usually matrix or inner-membrane proteins. Preproteins are processed by the MPP. The TIM22 complex is necessary for the membrane integration of proteins that contain internal targeting signals, as are carrier proteins of the inner mitochondrial membrane. The SAM/TOB complex, with Sam50 as the central component, facilitates membrane integration of b-barrel proteins [23–25]. Recently, metaxins have also been identified as functioning with Sam50 during integration of b-barrel proteins in mammalian mitochondria, though they probably exist in a protein complex separate from Sam50 [26]. The only one of these complexes with a bacterial counterpart is the SAM complex. Its core component, Sam50, shares sequence homology with Omp85, a protein of the Current Opinion in Microbiology 2008, 11:9–14
outer membrane of Neisseria sp. Similar to Sam50 in mitochondria, Omp85 facilitates the integration of neisserial porins into the outer bacterial membrane [27]. Omp85 homologs have since been identified in other bacteria, as well as in mitochondria and chloroplasts of all other organisms [28–30]. However, the main entry point of practically all proteins into mitochondria, the TOM complex, has no homologous machinery in bacteria [21].
Bacterial proteins with N-terminal mitochondrial targeting sequence Mitochondrial-associated protein (Map, former Orf19) and EspF from enteropathogenic E. coli (EPEC) are the best studied bacterial proteins that possess an Nterminal mitochondrial presequence with putative cleavage signal. Both proteins are injected into host cells via the type 3 secretion system (TTSS) and colocalize with host mitochondria [8,9,31]. Mitochondria-associated Map causes the dissipation of DCm and mitochondrial fragmentation, but appears to be dispensable for EPEC-induced host cell apoptosis [8,31]. A smaller form of Map can be found, possibly representing a mitochondrial imported form cleaved by the host MPP [8]. In vitro import assays with mitochondria from temperature-sensitive yeast mutants have shown that Map import depends on Tom22, Tom40, and www.sciencedirect.com
Bacterial proteins imported into mitochondria Kozjak-Pavlovic, Ross and Rudel 11
mtHsp70. Moreover, import of Map requires an intact DCm, typical for proteins imported into the mitochondrial matrix via the TIM23 complex [31]. It is not known whether homologs of Map, EPEC TrcA/TrcP and Shigella IpgB, have similar characteristics [8]. EPEC EspF has also a canonical N-terminal mitochondrial targeting signal and, upon infection, localizes to these organelles. EspF triggers apoptosis during EPEC infection by dissipating DCm, causing cytochrome c release and activating caspases 9 and 3. The N-terminal 34 amino acids of EspF fused to eGFP are sufficient to guide this construct to mitochondria. As for Map, a smaller form of EspF, possibly cleaved by the host MPP, can be detected in infected cells [9]. Recently, Nagai et al. demonstrated that the loss of DCm inhibited the mitochondrial import of EspF, suggesting a role of the inner membrane translocation machinery [32]. Results from our group indicate that EspFu, an EspF homologue from enterohaemorrhagic E. coli (EHEC), contains an N-terminal mitochondrial targeting sequence, which guides its translocation to mitochondria upon overexpression (V.K.-P., John Leong, T.R., unpublished data).
Mitochondria-targeted bacterial proteins with b-barrel structure Neisserial PorB proteins and Omp38 from Acinetobacter baumannii belong to this group [4,10–12]. These proteins are not secreted by classical bacterial secretion systems and the details of how they reach the host cell mitochondria during infection are still unknown. Omp38 of A. baumannii is shown to be targeted to mitochondria of Hep2 cells, where it induces the release of cytochrome c and apoptosis inducing factor (AIF), promoting apoptosis [12]. How Omp38 is recognized and taken up by mitochondria has not been investigated. The import of PorB from Neisseria gonorrhoeae has been studied in yeast mitochondria, where it was shown that this protein follows the import pathway similar to that of VDAC, being recognized by Tom20 and transported through the Tom40 pore [33]. In the light of the SAM complex discovery, it remains to be determined whether Sam50, a homolog of neisserial Omp85, plays a role in mitochondrial import of PorB. Gonococcal PorB expressed in human cells localizes to mitochondria and causes the dissipation of DCm, but not the release of cytochrome c. The previously determined outer membrane localization, however, does not explain the decoupling effect of PorB, leaving the possibility that some of the PorB molecules spontaneously integrate into the inner mitochondrial membrane where they dissipate DCm.
Proteins that do not belong to either category This large group of proteins consists mainly of bacterial toxins, which are secreted by the bacteria and enter the host cell by endocytosis. Two exceptions are the EPECwww.sciencedirect.com
translocated intimin receptor (Tir) and Salmonella SipB that are both injected into the host cell by TTSS. Host cell-associated Tir functions as a receptor for bacterial adherence. In an overexpression study, Tir, C-terminally tagged with YFP, colocalized with mitochondria and induced apoptosis. It remains to be shown whether Tir associates with host mitochondria during infection [34]. Salmonella enterica SipB targets host mitochondria during infection and supports autophagy-mediated cell death in caspase-1-deficient macrophages [17]. Transiently overexpressed SipB induces the formation of multivesicular fusion structures that contain marker proteins of endoplasmic reticulum and mitochondria. Although no known mitochondrial targeting sequence can be found, SipB seems to specifically target mitochondria, as also a mutant that has no fusogenic activity localizes to mitochondria [17]. Among the bacterial toxins associating with mitochondria, the vacuolating cytotoxin (VacA) from Helicobacter pylori has been extensively studied. Both purified and overexpressed VacA localized to mitochondria of mammalian cells and induced the loss of DCm and the release of cytochrome c [13,35]. Radiolabeled VacA was shown to be rapidly imported over the mitochondrial outer membrane, though it seems to lack a mitochondrial targeting sequence [13]. Cytotoxic activity of VacA may come from its tendency to form anion-selective channels in mitochondrial membranes [35]. However, in another report, VacA was found in late endosomes and it was demonstrated that H. pylori-induced cytotoxicity does not require a direct contact between VacA and mitochondria [36]. A range of other apoptosis-inducing toxins associate with host mitochondria if applied in purified form to host cells, inducing DCm dissipation and cytochrome c release. These include the Staphylococcus aureus a-toxin [15], the S. aureus Panton-Valentine leukocidin (PVL) [18], the Streptococcus pneumoniae pneumolysin [20], the Clostridium difficile Toxin A [14] and Toxin B [19], and the Clostridium sordellii Lethal toxin [16]. All these toxins added in purified form damage isolated mitochondria by unknown mechanisms. Considering this large group and the little mechanistic insight we have, the importance of the toxin–mitochondria association has clearly been underestimated in the past.
Conclusions Bacterial proteins targeting mitochondria comprise an emerging group of pathogenicity factors involved in the regulation of host cell death. The few examples investigated in detail unveiled an involvement of mitochondrial import machines in their uptake (Figure 2). Several proteins for inner-membrane transport and outer-membrane assembly (Sam50) have structural and functional bacterial counterparts, opening the possibility of a coCurrent Opinion in Microbiology 2008, 11:9–14
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Figure 2
Import of bacterial proteins into mitochondria. EPEC proteins EspF and Map enter mitochondria via the TOM complex and are subsequently sorted to the mitochondrial matrix. Uptake of PorB from N. gonorrhoeae by mitochondria requires the TOM complex. Consistent with the b-barrel topology, PorB may integrate into the outer membrane. A localization to the mitochondrial inner membrane would be consistent with the strong uncoupling activity observed for PorB. The entry routes for VacA from H. pylori and Omp38 from A. baumannii are until date unknown.
evolution of mitochondria-targeted bacterial effectors and mitochondrial import. Import studies were mostly performed on isolated yeast mitochondria, a model system that leaves much to be desired, considering that most of the pathogenic bacteria in question infect only mammalian cells. Clearly, the import routes for most of the
mitochondria-associated bacterial proteins are unknown and have to be investigated in relevant systems in future. The latter are mitochondria of the respective host cells, which are now generally accessible for import experiments by applying the new technology of RNA interference for specific loss of function studies [26].
Table 1 Bacterial pathogenicity factors targeting mitochondria Bacterial protein
Organism
EspF Map
EPEC EPEC
Tir SipB PorB
EPEC Salmonella N. gonorrhoeae
PorB Omp38 VacA a-Toxin PVL
N. meningitidis A. baumanii H. pylori S. aureus S. aureus
Pneumolysin Lethal toxin Toxin A Toxin B
S. pneumoniae C. sordelli C. difficile C. difficile
Mitochondrial phenotype
Biological effect
Targeting signal/Required mitochondrial import proteins
Loss of DCm Mitochondrial fragmentation, loss of DCm Unknown Membrane fusion Release of cytochrome c, loss of DCm Protection from DCm loss Release of cytochrome c, AIF Release of cytochrome c Release of cytochrome c Release of cytochrome c, Smac/DIABLO Release of AIF Release of cytochrome c Release of cytochrome c Swelling, release of cytochrome c
Induction of apoptosis Unknown
[9,32] [8,31]
Induction of apoptosis Induction of autophagy Induction of apoptosis
N-terminal leader/n.d. N-terminal leader/Tom40, Hsp70 Unknown Unknown b-Barrel protein/Tom20, Tom40
Inhibition of apoptosis Induction of apoptosis Induction of apoptosis Induction of apoptosis Induction of apoptosis
b-Barrel protein/n.d. b-Barrel protein/n.d. Unknown/n.d. b-Barrel protein/n.d. Unknown/n.d.
[10] [12] [13] [15] [18]
Induction Induction Induction Induction
Unknown/n.d. Unknown/n.d. Unknown/n.d. Unknown/n.d.
[20] [16] [14] [19]
Current Opinion in Microbiology 2008, 11:9–14
of of of of
cell death apoptosis apoptosis apoptosis
References
[34] [17] [11,33]
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Bacterial proteins imported into mitochondria Kozjak-Pavlovic, Ross and Rudel 13
In silico analyses supported by limited experimental evidence predicted a large number of mitochondria-targeting bacterial proteins [37]. Among these, bacterial pathogenicity factors may constitute a coherent group of proteins involved in the cross-talk between host and pathogen, awaiting their detection also in other bacteria. Such proteins may be identified in phenotypic screens. For example, a connection was established between chlamydial infection and mitochondria in a recent cytotoxicity screen in yeast. One of the proteins significantly impairing yeast growth was shown to target mitochondria [38]. So far, nearly all bacterial proteins identified to target mitochondria appear to play a role in apoptosis regulation (see Table 1). However, the few examples which could not be linked to cell death regulation indicate that there may be a broader function in pathogenicity, still to be discovered. For instance, several of the mitochondriatargeting bacterial proteins like Tir, Map, and EspF also interact with cytosolic host signaling factors involved in cytoskeleton regulation. Cytosolic depletion of such factors by mitochondrial import may influence their function on the host cell cytoskeleton. Modulation of the mitochondrial physiology by bacterial proteins, for example, to adjust the host cell to metabolite or energy source requirements of the pathogen is another possibility. The later assumption may be supported by a recent genome-wide RNA interference screen performed to identify host factors that, when depleted, inhibit the infection with the obligate intracellular bacterium Chlamydia caviae. Of these, members of TIM and TOM import machines were identified as necessary for C. caviae infection [39]. The specific chlamydial protein responsible for this effect remains to be identified. It is therefore a safe assumption that bacterial pathogens and host mitochondria as their distant relatives are still communicating in a much closer way than previously anticipated.
Acknowledgement We thank Dr Joachim Rassow for helpful comments on the manuscript.
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10. Massari P, Ho Y, Wetzler LM: Neisseria meningitidis porin PorB interacts with mitochondria and protects cells from apoptosis. Proc Natl Acad Sci U S A 2000, 97:9070-9075. 11. Muller A, Gunther D, Brinkmann V, Hurwitz R, Meyer TF, Rudel T: Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells. EMBO J 2000, 19:5332-5343. 12. Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Hyun SH, Kim SA, Lee SK, Lee JC: Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells. Cell Microbiol 2005, 7:1127-1138. 13. Galmiche A, Rassow J, Doye A, Cagnol S, Chambard JC, Contamin S, de Thillot V, Just I, Ricci V, Solcia E et al.: The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release. EMBO J 2000, 19:6361-6370. 14. He D, Hagen SJ, Pothoulakis C, Chen M, Medina ND, Warny M, LaMont JT: Clostridium difficile toxin A causes early damage to mitochondria in cultured cells. Gastroenterology 2000, 119:139-150. 15. Bantel H, Sinha B, Domschke W, Peters G, Schulze-Osthoff K, Janicke RU: Alpha-toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling. J Cell Biol 2001, 155:637-648. 16. Petit P, Breard J, Montalescot V, El Hadj NB, Levade T, Popoff M, Geny B: Lethal toxin from Clostridium sordellii induces apoptotic cell death by disruption of mitochondrial homeostasis in HL-60 cells. Cell Microbiol 2003, 5:761-771. 17. Hernandez LD, Pypaert M, Flavell RA, Galan JE: A Salmonella protein causes macrophage cell death by inducing autophagy. J Cell Biol 2003, 163:1123-1131. 18. Genestier AL, Michallet MC, Prevost G, Bellot G, Chalabreysse L, Peyrol S, Thivolet F, Etienne J, Lina G, Vallette FM et al.: Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J Clin Invest 2005, 115:3117-3127. 19. Matarrese P, Falzano L, Fabbri A, Gambardella L, Frank C, Geny B, Popoff MR, Malorni W, Fiorentini C: Clostridium difficile toxin B causes apoptosis in epithelial cells by thrilling mitochondria. Involvement of ATP-sensitive mitochondrial potassium channels. J Biol Chem 2007, 282:9029-9041. 20. Braun JS, Hoffmann O, Schickhaus M, Freyer D, Dagand E, Bermpohl D, Mitchell TJ, Bechmann I, Weber JR: Pneumolysin causes neuronal cell death through mitochondrial damage. Infect Immun 2007, 75:4245-4254. 21. Dolezal P, Likic V, Tachezy J, Lithgow T: Evolution of the molecular machines for protein import into mitochondria. Science 2006, 313:314-318. The paper reviews the current opinion on the evolution of protein import machines found in mitochondria. Current Opinion in Microbiology 2008, 11:9–14
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22. Hoogenraad NJ, Ward LA, Ryan MT: Import and assembly of proteins into mitochondria of mammalian cells. Biochim Biophys Acta 2002, 1592:97-105. 23. Stojanovski D, Johnston AJ, Streimann I, Hoogenraad NJ, Ryan MT: Import of nuclear-encoded proteins into mitochondria. Exp Physiol 2003, 88:57-64. 24. Wiedemann N, Frazier AE, Pfanner N: The protein import machinery of mitochondria. J Biol Chem 2004, 279: 14473-14476. 25. Neupert W, Herrmann JM: Translocation of proteins into mitochondria. Annu Rev Biochem 2007, 76:723-749. 26. Kozjak-Pavlovic V, Ross K, Benlasfer N, Kimmig S, Karlas A, Rudel T: Conserved roles of Sam50 and metaxins in VDAC biogenesis. EMBO Rep 2007, 8:576-582. 27. Bos MP, Robert V, Tommassen J: Biogenesis of the gramnegative bacterial outer membrane. Annu Rev Microbiol 2007, 61:191-214. 28. Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, Kahne D: Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 2005, 121: 235-245. 29. Clantin B, Delattre AS, Rucktooa P, Saint N, Meli AC, Locht C, Jacob-Dubuisson F, Villeret V: Structure of the membrane protein FhaC: a member of the Omp85-TpsB transporter superfamily. Science 2007, 317:957-961. 30. Bredemeier R, Schlegel T, Ertel F, Vojta A, Borissenko L, Bohnsack MT, Groll M, von Haeseler A, Schleiff E: Functional and phylogenetic properties of the pore-forming beta-barrel transporters of the Omp85 family. J Biol Chem 2007, 282:1882-1890. 31. Papatheodorou P, Domanska G, Oxle M, Mathieu J, Selchow O, Kenny B, Rassow J: The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology. Cell Microbiol 2006, 8:677-689. Using a genetic system, the authors demonstrate the mitochondrial import route of the EPEC Map protein. This is one of the few studies where import has been linked to factors of the mitochondrial protein import machines.
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32. Nagai T, Abe A, Sasakawa C: Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for bacterial pathogenesis: critical role of the 16th leucine residue in EspF. J Biol Chem 2005, 280:2998-3011. The authors demonstrate that an EspF point mutant deficient in mitochondrial targeting prevents mitochondrial damage during infection of cell culture models. The same mutant is attenuated in an animal model for EPEC infection. This is the first study to demonstrate that mitochondrial import of a bacterial protein affects pathogenicity in an in vivo animal model. 33. Muller A, Rassow J, Grimm J, Machuy N, Meyer TF, Rudel T: VDAC and the bacterial porin PorB of Neisseria gonorrhoeae share mitochondrial import pathways. EMBO J 2002, 21:1916-1929. 34. Malish HR, Freeman NL, Zurawski DV, Chowrashi P, Ayoob JC, Sanger JW, Sanger JM: Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events. Apoptosis 2003, 8:179-190. 35. Willhite DC, Blanke SR: Helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity. Cell Microbiol 2004, 6: 143-154. 36. Yamasaki E, Wada A, Kumatori A, Nakagawa I, Funao J, Nakayama M, Hisatsune J, Kimura M, Moss J, Hirayama T: Helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins Bax and Bak, leading to cytochrome c release and cell death, independent of vacuolation. J Biol Chem 2006, 281:11250-11259. 37. Lucattini R, Likic VA, Lithgow T: Bacterial proteins predisposed for targeting to mitochondria. Mol Biol Evol 2004, 21:652-658. 38. Sisko JL, Spaeth K, Kumar Y, Valdivia RH: Multifunctional analysis of Chlamydia-specific genes in a yeast expression system. Mol Microbiol 2006, 60:51-66. 39. Derre I, Pypaert M, Dautry-Varsat A, Agaisse H: RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection. PLoS Pathog 2007, 3:1446-1458. A genome-wide RNA interference screen identified 54 factors that, when depleted, inhibit C. caviae infection. Among these factors were Tim and Tom proteins suggesting a role for mitochondrial import in C. caviae infection. This is the first screen that identified mitochondrial import as crucial for bacterial infection.
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Import of bacterial pathogenicity factors into mitochondria Vera Kozjak-Pavlovic, Katharina Ross and Thomas Rudel Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane b-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors. Addresses Max Planck Institute for Infection Biology, Department of Molecular Biology, Research Group for Molecular Infection and Cancer Biology, Charite´platz 1, D-10117 Berlin, Germany Corresponding author: Kozjak-Pavlovic, Vera ([email protected]), Ross, Katharina ([email protected]) and Rudel, Thomas ([email protected])
Current Opinion in Microbiology 2008, 11:9–14 This review comes from a themed issue on Bacteria Edited by Thomas Meyer and Ilan Rosenshine Available online 14th February 2008 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.12.004
Introduction It is generally accepted that mitochondria have arisen from an endosymbiotic alpha-proteobacterial ancestor [1,2]. This fact is reflected in the protein content of mitochondria, many of mitochondrial proteins having counterparts in modern-day bacteria [3]. During the establishment of endosymbiosis, mitochondria have transferred the information for the majority of their proteins to the host cell nucleus. As a consequence, the import of nuclear-encoded proteins into mitochondria www.sciencedirect.com
made necessary the development of protein import machines. It is possible that one process was accelerated by the other, or that both processes happened simultaneously. Mitochondria have been identified as the target of an increasing number of bacterial proteins, which are transferred to the cell during infection, a process that often plays a crucial role in bacterial pathogenicity [4]. Consistent with a central role of mitochondria in apoptosis regulation [5], nearly all identified bacterial proteins targeting mitochondria modulate host cell death. They frequently induce the release of pro-apoptotic factors like cytochrome c and cause a breakdown of the mitochondrial membrane potential (DCm). Less is known about cell entry route and mitochondrial import, whereas mitochondrial phenotypes caused by bacterial effectors are well investigated. Multiple traits indicate parallels between bacterial and mitochondrial protein transport systems. For example, a peptidase from Rickettsia prowazekii with structural homology to mitochondrial processing peptidase (MPP) can cleave MPP substrates, the authentic signal sequences of proteins targeting mitochondria [6]. Additionally, certain bacterial signal peptides are recognized by mitochondrial import receptors and mediate protein transport into mitochondria [7]. Moreover, bacterial pathogenicity factors have been shown to contain N-terminal mitochondrial targeting signals [8,9] capable of directing to mitochondria any protein fused to it. Bacterial porins can also be targeted to host mitochondria [10–12]. They assume b-barrel topology and are therefore similar to mitochondrial porin, or voltage-dependent anion-regulated channel (VDAC). Remarkably, b-barrel proteins are found exclusively in outer membranes of bacteria, mitochondria, and chloroplasts, pointing to a common evolutionary origin of this protein class. Besides proteins with N-terminal mitochondrial presequence or b-barrel structure, a heterogeneous class of bacterial proteins including many bacterial toxins target mitochondria by so far unknown import routes [13–20]. Here, we will summarize the current view on the mitochondrial targeting and import of an emerging class of bacterial pathogenicity factors.
Protein import into mitochondria Protein import machines of mitochondria are complex and differ between organisms, though a core of each machine seems to be present in all eukaryotes [21]. Current Opinion in Microbiology 2008, 11:9–14
10 Bacteria
Figure 1
Import and sorting pathways of proteins in mammalian mitochondria. Proteins enter the mitochondrial intermembrane space via the translocase of the outer membrane (TOM) complex. The sorting and assembly machinery (SAM) complex mediates membrane insertion of b-barrel proteins. Proteins are sorted to the inner membrane by the translocase of the inner mitochondrial membrane (TIM) complexes TIM22 and TIM23. Proteins interacting with the TIM complexes are either integrated into the inner mitochondrial membrane or transported into the mitochondrial matrix.
Most of the components have been first described and functionally studied in fungal systems, to be later identified in mammals [22]. In the outer membrane, practically all proteins are first recognized by the translocase of the outer mitochondrial membrane (TOM) complex, which consists of the core component Tom40, receptors Tom20, Tom22, and Tom70, and various numbers of small Tom proteins (Figure 1). Proteins are afterwards transferred either to the TIM complexes in the inner mitochondrial membrane or to the SAM/TOB complex in the outer mitochondrial membrane. The TIM23 complex, with the PAM complex, is responsible for the import of proteins containing an N-terminal presequence, which are usually matrix or inner-membrane proteins. Preproteins are processed by the MPP. The TIM22 complex is necessary for the membrane integration of proteins that contain internal targeting signals, as are carrier proteins of the inner mitochondrial membrane. The SAM/TOB complex, with Sam50 as the central component, facilitates membrane integration of b-barrel proteins [23–25]. Recently, metaxins have also been identified as functioning with Sam50 during integration of b-barrel proteins in mammalian mitochondria, though they probably exist in a protein complex separate from Sam50 [26]. The only one of these complexes with a bacterial counterpart is the SAM complex. Its core component, Sam50, shares sequence homology with Omp85, a protein of the Current Opinion in Microbiology 2008, 11:9–14
outer membrane of Neisseria sp. Similar to Sam50 in mitochondria, Omp85 facilitates the integration of neisserial porins into the outer bacterial membrane [27]. Omp85 homologs have since been identified in other bacteria, as well as in mitochondria and chloroplasts of all other organisms [28–30]. However, the main entry point of practically all proteins into mitochondria, the TOM complex, has no homologous machinery in bacteria [21].
Bacterial proteins with N-terminal mitochondrial targeting sequence Mitochondrial-associated protein (Map, former Orf19) and EspF from enteropathogenic E. coli (EPEC) are the best studied bacterial proteins that possess an Nterminal mitochondrial presequence with putative cleavage signal. Both proteins are injected into host cells via the type 3 secretion system (TTSS) and colocalize with host mitochondria [8,9,31]. Mitochondria-associated Map causes the dissipation of DCm and mitochondrial fragmentation, but appears to be dispensable for EPEC-induced host cell apoptosis [8,31]. A smaller form of Map can be found, possibly representing a mitochondrial imported form cleaved by the host MPP [8]. In vitro import assays with mitochondria from temperature-sensitive yeast mutants have shown that Map import depends on Tom22, Tom40, and www.sciencedirect.com
Bacterial proteins imported into mitochondria Kozjak-Pavlovic, Ross and Rudel 11
mtHsp70. Moreover, import of Map requires an intact DCm, typical for proteins imported into the mitochondrial matrix via the TIM23 complex [31]. It is not known whether homologs of Map, EPEC TrcA/TrcP and Shigella IpgB, have similar characteristics [8]. EPEC EspF has also a canonical N-terminal mitochondrial targeting signal and, upon infection, localizes to these organelles. EspF triggers apoptosis during EPEC infection by dissipating DCm, causing cytochrome c release and activating caspases 9 and 3. The N-terminal 34 amino acids of EspF fused to eGFP are sufficient to guide this construct to mitochondria. As for Map, a smaller form of EspF, possibly cleaved by the host MPP, can be detected in infected cells [9]. Recently, Nagai et al. demonstrated that the loss of DCm inhibited the mitochondrial import of EspF, suggesting a role of the inner membrane translocation machinery [32]. Results from our group indicate that EspFu, an EspF homologue from enterohaemorrhagic E. coli (EHEC), contains an N-terminal mitochondrial targeting sequence, which guides its translocation to mitochondria upon overexpression (V.K.-P., John Leong, T.R., unpublished data).
Mitochondria-targeted bacterial proteins with b-barrel structure Neisserial PorB proteins and Omp38 from Acinetobacter baumannii belong to this group [4,10–12]. These proteins are not secreted by classical bacterial secretion systems and the details of how they reach the host cell mitochondria during infection are still unknown. Omp38 of A. baumannii is shown to be targeted to mitochondria of Hep2 cells, where it induces the release of cytochrome c and apoptosis inducing factor (AIF), promoting apoptosis [12]. How Omp38 is recognized and taken up by mitochondria has not been investigated. The import of PorB from Neisseria gonorrhoeae has been studied in yeast mitochondria, where it was shown that this protein follows the import pathway similar to that of VDAC, being recognized by Tom20 and transported through the Tom40 pore [33]. In the light of the SAM complex discovery, it remains to be determined whether Sam50, a homolog of neisserial Omp85, plays a role in mitochondrial import of PorB. Gonococcal PorB expressed in human cells localizes to mitochondria and causes the dissipation of DCm, but not the release of cytochrome c. The previously determined outer membrane localization, however, does not explain the decoupling effect of PorB, leaving the possibility that some of the PorB molecules spontaneously integrate into the inner mitochondrial membrane where they dissipate DCm.
Proteins that do not belong to either category This large group of proteins consists mainly of bacterial toxins, which are secreted by the bacteria and enter the host cell by endocytosis. Two exceptions are the EPECwww.sciencedirect.com
translocated intimin receptor (Tir) and Salmonella SipB that are both injected into the host cell by TTSS. Host cell-associated Tir functions as a receptor for bacterial adherence. In an overexpression study, Tir, C-terminally tagged with YFP, colocalized with mitochondria and induced apoptosis. It remains to be shown whether Tir associates with host mitochondria during infection [34]. Salmonella enterica SipB targets host mitochondria during infection and supports autophagy-mediated cell death in caspase-1-deficient macrophages [17]. Transiently overexpressed SipB induces the formation of multivesicular fusion structures that contain marker proteins of endoplasmic reticulum and mitochondria. Although no known mitochondrial targeting sequence can be found, SipB seems to specifically target mitochondria, as also a mutant that has no fusogenic activity localizes to mitochondria [17]. Among the bacterial toxins associating with mitochondria, the vacuolating cytotoxin (VacA) from Helicobacter pylori has been extensively studied. Both purified and overexpressed VacA localized to mitochondria of mammalian cells and induced the loss of DCm and the release of cytochrome c [13,35]. Radiolabeled VacA was shown to be rapidly imported over the mitochondrial outer membrane, though it seems to lack a mitochondrial targeting sequence [13]. Cytotoxic activity of VacA may come from its tendency to form anion-selective channels in mitochondrial membranes [35]. However, in another report, VacA was found in late endosomes and it was demonstrated that H. pylori-induced cytotoxicity does not require a direct contact between VacA and mitochondria [36]. A range of other apoptosis-inducing toxins associate with host mitochondria if applied in purified form to host cells, inducing DCm dissipation and cytochrome c release. These include the Staphylococcus aureus a-toxin [15], the S. aureus Panton-Valentine leukocidin (PVL) [18], the Streptococcus pneumoniae pneumolysin [20], the Clostridium difficile Toxin A [14] and Toxin B [19], and the Clostridium sordellii Lethal toxin [16]. All these toxins added in purified form damage isolated mitochondria by unknown mechanisms. Considering this large group and the little mechanistic insight we have, the importance of the toxin–mitochondria association has clearly been underestimated in the past.
Conclusions Bacterial proteins targeting mitochondria comprise an emerging group of pathogenicity factors involved in the regulation of host cell death. The few examples investigated in detail unveiled an involvement of mitochondrial import machines in their uptake (Figure 2). Several proteins for inner-membrane transport and outer-membrane assembly (Sam50) have structural and functional bacterial counterparts, opening the possibility of a coCurrent Opinion in Microbiology 2008, 11:9–14
12 Bacteria
Figure 2
Import of bacterial proteins into mitochondria. EPEC proteins EspF and Map enter mitochondria via the TOM complex and are subsequently sorted to the mitochondrial matrix. Uptake of PorB from N. gonorrhoeae by mitochondria requires the TOM complex. Consistent with the b-barrel topology, PorB may integrate into the outer membrane. A localization to the mitochondrial inner membrane would be consistent with the strong uncoupling activity observed for PorB. The entry routes for VacA from H. pylori and Omp38 from A. baumannii are until date unknown.
evolution of mitochondria-targeted bacterial effectors and mitochondrial import. Import studies were mostly performed on isolated yeast mitochondria, a model system that leaves much to be desired, considering that most of the pathogenic bacteria in question infect only mammalian cells. Clearly, the import routes for most of the
mitochondria-associated bacterial proteins are unknown and have to be investigated in relevant systems in future. The latter are mitochondria of the respective host cells, which are now generally accessible for import experiments by applying the new technology of RNA interference for specific loss of function studies [26].
Table 1 Bacterial pathogenicity factors targeting mitochondria Bacterial protein
Organism
EspF Map
EPEC EPEC
Tir SipB PorB
EPEC Salmonella N. gonorrhoeae
PorB Omp38 VacA a-Toxin PVL
N. meningitidis A. baumanii H. pylori S. aureus S. aureus
Pneumolysin Lethal toxin Toxin A Toxin B
S. pneumoniae C. sordelli C. difficile C. difficile
Mitochondrial phenotype
Biological effect
Targeting signal/Required mitochondrial import proteins
Loss of DCm Mitochondrial fragmentation, loss of DCm Unknown Membrane fusion Release of cytochrome c, loss of DCm Protection from DCm loss Release of cytochrome c, AIF Release of cytochrome c Release of cytochrome c Release of cytochrome c, Smac/DIABLO Release of AIF Release of cytochrome c Release of cytochrome c Swelling, release of cytochrome c
Induction of apoptosis Unknown
[9,32] [8,31]
Induction of apoptosis Induction of autophagy Induction of apoptosis
N-terminal leader/n.d. N-terminal leader/Tom40, Hsp70 Unknown Unknown b-Barrel protein/Tom20, Tom40
Inhibition of apoptosis Induction of apoptosis Induction of apoptosis Induction of apoptosis Induction of apoptosis
b-Barrel protein/n.d. b-Barrel protein/n.d. Unknown/n.d. b-Barrel protein/n.d. Unknown/n.d.
[10] [12] [13] [15] [18]
Induction Induction Induction Induction
Unknown/n.d. Unknown/n.d. Unknown/n.d. Unknown/n.d.
[20] [16] [14] [19]
Current Opinion in Microbiology 2008, 11:9–14
of of of of
cell death apoptosis apoptosis apoptosis
References
[34] [17] [11,33]
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Bacterial proteins imported into mitochondria Kozjak-Pavlovic, Ross and Rudel 13
In silico analyses supported by limited experimental evidence predicted a large number of mitochondria-targeting bacterial proteins [37]. Among these, bacterial pathogenicity factors may constitute a coherent group of proteins involved in the cross-talk between host and pathogen, awaiting their detection also in other bacteria. Such proteins may be identified in phenotypic screens. For example, a connection was established between chlamydial infection and mitochondria in a recent cytotoxicity screen in yeast. One of the proteins significantly impairing yeast growth was shown to target mitochondria [38]. So far, nearly all bacterial proteins identified to target mitochondria appear to play a role in apoptosis regulation (see Table 1). However, the few examples which could not be linked to cell death regulation indicate that there may be a broader function in pathogenicity, still to be discovered. For instance, several of the mitochondriatargeting bacterial proteins like Tir, Map, and EspF also interact with cytosolic host signaling factors involved in cytoskeleton regulation. Cytosolic depletion of such factors by mitochondrial import may influence their function on the host cell cytoskeleton. Modulation of the mitochondrial physiology by bacterial proteins, for example, to adjust the host cell to metabolite or energy source requirements of the pathogen is another possibility. The later assumption may be supported by a recent genome-wide RNA interference screen performed to identify host factors that, when depleted, inhibit the infection with the obligate intracellular bacterium Chlamydia caviae. Of these, members of TIM and TOM import machines were identified as necessary for C. caviae infection [39]. The specific chlamydial protein responsible for this effect remains to be identified. It is therefore a safe assumption that bacterial pathogens and host mitochondria as their distant relatives are still communicating in a much closer way than previously anticipated.
Acknowledgement We thank Dr Joachim Rassow for helpful comments on the manuscript.
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