Article https://doi.org/10.1038/s41467-023-36629-3 A trans-kingdom T6SS effector induces the fragmentation of themitochondrial network andactivates innate immune receptorNLRX1 to promote infection Joana Sá-Pessoa 1, Sara López-Montesino2, Kornelia Przybyszewska1, Isabel Rodríguez-Escudero2, Helina Marshall1, Adelia Ova 1, Gunnar N. Schroeder1, Peter Barabas1, María Molina2, Tim Curtis1, Víctor J. Cid 2 & José A. Bengoechea 1 Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytos- keleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mito- chondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by mod- ulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFβ-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria. Mitochondria originated from an endosymbiont α-Proteobacterium related to the genus Rickettsia more than 1.45 billion years ago1. Con- sistent with this bacterial origin, mitochondria are delimitated by a double membrane with an inner membrane characterized by the pre- sence of cardiolipin and absence of cholesterol; they house a small circular genome referred as mitochondrial DNA (mtDNA), and a functional machinery for protein synthesis2. However, and in contrast to most bacteria, mitochondria are highly dynamic, and their morphology is balanced between two tightly regulated opposite events, fission and fusion. These processes are essential to maintain functional mitochondria when cells experience metabolic or environ- mental stresses3. Mitochondria are fundamental to eukaryotic cell function. They are central for the energy production of the cell upon oxidation of tricarboxylic acid (TCA) cycle intermediates which involves the crea- tion and harnessing of a membrane potential across the inner Received: 19 December 2022 Accepted: 9 February 2023 Check for updates 1Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, UK. 2Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias, Madrid, Spain. e-mail: j.bengoechea@qub.ac.uk Nature Communications | (2023) 14:871 1 12 34 56 78 9 0 () :,; 12 34 56 78 9 0 () :,; http://orcid.org/0000-0002-0038-2120 http://orcid.org/0000-0002-0038-2120 http://orcid.org/0000-0002-0038-2120 http://orcid.org/0000-0002-0038-2120 http://orcid.org/0000-0002-0038-2120 http://orcid.org/0000-0002-8576-6923 http://orcid.org/0000-0002-8576-6923 http://orcid.org/0000-0002-8576-6923 http://orcid.org/0000-0002-8576-6923 http://orcid.org/0000-0002-8576-6923 http://orcid.org/0000-0002-3933-2016 http://orcid.org/0000-0002-3933-2016 http://orcid.org/0000-0002-3933-2016 http://orcid.org/0000-0002-3933-2016 http://orcid.org/0000-0002-3933-2016 http://orcid.org/0000-0002-9677-8751 http://orcid.org/0000-0002-9677-8751 http://orcid.org/0000-0002-9677-8751 http://orcid.org/0000-0002-9677-8751 http://orcid.org/0000-0002-9677-8751 http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-36629-3&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-36629-3&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-36629-3&domain=pdf http://crossmark.crossref.org/dialog/?doi=10.1038/s41467-023-36629-3&domain=pdf mailto:j.bengoechea@qub.ac.uk mitochondrial membrane4. Mitochondria also participate in the homeostasis of intracellular Ca2+5. High concentrations of Ca2+ can accumulate in the mitochondrial matrix due to the physical proximity to the endoplasmic reticulum (ER) and the presence of a regulatedCa2+ channel, the mitochondrial calcium uniporter (MCU) complex5. This Ca2+ entry may be associated with cell death via apoptosis5. Finally, mitochondria also contribute to cell intrinsic defence through ROS and by sensing infections6. Because of this central role in cellular processes, mitochondria are emerging as a prime target for pathogens7. The bacterial origin of the mitochondria evokes an intriguing question: do pathogens interact with mitochondria employing the same systems they do deploy as defence against other bacterial competitors? For example, bacteria use the type VI secretion system (T6SS) to restrict the growth of other bacteria by delivering effectors into a target cell in a one-step process8. These antimicrobial T6SS effectors target essential elements of the bacterial cell such as the peptidoglycan, nucleic acids, or membrane phospholipids8. There are also a few examples of T6SS effectors delivered intomammalian cells9. These effectors mostly target the cytoskeleton and affect intracellular survival and bacterial internalization9. Yet surprisingly, no T6SS effectors targeting the mitochondria have been reported to date. Recently, we have dissected the T6SS of the hypervirulent Kleb- siella pneumoniae strain CIP52.145 (hereafter Kp52145). This strain encodes all the factors found in invasive K. pneumoniae strains10,11. We demonstrated the role of the T6SS in intra and inter bacterial species, and anti-fungal competition12. We characterized VgrG4 as new trans- kingdomT6SS effector intoxicating bacteria, yeast and fungi12. vgrG4 is encoded in more than 10% of the Klebsiella genomes listed in NCBI (total 812), including isolates associated with human invasive infec- tions and multidrug resistant strains. Truncation experiments demonstrated that the region of VgrG4 containing the domain DUF2345 is sufficient to exert microbial toxicity via ROS induction12. We termed this domain as ROS toxic domain (RTD)12 which is present in other VgrGs from Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains. The trans-kingdom nature of VgrG4 led us to investigate whether K. pneumoniaemayexploit VgrG4 to control host cell biology.Here, we demonstrate that VgrG4 exerts a profound effect on mitochondrial dynamics in yeast and epithelial cells, resulting in fragmentation of the mitochondria network in lung cells. Strikingly, this fragmentation has no major effect on mitochondrial function. We demonstrate that VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria to activate Drp1 to fragment the mitochondria. VgrG4-triggered transfer of Ca2+ also activates the mitochondria located innate immune recep- tor NLRX1. We show that VgrG4-dependent activation of NLRX1 limits the activation of inflammatory responses in a ROS-dependent manner in a process abrogating the post-translational modification of cullin-1 with NEDD8. Collectively, our findings identify a T6SS effector tar- geting themitochondria and uncover a previously unknown signalling cascade exploited by a pathogen to evade innate responses. Results K. pneumoniae VgrG4 RTD domain induces oxidative stress and mitochondrial condensation in Saccharomyces cerevisiae We have demonstrated that VgrG4-induced antibacterial lethality is ROS-dependent being the 518-837 stretch of VgrG4 (RTD, see Fig. 1A) sufficient12. The fact that VgrG4 inhibits growth of the eukaryotic yeast model S. cerevisiae when endogenously expressed12 led us to assess whether VgrG4 expression was related to ROS production in yeast. Cells were stained with the ROS reporter dihydroethidium (DHE) and analysed by flow cytometry. Overexpression of GST-tagged full-length VgrG4 led to significant ROS production over control cells expressing GST alone in yeast cells. Among the truncated versions expressed, the one corresponding to the RTD domain alone led to higher ROS levels, whereas non-toxic versions, lacking the RTD, led to lower ROS levels (Supplementary Fig. 1A). Oxidative stress is directly associated with mitochondria dys- function in eukaryotic cells13, prompting us to investigate mitochon- dria morphology in yeast cells expressing different versions of VgrG4. To visualise yeast mitochondria in VgrG4-expressing cells, we used an Ilv6-mCherry fusion as amitochondrialmarker. GST-VgrG4 induced an aberrant mitochondrial morphology. Instead of a normal mitochon- drial network extended along the cell, most cells displayed condensed mitochondrial clumps displaced to one side of the yeast cell (Fig. 1B). TheVgrG4RTD regionwas sufficient andnecessary to cause this effect, as the truncated versions testedbearing theRTD,GST-VgrG4518-899 and GST-VgrG4518-837 were as efficient as full-length VgrG4 provoking such effect, while a version deleted in the 518-837 region, named ΔRTD, caused no mitochondrial condensation (Fig. 1B). Moreover, toxicity of VgrG4 in yeast was partially abrogated by deletion of the RTD stretch (Supplementary Fig. 1B). The different effect on mitochondria or growth did not reflect differences in expression or stability among the fusions, as determined by immunoblot (Supplementary Fig. 1C). Themitochondria phenotype was reminiscent of that observed in a dnm1Δ mutant, lacking the mitochondrial dynamin-related GTPase Dnm1 (called Drp1 in mammals), in which mitochondria have been reported to collapse into one or more structures and nets14. Dnm1 is essential for propermitochondrialfission in yeast. Dnm1 is recruited to the mitochondrial outer membrane (MOM), where it associates with Fis1, which is anchored to the MOM15–17. Once on the membrane Dnm1 triggers the division of the mitochondrial tubular structure18,19. As shown in Supplementary Fig. 1D, the condensed mitochondria phe- notype of dnm1 and fis1 mutants was identical to that of cells expres- sing VgrG4. Moreover, VgrG4 expression did neither prevent nor aggravate the phenotype of dnm1 or fis1 deletants. The lack of an epistatic relation suggested that VgrG4 effect in the cell might involve the mitochondrial fission machinery. VgrG4 colocalises with the yeast ERMES component Mdm34 through its VgrG domain A number of studies have revealed a close contact between the mito- chondria and ER20,21, and that interactions with the ER promote mito- chondrial fission preferentially at the contact sites of the organelles22. To assess if VgrG4 associates with and affects ER-mitochondria inter- actions, we analysed the subcellular localization of VgrG4 in relation to both the ER and themitochondria. We constructed GFP fusions of full- lengthVgrG4, its VgrGdomain alone (VgrG41-517) and its C-terminal half including the RTD (VgrG4518-899). These fusions were efficiently expressed in yeast, as determined by immunoblot (Supplementary Fig. 1E). Ilv6-mCherry and DsRed-HDEL were used as a mitochondrial and ER markers, respectively. Fluorescence microscopy revealed that both GFP-VgrG4 and VgrG41-517 localised at discrete cytoplasmic puncta (Fig. 1C–E and Supplementary Fig. 1F). Relative to mitochon- dria, full-length VgrG4 puncta were interspersed in the condensed mitochondrial mesh, whereas the puncta of the VgrG N-terminal domain alone were distributed along the cell, often adjacent or to the intact mitochondrial network (Fig. 1C). GFP-VgrG4518-899 was found at larger and more diffuse irregular areas, often in proximity to the pole where the bud was emerging in the mother cell (Fig. 1C–E and Sup- plementary Fig. 1F). Such GFP-VgrG4518-899 areas were enclosed by mitochondrialmembranes but did not overlap (Fig. 1C). Relative to the ER, GFP-VgrG4 and VgrG41-517 colocalised with ER structures, often different to theperinuclear ER (nER) and cortical ER (cER) (Fig. 1D). The diffuse areas where GFP-VgrG4518-899 accumulated were also diffusely stained by the DsRed-HDEL marker, suggesting that they may derive from cytoplasmic ER membranes (Fig. 1D). Collectively, these findings indicate proximity of VgrG4 with the ER and the mitochondria. Fur- thermore, the non-toxicN-terminal VgrGdomain is responsible for the formation of tight puncta, whereas its elimination leads to the toxic Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 2 RTD domain to mark larger areas, still in the proximity of both mito- chondria and ER membranes (Fig. 1C, D). In S. cerevisiae, mitochondria are linked to the ER through the ERMES complex, which supports inter-organellar exchange of Ca2+ and lipids, and is essential for Dnm1-triggered mitochondrial fission23. The ERMES complex consists of four core components, which include the ERmembrane protein Mmm1, and the MOM proteinMdm3424. We sought to determine whether VgrG4 co-localised with ERMES components. Thus, we co-expressed GFP-VgrG4, GFP-VgrG41-517 or GFP-VgrG4518-899 with Mdm34-DsRed and observed that VgrG4 and GFP-VgrG41-517 puncta tightly co-localisedwithMdm34 (Fig. 1E; 89.8%± 3.8 of GFP-VgrG4 spots and 87.1% ± 1.3 spots overlapped Mdm34- DsRed spots). The more diffuse GFP-VgrG4518-899 also colocalised with Mdm34, but only partially (Fig. 1E; 40.0% ± 3.0). In contrast, as shown c)a) b) VgrG4 1 518 838 899 VgrG domain DUF2345 (RTD) VgrG4-specific C-terminus d) e) f) + + + - - - - - - - - - -- +- + + + - - - - - - - - - -- +- Lysates coIP GST G ST -V gr G 4 FL 1-517 518-899 Mdm34-HA GST FL 518-899 1-517G ST -V gr G 4 MergeIlv6- mCherry GFP- VgrG4 FL 518-899 1-517 DsRed- HDEL MergeGFP- VgrG4 FL 518-899 1-517 Mdm34-DsRed GFP-VgrG4 FL 518-899 1-517 Merge GS T FL 1-5 17 51 8-8 37 51 8-8 99 83 7-8 99 ∆R TD 0 20 40 60 80 100 % of ce lls w ith co nd en se d m ito ch on d r ia GST-VgrG4 0.015 <0.001 0.002 - 100 kDa - 35 kDa - 25 kDa - 63 kDa - 48 kDa - 63 kDa Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 3 in (Supplementary Fig. 1F), VgrG4 versions only partially co-localised with Mmm1-DsRed, which in our hands led to a more dispersed cyto- plasmic localization as compared to that of Mdm34, suggesting that Mmm1 overexpression from a plasmid led to localisations alternative to the ERMES. Still, most VgrG4 and VgrG41-517 were adjacent to these Mmm1 disperse structures, and some VgrG4518-899 spots overlapped. To validate independently the colocalisationof VgrG4withMdm34,we performed GST pull-down experiments. For this purpose, we con- structed anHA-tagged version of Mdm34 and pulled it downwith GST fusions of VgrG4, VgrG41-517 and VgrG4518-899. Interestingly, Mdm34- HA co-purified with all three GST-VgrG4 versions (Fig. 1F). In sum, our results in the yeast model uncover that VgrG4 inter- feres with mitochondrial morphology via its RTD domain, and that VgrG4 colocalises with the yeast ERMES component Mdm34, mainly through its VrgG domain. VgrG4 is translocated into human lung epithelial cells and induces mitochondria fragmentation We next sought to establish whether VgrG4 could also target mito- chondria in mammalian cells. We focused on human lung cells as they form the first line of defence against infections during pneumonia. K. pneumoniae does not invade these cells25,26 and, therefore, we asked whether K. pneumoniae injects VgrG4 into the cells in a T6SS- dependent manner. To address this question, VgrG4 was tagged with a 13-residue phosphorylatable peptide tag (GSK) derived from the human GSK-3β kinase27. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phos- phorylation of the tag, which can be detected with phospho-specific GSK3β antibodies27. Likewise other pathogens27, Kp52145 did not phosphorylate the GSK-tag (Fig. 2A). Infection of A549 cells with Kp52145 expressing VgrG4-GSK resulted in the translocation of the protein that was subsequently phosphorylated (Fig. 2A). Translocation of VgrG4-GSK was T6SS-dependent because we could not detect any phosphorylated VgrG4 in cells infected with the clpV mutant expres- sing VgrG4-GSK (Fig. 2A). A clpVmutant cannot assemble a functional T6SS12. We next investigated the ultrastructural changes of the mito- chondria after Klebsiella infection using transmission electron micro- scopy (TEM). Non-infected cells exhibited mitochondria with normal- appearing cristae (Fig. 2B). In contrast, in Klebsiella infected cells mitochondria were elongated with increase surface area (Fig. 2B). This phenotype is indicative of mitochondria fragmentation. Confocal microscopy experiments showed that Kp52145 inducedmitochondrial network fragmentation in A549 cells (Fig. 2C). Similar phenotype was observed in cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Supplementary Fig. 2); this drug triggers mitochondria fragmentation28–30. In contrast, the phenotype was not observed in cells infected with the vgrG4 mutant (Fig. 2C). Complementation of vgrG4 restored the fragmentation of the mito- chondrial network, demonstrating that the phenotype is VgrG4 dependent. The mitochondrial network was analysed using a semi- automated ImageJ plug31. The mitochondrial network of Kp52145- infected cells presented higher number of branches and junctions than non-infected cells or cells infected with the vgrG4mutant, which were indistinguishable (Fig. 2C and Supplementary Fig. 3A). The total length of the brancheswas reduced inKp52145-infected cells compare to non- infected cells or cells infected with the vgrG4 mutant (Supplementary Fig. 3A). Complementation of the mutant restored the mitochondrial network (Supplementary Fig. 3A). No differences were found on the mean length diameter of thebranches (Supplementary Fig. 3A). VgrG4- induced mitochondrial fragmentation was also observed in human alveolar primary lung NuLi-1 cells (Supplementary Fig. 3B), indicating that the phenotype was not cell line dependent. Because VgrG proteins are also known to act as cargo for other T6SS effectors8, the possibility exists that the mitochondrial frag- mentation could be mediated by other T6SS effector(s) delivered by VgrG4. To exclude this possibility, we took advantage of a Yersinia toolbox which allows to study the cellular effects of single bacterial effectors following Yersinia type 3 secretion system (Ysc)-T3SS-medi- ated injection into eukaryotic cells32. Control experiments confirmed the secretion of VgrG4 tagged with a VSV-G epitope in conditions in which the T3SS is active (Supplementary Fig. 3C). Cells infected with Y. enterocolitica encoding vgrG4 (hereafter YeVgrG4) presented frag- mentation of the mitochondrial network in contrast to cells infected with the Y. enterocolitica control strain (Fig. 2D). Analysis of the mito- chondrial network showed that YeVgrG4 induced similar changes as those triggered by Kp52145 (Fig. 2D). The assessment of truncated variants of VgrG4 confirmed that the C-terminal region containing the RTD domain was essential and sufficient to trigger the fragmentation of the mitochondrial network (Fig. 2D). Recently, we have demonstrated that there is considerable diversity in the T6SS loci amongst K. pneumoniae strains12. We rea- soned that strains encoding vgrG4 should trigger the fragmentation of the mitochondrial network similarly to Kp52145. In good agreement, vgrG4 positive strains, ATCC43816 and NTUH-K2044, induced the fragmentation of mitochondria whereas this was not the case for the vgrG4 negative strains SGH10 and MGH78578 (Fig. 2E). Analysis of the mitochondrial network revealed that only vgrG4 positive strains induced similar changes as those observed in cells infected with Kp52145 (Fig. 2E). Collectively, these results demonstrate that K. pneumoniae indu- ces the fragmentation of the mitochondrial network in human lung epithelial cells upon T6SS-dependent injection of VgrG4. VgrG4-inducedmitochondrial fragmentation is Drp1-dependent Mitochondrial fragmentation could result fromenhancedfission33. The GTPase dynamin-related protein 1 (Drp1) is critically essential for mitochondrial fission34. Phosphorylation of Drp1 plays a crucial role in its activity with Ser616 phosphorylation promoting fission35. We therefore investigated whether VgrG4 triggers the phosphorylation of Fig. 1 | K. pneumoniae VgrG4 RTD domain causes mitochondrial condensation in S. cerevisiae, whereas its VgrG domain is sufficient to drive co-localization with the mitochondrial ERMES component Mdm34. a Scheme of the primary structure of VgrG4, depicting the domains considered in the development of truncated versions used in this work. b Yeast cells expressing the RTD domain of VgrG4 display condensedmitochondria. Cells were co-transformedwith YEplac112- Ilv6-mCherry, a construct expressing a mCherry fusion to the mitochondrial Ilv6 protein, and the indicated GST-VgrG4 fusions or the empty pEG(KG) vector as a control (GST). Representative cells are shown and averagepercentages of cells with alteredmitochondria with standard deviation error bars are shown in the graph; at least a total of 300 cells were analysed per smaple in three independent experi- ments. Two-tailed student’s T-test was applied for statistical significance between yeast cells harbouring the empty vector and the VgrG4 constructs with no adjust- ment for multiple comparison (p values indicated in the graph). c Fluorescence microscopy of co-expressed GFP-VgrG4 versions and the mitochondrial marker Ilv6-mCherry. Representative YPH499 strain transformants induced in galactose- containing media for 5 h are shown. d Co-localisation of GFP-VgrG4 spots and ER membranes. VHY87 cells, constitutively expressing the DsRed-HDEL marker, were transformed with pYES2-GFP-VgrG4 plasmids expressing the indicated versions, induced in galactose-containing media for 5 h and observed. e Bright field and fluorescence microscopy of the SLY001 strain co-transformed with the plasmids pAG424-Mdm34-DsRed (Mdm34 DsRed) and pYES2GFP-VgrG4 (FL) or pYES2GFP- VgrG4 1-517 (1-517) or pYES2GFP-VgrG4 518-899 (518-899). White arrows indicate co-localization events. Scales bars represent 5 µm in c, d, and e. f Immunoblots corresponding to a representative GSTpull-down experiment on lysates fromyeast cultures co-expressing Mdm34-HA. Images correspond to the same membrane hybridized with anti-HA (top panels) and anti-GST (lower panes) primary anti- bodies. Images are representative of three independent experiments. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 4 Drp1. Immunoblotting experiments showed that Kp52145 and YeVgrG4 induced the phosphorylation of Drp1 (Fig. 2F, G, respec- tively), indicating that VgrG4 was sufficient to trigger the activation of Drp1. The quantification of the blots is shown in (Supplementary Fig. 4). To determine whether VgrG4-induced mitochondrial frag- mentation is Drp1-dependent, we infected cells in which drp1 was silenced by siRNA. Kp52145-inducedmitochondrial fragmentation was not observed in drp1 knockdown cells in contrast to cells treated with All Stars control siRNA (Fig. 2H). drp1 knockdown efficiencywas higher than 60% (Supplementary Fig. 5A, B). To substantiate further the role of Drp1, we used Mdivi-1, a compound that inhibits Drp1 function during mitochondrial fission36 although some caveats on the specifi- city of the inhibitor have been reported37. When Mdivi-1-treated cells were infected with Kp52145, we did not observe any mitochondrial a) b) c) d) e) h) f) g) i) n.i. Kp52145 ΔvgrG4 comp 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m i to c h on dr ia <0.0001 <0.0001 n.i . Ye Ye Vg rG 4 Ye Vg rG 4 1- 51 7 Ye Vg rG 4 51 8-8 99 0.8 1.0 1.2 1.4 1.6 B ra nc he s /m ito ch on dr ia <0.0001 <0.0001 ni Kp52 Kp43 NTUH MGH SGH 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia <0.0001 <0.0001 <0.0001 n.i. Kp52145 n.i. Kp52145 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia <0.0001 AS Drp1 siRNA n.i. Kp52145 n.i. Kp52145 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia DMSO Mdivi-1 <0.0001 n.i. 1h 3h 5h Drp1 Kp52145 p-Drp1S616 n.i. Ye YeVgrG4 Drp1 p-Drp1S616 n.i. Kp52145 - 95 kDa - 72 kDa - 95 kDa - 72 kDa - 95 kDa - 72 kDa - 72 kDa - 95 kDa - 95 kDa - 43 kDa Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 5 fragmentation (Fig. 2I). As anticipated, YeVgrG4-triggered mitochon- drial fragmentation was not detected in Mdivi-1-treated cells (Supple- mentary Fig. 3D), and in cells in which drp1 was silenced by siRNA (Supplementary Fig. 3E). Altogether, our results show that VgrG4- induced fragmentation of mitochondria depends on the activity of Drp1. VgrG4-induced mitochondrial fragmentation does not impair mitochondria physiology We next investigated the physiology of the mitochondria in Klebsiella- infected cells.We first assessedwhether VgrG4-inducedmitochondrial fragmentation affects the contribution of the mitochondria to cellular bioenergetics using a Seahorse XFe96 analyser. The oxygen con- sumption rate (OCR) increased following infection with Kp52145 (Supplementary Fig. 6A), indicating an increase in mitochondrial basal respiration (Supplementary Fig. 6B). Addition of oligomycin triggered a decrease of cellular OCR, however the OCR was still significantly higher in Kp52145 infected cells compared to non-infected ones indi- cating that not all oxygen consumption is used for ATP production in K. pneumoniae-infected cells (Supplementary Fig. 6A, B). ATP levels were higher in Kp52145-infected cells than in non-infected cells (Sup- plementary Fig. 6B). The fact that the proton leak was not different between non-infected cells and Kp52145-infected ones indicates no differences in the basal respiration not coupled to ATP production (Supplementary Fig. 6B). Subsequent addition of FCCP, which stimu- lates respiration, showed that the maximal respiration capacity was similar between non-infected and Kp52145-infected cells (Supple- mentary Fig. 6A, B). The spare respiratory capacity was not sig- nificantly different between Kp52145-infected cells and non-infected ones (Supplementary Fig. 6A, B), indicating that K. pneumoniae infec- tion does not deplete the cellular energy via an increased oxidative phosphorylation. The fact that OCR was higher in Kp52145-infected cells as compared to non-infected ones after the addition of rotenone and antimycin A indicates that the non-mitochondrial respiration is increased in K. pneumoniae-infected cells (Supplementary Fig. 6A, B). Altogether, these findings demonstrate that Kp52145 infection does not impair themitochondria bioenergetics. The fact that thewild-type, the vgrG4 mutant and the complemented strains triggered similar changes (Supplementary Fig. 6A, B) demonstrates that K. pneumoniae- triggered metabolic changes are not affected by VgrG4-induced frag- mentation of the mitochondrial network. The release of mtDNA to the cytosol of cells has been associated with the fragmentation of mitochondria38. Therefore, we assessed whether VgrG4 will trigger the release of mtDNA to the cytosol by determining the cytoplasmic levels of the mitochondria-encoded cytochromec oxidase I gene (mt-CO1). qPCR experiments showed that YeVgrG4 infection resulted in the presence of mtDNA in the cytosol of A549 and NuLi-1 cells (Supplementary Fig. 7A, B, respectively). The presence ofmtDNA in the cytosol results in the activation of the cGAS- STING signalling pathway leading to the upregulation of the expres- sion of type I IFN and interferon-stimulated genes (ISGs)38. To inves- tigate whether VgrG4-induced release of mtDNA activates the STING signalling pathway, we challenged NuLi-1 cells and measured tran- scription of target genes. A549 cells do not express STING39 and, therefore, they are not suitable for this analysis. As we expected, YeVgrG4 induced the expression of ifnb, and the ISGs irf3, isg15 and isg54 in NuLi-1 cells (Supplementary Fig. 7C). However, this was not the case in cells treated with the STING antagonist H15140. Combined this suggests that VgrG4 induces mtDNA release which stimulates STING- governed signalling. Mitochondrial fragmentation and release of mtDNA have been linked to the dissipation of themitochondriamembrane potentialΔΨ, important for the generation of ATP, transport across the mitochon- dria membrane41, and cell death38. However, we found that neither infection with K. pneumoniae nor with YeVgrG4 caused any change in ΔΨ, in contrast to cells treated with the membrane uncoupler CCCP (Supplementary Fig. 7D). In line with this, we observed no differences in the levels of intracellular ATP between non-infected cells, and those infected with K. pneumoniae or YeVgrG4 (Supplementary Fig. 4E). We also observed no differences in cell viability between non-infected and infected cells (Supplementary Fig. 7F), finding consistent with the lack of change in the ΔΨ. Altogether, these data suggest that VgrG4-inducedmitochondrial fragmentation results in the release of mtDNA to the cytosol but without perturbation of the functionality of mitochondria. VgrG4 colocalises with the ER protein mitofusin 2 We next sought to establish the colocalisation of VgrG4 within mam- malian cells. VgrG4 delivered by Yersinia showed a puncta distribution within A549 cells; however these puncta did not colocalise with mitochondria although they were found in closed proximity (Supple- mentary Fig. 8). The colocalisation of VgrG4 with the ER in yeast led us to examine whether VgrG4 colocalises with the ER inmammalian cells. Confocal microscopy experiments revealed the colocalisation of VSV- G tagged VgrG4 puncta with the ER (Fig. 3A). FLAG-tagged VgrG4 injected by Kp52145 also showed a puncta distribution, and we observed its colocalisation with the ER (Fig. 3A). Fractionation experiments corroborated that Yersinia-injected VgrG4 was present in the ER but not in the mitochondria fraction (Fig. 3B). The VgrG4 C-terminal region containing the RTD domain was sufficient for VgrG4 colocalisation with the ER (Fig. 3C) whereas this was not observed in cells injected with the N-terminal region of VgrG4 (Fig. 3C). In yeast, VgrG4 colocalised with ERMES component Mdm34. However, nomammalianhomologueexists of the ERMEScomponents. Instead, a number of tethering proteins have been reported to main- tain the ER-mitochondriaMCS, including vesicle-associatedmembrane Fig. 2 | VgrG4 is translocated into A549 cells in a T6SS-dependent manner and induces Drp1 dependent mitochondria fragmentation. Translocation of VgrG4 into lung epithelial cells was analysed by immunoblot of phosphorylated GSK3β, GSK3β-tag and total GSK3β levels (a) in lysates of A549 cells infected with Kp52145 (Kp52) and the T6SS inactive clpV mutant (strain ΔclpV) for 2 hours or left unin- fected (n.i.). Bacterial lysates (1 × 106 bacteria) were run for the same antibodies. Mitochondria ultrastructure was analysed by transmission electron microscopy (TEM) (b) in A549 cells infected with Kp52145 for 5 hours or left uninfected (n.i.) (magnification 20,000×, scale bar 200nm). Mitochondria fragmentation (c) was analysedby confocalmicroscopyofA549 cells treatedwithmitotracker red (50μM, 30minbefore infection, in red) and infected for 3 hwith Kp52145, the vgrG4mutant (ΔvgrG4), or the complemented strain (comp, ΔvgrG4/pBAD30vgrG4) or left uninfected (n.i.). The number of branches/mitochondria was determined with the Mitochondria Analyzer plugin for ImageJ. The graph is the result of the analysis of twenty images per condition of three independent experiments representing average with standard deviation error bars; at least 100 cells were analysed per sample. Fragmentation was also analysed following infection with different Y. enterocolitica strains for 90min (d) and following infection with different wild-type K. pneumoniae strains for 3 h (e); at least 100 cells were analysed per sample. Nuclei were stained with Hoechst (DAPI, in blue). All images and immunoblots are representative of three independent experiments. f Immunoblot analysis of the levels of phosphorylated Drp1 (S616) and total Drp1 in lysates of A549 cells infected with Kp52145 for the indicated times. Phosphorylation of Drp1 was also analysed in lysates of A549 cells infectedwith Y. enterocolitica (Ye) and YeVgrG4 for 90min (g). h Confocalmicroscopy of A549 cells transfected with a non-silencing control (AS – All Stars) or with a Drp1 siRNA, and treated with mitotracker red (50μM, 30min, in red), and infected with Kp52145 for 3 h. To confirm further the role of Drp1 in VgrG4-induced mitochondria fragmentation, cells were treated with the Drp1 inhibitor Mdivi-1 (10μM, 2 h before infection) or DMSO (vehicle solution) and infected with Kp52145 for 3 h (i); at least 100 cells were analysed per sample. One- way ANOVA with Tukey’s test for multiple comparison was applied for statistical significance for all the indicated comparisons (p values indicated in the graph). Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 6 protein B (VAPB), protein tyrosine phosphatase-interacting protein-51 (PTPIP51), Mitofusin (Mfn) 1/2, inositol 1,4,5-trisphosphate receptor (IP3R), glucose-regulated protein 75 (GPR75), voltage-dependent anion channel (VDAC1) complex, and B-cell receptor-associated pro- tein 31 (Bap31)42. Recent work strongly indicates that VAPB and Mfn2 are the main ER-mitochondria tethering proteins43. To determine whether theseproteins are needed for the colocalisationof VgrG4with the ER, their expression was reduced by siRNA and the colocalisation of VgrG4 with the ER assessed by confocal microscopy. Control experiments confirmed the knockdown of VAPB and Mfn2 (Supple- mentary Fig 5). Figure 3D shows that the colocalisation of VgrG4 with the ER was lost only in cells deficient for Mfn2. Furthermore, in cells c) a) b) Calnexin VSV-G Tom20 n.i. YeVgrG4 Endoplasmic Re�cu n.i. Mitochondria YeVgrG4 e) d) f) GFP n.i. VSV-G YeIgG YeVgrG4 - 95 kDa - 72 kDa - 26 kDa - 95 kDa - 95 kDa Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 7 transfected with an YFP fusion ofMfn2, we detected the colocalisation of Mfn2-YFP with VgrG4 by confocal microscopy (Fig. 3E). Moreover, Mfn2-YFP co-immunoprecipitated VSV-G tagged VgrG4 (Fig. 3F). Altogether, these results establish that VgrG4 colocalises with the ER protein Mfn2. The C-terminal region containing the RTD domain mediates the colocalisation of VgrG4 with the ER and Mfn2. VgrG4 triggers the transfer of Ca2+ to the mitochondria from the ER The ER-mitochondria contact sites are crucial in the regulationof inter- organellar Ca2+ exchange44. We examined whether VgrG4 affects the transfer of Ca2+ from the ER to the mitochondria. To detect the accu- mulation of Ca2+in the mitochondria, we used the cell permeable mitochondria probe Rhod-2 AM whose fluorescence increases upon binding Ca2+. Kp52145 infection increased the fluorescence of Rhod-2 in the mitochondria (Fig. 4A). VgrG4 was sufficient to evoke the accumulation of Ca2+in the mitochondria as YeVgrG4 infection also increased Rhod-2 fluorescence (Fig. 4A). Time course experiments showed an increase in Rhod-2 fluorescence over time following YeVgrG4 infection (Supplementary Fig. 9A), being the RTD containing domain of VgrG4 sufficient to increase the fluorescence of Rhod-2 (Fig. 4B). The colocalisation of VgrG4 with the ER Mfn2 is essential to evoke the Ca2+ accumulation in the mitochondria because no increase of Rhod-2 fluorescence was found in mfn2 knockdown cells by siRNA (Fig. 4C). Others have shown that silencing of mfn2 does not affect agonist-induced ER Ca2+ release45. To determine the receptors and channels responsible for VgrG4- induced accumulation of Ca2+ in the mitochondria, we took advantage of a panel of well-established inhibitors affecting ER-mitochondria Ca2+ exchange. Blocking of ryanodine receptors using ryanodine had no effect onCa2+ accumulation following infectionwithYeVgrG4 (Fig. 4D). Likewise, blocking of IP3-mediated Ca2+ release with Xestospongin C or inhibition of both IP3 receptors and TRP channels with 2-Aminoethoxydiphenyl borate (2APB) had no effect on Rhod-2 fluorescence (Fig. 4D). On the contrary, thapsigargin, a non- competitive inhibitor of the sarco/endoplasmic reticulum Ca-ATPase (SERCA), abrogated YeVgrG4-mediated increase in Rhod-2 fluores- cence (Fig. 4D), demonstrating that VgrG4-induced Ca2+ accumulation in the mitochondria is dependent on the activation of the ER SERCA pumps. The fact that the inhibitor ruthenium red decreased VgrG4- induced increase of Rhod-2 fluorescence (Fig. 4D) indicates that mitochondria Ca2+ accumulation is dependent on the mitochondrial calcium uniporter complex (MCU). Further substantiating this result, silencing of mcu with siRNA resulted in no increase in Rhod-2 fluor- escence following YeVgrG4 infection (Fig. 4E). Collectively, these findings demonstrate that VgrG4 triggers the accumulationofCa2+ in themitochondria via themitochondria calcium uniporter complex upon activation of the ER SERCA pumps. VgrG4-inducedCa2+ accumulation in themitochondriamediates the fragmentation of the mitochondria network Ca2+ levels have been implicated in the regulation of mitochondria morphology46. Therefore, we sought to establish whether VgrG4- induced Ca2+ accumulation in the mitochondria results in the fragmentation of the mitochondria network. Confocal microscopy experiments uncovered that inhibition of the ER SERCA pumps with thapsigargin abrogated YeVgrG4 and Kp51245-induced mitochondrial fragmentation (Fig. 4F) whereas the inhibition of ryanodine receptors, IP3 receptors and TRP channels with ryanodine, Xestospongin C, and 2APB, respectively had no effect on YeVgrG4-triggered fragmentation of the network (Supplementary Fig. 9B). YeVgrG4 did not affect the mitochondria network in cells in whichmcu was silenced using siRNA (Fig. 4G). Moreover, treatment of cells with thapsigargin also abro- gated YeVgrG4-induced phosphorylation of Drp1 (Fig. 4H and Sup- plementary Fig. 4), uncovering that the transfer of Ca2+ to the mitochondria is the signal triggering the activation of Drp1 in VgrG4- treated cells. To sustain further that Ca2+ release from the ER is sufficient to induce the fragmentation of the mitochondria, cells were treated with carbachol that triggers the release of Ca2+ from the ER47. Control experiments showed that carbachol treatment increased the fluores- cence of Rhod-2 (Supplementary Fig. 10A). Carbachol treatment resulted in the fragmentation of the mitochondrial network (Supple- mentary Fig. 10B), and the phosphorylation of Drp1 (Supplementary Fig. 10C). The fact that treatment of cells with the Drp1 inhibitorMdivi- 1 abrogated carbachol-induced mitochondria fragmentation (Supple- mentary Fig. 10B) demonstrates that carbachol-induced fragmentation is Drp1-dependent. Therefore, carbachol treatment recapitulates VgrG4 action. Altogether, these results strongly support the notion that VgrG4- induced mitochondrial Ca2+ accumulation from the ER mediates the activation of Drp1 responsible for VgrG4-induced fragmentation of the mitochondria network. VgrG4 induces mitochondria ROS upon activation of the innate receptor NLRX1 Mitochondria produce ROS (mtROS) due to electron slippage during oxidative phosphorylation48, and there are indications of a crosstalk between Ca2+ levels in the mitochondria and mtROS production44. We then investigated whether VgrG4 induces mtROS by assessing the oxidation of the mitochondria localized fluorescent dye mitoSOX. Kp52145 and YeVgrG4 infections resulted in the oxidation of mitoSOX that colocalised with mitochondria (Fig. 5A). Quantification of mito- SOX oxidation corroborated that Kp52145 infection increased the levels of mtROS whereas the vgrG4 mutant did not increase mtROS levels over those of the non-infected cells (Fig. 5B). Complementation experiments confirmed that mtROS induction is VgrG4-dependent (Fig. 5B). The fact that YeVgrG4 also induced mtROS (Fig. 5A, B) sug- gests that VgrG4 translocation to cells is sufficient to elicit mtROS. In mfn2 knockdown cells, YeVgRG4 did not induce mtROS, indicating that the ER colocalization of VgrG4 is essential for the production of mtROS (Fig. 5C). We next assessed whether VgrG4-induced Ca2+ accumulation in the mitochondria mediates mtROS induction. Thapsigargin and ruthenium red, two drugs that inhibited the VgrG4-triggered transfer of Ca2+ into the mitochondria (Fig. 4D), abrogated YeVgrG4-induced mtROS (Fig. 5D). In contrast, ryanodine, an inhibitor that did not affect VgrG4-triggered Ca2+ accumulation in Fig. 3 | VgrG4 colocalizes with the ER. VgrG4 localisation within the cells was analysed by confocal microscopy of A549 mCherryER cells (ER in red). Cells were infectedwith YeVgrG4 for 90min, or Kp52145 for 3 h, or left uninfected (n.i.) VgrG4 was stained with VSV-G or FLAG (in green) (a). b Immunoblot analysis of VgrG4- tagged with VSV-G, Calnexin (ER marker) and Tom20 (mitochondria marker) in fractionated lysates of A549 cells infected with YeVgrG4 for 90min or left unin- fected (n.i.). c Confocal microscopy of A549 mCherryER cells infected with Y. enterocolitica encoding N- and C-terminal truncated forms of VgrG4 tagged with VSV-G for 90min. To determine the tethering protein associatingwith VgrG4, A549 mCherryER cells were transfected with siRNAs for VAPB orMFN2 and infected with YeVgrG4 for 90min (d). e Confocal microscopy of A549 cells transfected with the MFN2-YFP plasmid (MFN2 in green) and infected with YeVgrG4 for 90min. VgrG4 was stained with VSV-G. f Immunoblot analysis of VSV-G (VgrG4) and GFP (MFN2) levels in immunoprecipitates of A549. 24 h after transfection with a MFN2-YFP plasmid, cells were infected with YeVgrG4 for 90min. Lysates were immunopre- cipitated using anti-GFP antibody, andmembranes were first probed with antibody against VSV-G and subsequentlywith antibody against GFP. Pre-immunemouse IgG served as negative control. All images and immunoblots are representative of three independent experiments. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 8 mitochondria (Fig. 4D), did not reduce VgrG4-induced mtROS (Fig. 5D). Carbachol treatment elicited mtROS (Supplementary Fig. 10D), substantiating that the accumulation of Ca2+ into the mitochondria induces mtROS. Altogether, these results support the notion that VgrG4-stimulated Ca2+ accumulation is responsible for mtROS levels. However, VgrG4-induced mtROS is independent of the activation of Drp1 because treatment of cells with Mdivi-1 had no effect on YeVgrG4-mediated oxidation of mito- SOX (Fig. 5D). We next sought to provide mechanistic insights into the mole- cular basis connecting VgrG4-induced Ca2+ accumulation and mtROS. NLRX1 is a mitochondrial NOD-like receptor that has been suggested to interact with the electron transport chain and potentiate the pro- duction of mtROS49. Therefore, we asked whether VgrG4-induced a) b) n.i . Ye Ye Vg rG 4 Ye Vg rG 4 1- 51 7 Ye Vg rG 4 51 8-8 99 40000 60000 80000 100000 R ho d2 (R FU ) <0.0001 <0.0001 c) e)d) ni Ye YeVgrG4 ni Ye YeVgrG4 40000 50000 60000 70000 80000 Rh od 2 (R FU ) <0.0001 <0.0001 AS MCU siRNA n.i. YeVgrG4 n.i. YeVgrG4 40000 60000 80000 100000 Rh od 2 (R FU ) <0.0001 AS MFN2 siRNA no n- in fe ct ed D M SO R ya no di ne Th ap si g a rg in R ut he ni um R ed 2A PB Xe st os po ng in C 60000 90000 Rh od 2 (R FU ) <0.0001 <0.0001 <0.0001 YeVgrG4 g) h) f) K p5 21 45 Ye Vg rG 4 K p5 21 45 Ye Vg rG 40.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia <0.0001 DMSO Thapsigargin <0.0001 n.i. YeVgrG4 n.i. YeVgrG4 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia AS MCU siRNA <0.0001 n.i. YeVgrG4 Drp1 p-Drp1S616 n.i. YeVgrG4 DMSO Thapsigargin - 95 kDa - 72 kDa - 72 kDa Fig. 4 | VgrG4 triggers the transfer of Ca2+ from the ER to the mitochondria causing fragmentation. a Confocal microscopy of A549 cells treated with mito- tracker green (50μM, 30min before infection, in green) and then infected with Y. enterocolitica strains for 90min, orwith Kp52145 for 3 h. After 90min or 3 h, Rhod2 in HBSS without calcium (50 µM, in red) was added for 30min to stain the calcium accumulation in the mitochondria. b Rhod2 fluorescence was measured at 90min post infection as indicated before in cells infected with Y. enterocolitica strains encoding truncated versions of VgrG4. Rhod2 accumulation was measured in cells knockdown forMFN2 (20 nM)by siRNA transfection and infectedwith YeVgrG4 (c). Cells were treated with ryanodine (100nM), thapsigargin (1μM), ruthenium red (100μM), 2APB (10μM) or Xestospogin C (10μM) or treated with vehicle control (DMSO, no inhibitor) 60min post infection. Rhod2 fluorescence was measured as previously described (d). e Rhod2 fluorescence was measured in cells transfected with siRNA for the mitochondria calcium uniporter (MCU) and infected for 2 h as described in b. f Confocal microscopy of A549 cells treated with mitotracker red (50μM, 30min, in red) and infected with YeVgrG4 for 90min or Kp52145 for 3 h. Nuclei were stained with Hoechst (DAPI, in blue), and at least 100 cells were ana- lysed per sample. Cells were treated with thapsigargin (1μM, 30min before end of infection) or DMSO (vehicle solution). Mitochondria fragmentation was assessed by confocal microscopy in cells transfected with siRNA for MCU (20 nM) or AS control and infected with YeVgrG4 (g); at least 100 cells were analysed per sample. The levels of S616 phosphorylated Drp1 and tubulin were determined by immu- noblotting in lysates of YeVgrG4-infected A549 cells treated with thapsigargin (1μM) or vehicle control (DMSO) for 30min before the end of the infection (h). One-way ANOVA with Tukey’s multiple comparison test was applied for statistical significance (p values indicated in the graph). Images are representative of three independent experiments. Data in graphs are presented as the mean ± SD of five independent experiments measured in duplicate. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 9 mtROS is dependent on NLRX1 using the oxidation of the fluorescence dye dichlorofluorescein (DCF) as read-out. Supplementary Fig. 11A shows that YeVgrG4 induced fluorescence levels of DCF were sig- nificantly lower in nlrx1 knockdown cells using siRNA than in cells treated with non-silencing control All Stars siRNA. Interestingly car- bachol did not elicit mtROS in cells in which nlrx1 was silenced (Sup- plementary Fig. 10E), demonstrating that the accumulation ofCa2+ into the mitochondria triggers mtROS in a NLRX1-dependent manner. We next questioned whether NLRX1-induced mtROS affects the mitochondria morphology. Scavenging mtROS with mitoTEMPO did not abrogate Kp52145-induced fragmentation of the mitochondria network (Fig. 5E). Reinforcing the notion that ROS had no effect on mitochondriamorphology, the potent ROS inducerH2O2 had no effect on the mitochondria network (Supplementary Fig. 6F). YeVgrG4 still induced the fragmentation of the mitochondria network in nlrx1 knockdown cells, and the fragmentation was not different to that induced by the infection in non-silencing control All Stars siRNA treated cells (Supplementary Fig. 11B). Control experiments confirmed that therewere no differences in the fluorescence of Rhod-2 in All stars treated and nlrx1 knockdown cells infected with YeVgrG4 (Supple- mentary Fig. 11C). Altogether, these results are consistent with the notion that VgrG4-induced mtROS does not affect the mitochondria network, and thatNLRX1does not control VgrG4-induced influxofCa2+ to the mitochondria. Collectively, these data demonstrate that VgrG4-triggered Ca2+ accumulation in the mitochondria induces mtROS in a NLRX1- dependent manner. NLRX1 elicited ROS mediates cullin 1 deNEDDylation to mod- ulate epithelial signalling Extracellular commensal bacteria exploit ROS to attenuate the acti- vation of the immunoregulatory NF-κB pathway50 that governs host defence pathways against pathogens including K. pneumoniae25,51–53. We hypothesized that Klebsiella exploits NLRX1-induced mtROS to limit the activation of NF-κB. In cells pre-treated with mitoTEMPO we observed an increase in the nuclear translocation of theNF-κBp65 unit following infection with Kp52145 (Fig. 6A). A similar result was obtained following infection of nlrx1 knockdown cells (Fig. 6B). These results suggested that VgrG4 limits the activation of NF-κB. Indeed, and in sharp contrast to thewild-type strain, the vgrG4mutant induced the nuclear translocation of p65 (Fig. 6C). Complementation of the mutant restored the wild-type phenotype (Fig. 6C). Moreover, VgrG4 was sufficient to reduce the p65 translocation induced by Y. enter- ocolitica (Supplementary Fig. 12A). However, this was not the case in cells pre-treated with mitoTEMPO or in cells knockdown for nlrx1 (Supplementary Fig. 12B and 12C, respectively). Consistentwith VgrG4- mediated control of NF-κB activation, the vgrG4 mutant induced higher levels of IL8 than the wild-type strain (Fig. 6D). Moreover, b) c) n.i. YeVgrG4 n.i. YeVgrG4 0 1000 2000 3000 4000 RO S (R FU ) <0.0001 AS MFN2 siRNA n.i . Kp 52 14 5 Δv grG 4 co mp Ye Ye Vg rG 4 1500 2000 2500 3000 3500 M ito So x (R FU ) <0.0001 <0.0001 <0.0001 Ye Ye Vg rG 4 Th ap si g a rg in R ut he ni um R ed R ya no di ne D M SO M di vi -1 0 2000 4000 6000 RO S (R FU ) <0.0001 <0.0001 <0.0001 YeVgrG4 d) e) a) n.i. Kp52145 n.i. Kp52145 0.8 1.0 1.2 1.4 1.6 B ra nc he s/ m ito ch on dr ia DMSO mitoTEMPO <0.0001 <0.0001 Fig. 5 | VgrG4 induces mitochondrial ROS production. a Mitochondrial ROS (mtROS) was detected in infected A549 cells by confocal microscopy. Cells were treatedwithmitotrackergreen (50μM,30min, in green), infectedwith YeVgrG4 for 90min or with Kp52145 for 3 h, and treated with mitoSOX red mitochondrial superoxide indicator (10 µM for 30min incubated in calcium free HBSS). Fluores- cence was measured (b) in A549 cells upon infection with Y. enterocolitica strains for 90min or K. pneumoniae strains for 3 h, followed by incubation with mitoSOX (10 µM incubated in calcium free HBSS for 30min prior to measurement). c To demonstrate that VgrG4 localisation is important for ROS production, 2’,7’- dichlorofluorescein (DCF) was used to measure ROS production by A549 cells transfected with MFN2 siRNA or non-silencing (AS) control and infected with YeVgrG4 for 90min. d DCF fluorescence was measured in A549 cells pre-treated with Mdivi-1 (10 µM, 2 h) or vehicle control (DMSO), and infected with YeVgrG4 for 90min. Cells were treated with thapsigargin (1μM), ruthenium red (100μM), rya- nodine (100nM) 30min before the end of infection. Mitochondria fragmentation was analysed by confocal microscopy of A549 cells treated with mitotracker red (50μM, 30min, in red) and stained with Hoechst (in blue). Cells were pre-treated with mitoTEMPO, a mitochondrial superoxide scavenger (10μM, 2 h), and infected with Kp52145 for 3 h (e); at least 100 cells were analysed per sample. One-way ANOVA with Tukey’s multiple comparison test was applied for statistical sig- nificance (p values indicated in the graph). Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 10 a) b) c) n.i. Kp52145 0 20 40 60 80 100 p6 5 nu cl ea rl oc al is at io n (% ) DMSO mitoTEMPO 0.0203 1357 1714 1792 1169 n.i. Kp52145 0 20 40 60 80 p6 5 nu cl ea rl oc al is at io n (% ) AS NLRX1 siRNA 0.0011 1367 1335 1499 1126 d) ni Kp 52 14 5 Δv grG 4 co mp ni Kp 52 14 5 Δv grG 4 co mp 0 100 200 300 400 hI L8 [p g/ m l] AS NLRX1 siRNA 0.0030 <0.0001 <0.0001 <0.0001 0.0167 e) g) h) i) n.i. 1h 3h 5h p-Iκκ Ub48 IκBα n.i. Kp52145IgG MG262 n.i. 1h 3h 5h p-IκBα Tubulin Kp52145 Tubulin CUL1 CUL1-NEDD8 n.i. Kp52145 H2O2 Kp52145 Tubulin f) n.i. 1h 3h 5h Kp52145 IκBα Tubulin k) l) j) n.i. Kp52145 ΔvgrG4 comp Cul1 Tubulin Cul1 Tubulin n.i. Kp52145 n.i. AS NLRX1 siRNA Kp52145 NEDD8 Ubc12 n.i. Kp52145 MitoTEMPO IgG n.i. Kp52145 DMSO IgG n.i. Kp52145 ΔvgrG4 comp 0 20 40 60 80 p6 5 nu cl ea rl oc al is at io n (% ) n=478 n=563 n=611 n=405 0.0077 - 55 kDa - 55 kDa - 72 kDa - 55 kDa - 55 kDa - 95 kDa - 72 kDa - 55 kDa - 43 kDa - 55 kDa - 80 kDa - 55 kDa Cul1●NEDD8 Cul1 - 80 kDa - 55 kDa Cul1●NEDD8 Cul1 - 80 kDa - 55 kDa Cul1●NEDD8 Cul1 - 17 kDa - 10 kDa Fig. 6 | NLRX1-mediated ROS controls NF-κB signalling and affects cullin 1 NEDDylation. Immunofluorescence microscopy of A549 cells stained with anti- body for the p65NF-κB subunit. Cells were pre-treatedwithmitoTEMPO (10μM, 2 h pre-infection), orwith vehicle control (DMSO) and infectedwithKp52145 for 3 h (a). b Cells were transfected with NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with Kp52145 for 3 h. c Cells were infected with Kp52145, the vgrG4 mutant (ΔvgrG4) or the complemented strain (comp, ΔvgrG4/pBAD30vgrG4) for 3 h. The percentage of p65 NF-κB localised in the nucleus is represented on the graph in a–c and it is the result of counting ofminimumof hundred cells from each of three independent experiments. The total number of counted cells is indicated on top of each bar. ELISA of IL-8 secreted by A549 cells transfected with either NLRX1 siRNA (50 nM) or a non-silencing control (AS) and infected with K. pneu- moniae strains. After 3 h of contact, the medium was replaced with medium con- taining gentamicin (100 µg/mL) to kill extracellular bacteria, and after 2 h the medium was collected (d). Images are representative of three independent experiments. Data in graphs are presented as the mean ± SD of three independent experiments. Two way-ANOVA with Holm-Sidak’s multiple comparisons test was used for statistical significance (p values indicated in the graph). e Immunoblot analysis of total IκBα and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. f Immunoblot analysis of phosphorylated IκBα and tubulin levels in lysates of A549 cells infected with Kp52145 for the indicated times. g Levels of phosphorylated Iκκα/β and tubulin in cells infectedwith Kp52145 for the indicated times. IκBα immunoprecipitation and immunoblot for K48-linkage spe- cific polyubiquitin (Ub48) in cells treated with the proteasome inhibitor MG262 (5μM, 2 h before infection), and infected with Kp52145 for 3 h or left uninfected (n.i.). Pre-immune mouse IgG was used as a control for immunoprecipitation (h). Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549cells infectedwith Kp52145 for 5 h, or treated with H2O2 (5μM, 5min). Cul-1 appears as a doublet, with the higher molecular band representing the NEDDylated form of Cul-1 (i). j Immunoblot analysis of Cul-1 and tubulin levels in lysates of A549 cells transfected with NLRX1 siRNA (50nM) or a non-silencing control (AS), infected with Kp52145 for 5 h. k Cells were infected with Kp52145, the vgrG4 mutant (ΔvgrG4) or the complemented strain (comp) for 5 h, and the levels of Cul-1 and tubulin assessed by western blot. lCells were pre-treated withmitoTEMPO (10μM, 2 h pre-infection) or a vehicle control (DMSO), infected with Kp52145 for 5 h, and Ubc12 was immuno- precipitated followed by detection of NEDD8 and Ubc12 levels by immunoblotting. Images are representative of three independent experiments. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 11 translocation of VgrG4 was sufficient to reduce Y. enterocolitica- induced IL8 levels (Supplementary Fig. 12D). No differences between strains were observed in nlrx1 knockdown cells (Fig. 6D and Supple- mentary Fig. 12D). Altogether, these results support the notion that VgrG4-induced NLRX1-dependent mtROS negative regulates the acti- vation of NF-κB with a concomitant reduction in inflammation. We next addressed at which level of the NF-κB signalling cas- cade VgrG4 exerts its negative effect via NLRX1-induced ROS. In the canonical NF-κB pathway, nuclear translocation of NF-κB is pre- ceded by phosphorylation and subsequent degradation of IκBα by the ubiquitin proteasome54. Experiments confirmed that Kp52145 infection did not affect the levels of IκBα (Fig. 6E). However, we noted that infection did result in the phosphorylation of IκBα (Fig. 6F), suggesting that the signalling cascade leading to the phosphorylation of IκBα is not affected by VgrG4. Supporting this notion further, Kp52145 infection resulted in the phosphorylation of the kinase Iκκα/β (Fig. 6G) that controls the phosphorylation of IκBα. These results led us to investigate whether the poly- ubiquitination of IκBα is affected in K. pneumoniae-infected cells. Indeed, we found a decrease in the levels of ubiquitinated IκBα in Kp52145-infected cells (Fig. 6H). Interestingly, this is not specific of IκBα because we also observed a decrease in the levels of ubiquiti- nation of β-catenin (Supplementary Fig. 13A), whose levels are controlled also by the ubiquitin proteasome. The reduced ubiqui- tination of β-catenin resulted in increased levels of β-catenin in infected cells (Supplementary Fig. 13B) with a concomitant increase in the expression of β-catenin-controlled genes (Supplementary Fig. 13C). Control experiments showed that that expression of β- catenin was not increased by Kp52145 (Supplementary Fig. 13C). The ubiquitination of proteins such as IκBα and β-catenin for their subsequent degradation by the ubiquitin proteasome is mediated by a common ubiquitin ligase, E3-SCFβ−TrCP 55. Therefore, we investigated whether K. pneumoniae may target E3-SCFβ-TrCP. The covalent mod- ification with NEDD8, termed neddylation, of the cullin-1 (Cul-1) sub- unit of the E3-SCFβ−TrCP is essential for the function of the ligase and, therefore, it is emerging as central regulatory event in cellular pro- cesses governed by the ubiquitin proteasome degradation of proteins56. Immunoblotting experiments revealed an overall decrease in the pattern of NEDD8-conjugated proteins in Kp52145-infected cells (Supplementary Fig. 13D).Moreover, Kp52145 induced the loss of Cul-1 neddylation (Fig. 6I). This was dependent on ROS and NLRX1 because Kp52145-indcued loss of Cul-1 neddylation was not observed in cells treated with the ROS inhibitor DPI or in nlrx1 silenced cells (Supple- mentary Figs. 13E and Fig. 6J, respectively). As we anticipated, the neddylation of Cul-1 was not affected in cells infected with the vgrG4 mutant (Fig. 6K); complementation of the mutant resulted in the loss of Cul-1 neddylation (Fig. 6K). Altogether, these findings support the notion that VgrG4 causes a loss in Cul-1 neddylation in a NLRX1-ROS- dependent manner, thereby affecting the function of E3-SCFβ-TrCP. We next questioned how NLRX1-ROS triggers the loss of Cul-1 neddylation. ROS signals can be transduced via the transient oxidative inactivation of catalytic cysteine residues present in certain regulatory enzymes57. Inspection of the enzymes of the neddylation machinery revealed the presence of putative redox-sensitive residues in Ubc12. This enzyme catalyses the transfer of a thioesterified NEDD8 moiety from its own active site to the Lys720 of cullin substrates58, making then Ubc12 a potential target of VgrG4-induced ROS to abolish Cul1- ned- dylation. Providing initial support to this hypothesis, studies using Fig. 7 | K. pneumoniae targets the mitochondria via its T6SS to promote infection. Working model of K. pneumoniae manipulation of the mitochondria in epithelial cells by exploiting its T6SS. Kp52145 injects the trans-kingdom T6SS effector VgrG4 into epithelial in a T6SS-dependent manner. VgrG4 colocalises with the ER-mitochondria tethering protein mitofusin 2 (MFN2). The ER localization of VgrG4 results in the influx of calcium from the ER to themitochondria via theMCU channel. Mitochondria calcium induces the activation of Drp1 leading to the frag- mentation of the mitochondria network. Additionally, mitochondria calcium activates the mitochondria innate receptor NLRX1 to produce ROS. NLRX1-elicted ROS abrogates the NEDDylation of Cul-1, essential for the function of the ubiquitin ligase, E3-SCFβ−TrCP responsible for the ubiquitination of the IκBα after its phos- phorylation by the Iκκα/β kinase. The degradation of IκBα by the ubiquitin pro- teasome allows the nuclear translation of NF-κB to launch an antimicrobial programme. Therefore, by targeting the NEDDylation of Cul-1, VgrG4 subverts the activation of host defences. Created with BioRender.com. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 12 purified Ubc12 have demonstrated a NEDD8∼Ubc12 thioester form of Ubc12 is sensitive to oxidation59. We therefore evaluated the effects of K. pneumoniae on endogenous Ubc12. Immunoprecipitation experi- ments revealed the absence of Ubc12 modified with NEDD8 in Kp52145-infected cells (Fig. 6L). This was dependent on mtROS because the modification with NEDD8 was detected in infected cells pre-treated with mitoTEMPO (Fig. 6L). These data together with the published evidence indicate that VgrG4-induced NLRX1-mtROS abol- ishes the loading of Ubc12 with NEDD8 substrate. Collectively, this evidence establishes that VgrG4-induced NLRX1- mtROS inhibits the activation of NF-κB. Mechanistically, NLRX1-ROS abolishes the neddylation of Cul-1 by oxidative inactivation of Ubc12, theNEDD8-conjugating enzyme, limiting the activity the E3-SCFβ-TrCP and thus the ubiquitination of IκBα, which, consequently, it is not degraded by the ubiquitin proteasome. Discussion Here, we establish that bacteria utilize the T6SS to target the mito- chondria in yeast and mammalian cells. We demonstrate that K. pneumoniae exploits the trans-kingdom T6SS effector VgrG4 to frag- ment the mitochondria network in a Drp1-dependent manner (Fig. 7). Mechanistically, Drp1 activation results from VgrG4-triggered transfer of Ca2+ into the mitochondria from the ER (Fig. 7). Moreover, VgrG4- induced Ca2+ accumulation in the mitochondria also activates the innate receptor NLRX1 to produce mtROS. VgrG4-induced mtROS perturbs the ubiquitin ligase complex E3-SCFβ-TrCP to limit the activa- tion of inflammatory responses governed by NF-κB (Fig. 7). Collec- tively, our findings unveil a hitherto unknown anti-eukaryotic function of the T6SS, expanding substantially the roles of this secretion system. Furthermore, this work illustrates a new anti-immunology axis exploited by a human pathogen to govern the activation of inflam- matory responses. K. pneumoniae exemplifies the global threat posed by antibiotic resistant bacteria. K. pneumoniae-triggered pulmonary infection has a high mortality rate reaching 50% even with antimicrobial therapy and may approach 100% for patientswith alcoholismanddiabetes. There is a wealth of knowledge on how K. pneumoniae develops resistance to different antibiotics60, however we still lack a complete understanding of what makes K. pneumoniae a successful pathogen. Klebsiella does not encode type III or IV secretion systems known to deliver effectors into immune cells, or any of the toxins affecting cell biology, making then interesting to identify the factor(s) utilized by the pathogen to manipulate cells. Our work revealed VgrG4 as a new type of anti- immunology weapon. However, we cannot rule that Klebsiella may inject additional T6SS effectors into cells. Future studies arewarranted to elucidate whether Klebsiella exploits additional T6SS effectors to manipulate cells. Compared to the number of antibacterial T6SS effectors, there are few anti-eukaryotic effectors characterized9. Additionally, they do not share any structural characteristic or target similar pathways, reflecting the versatility of the T6SS, facilitating a panoply of interac- tions with microbes and eukaryotic cells. VgrG4 showed a puncta distribution in epithelial cells, consistent with the aggregation of a number of VgrG4 monomers. This is in agreement with the biochem- istry and structural data showing that VgrG proteins form at least tri- meric structures61 thatmay contain even higher number of monomers inside the cell. Given the effect on mitochondria dynamics, it was somewhat unexpected to find out that VgrG4 did not colocalise with the mitochondria. Instead, our results demonstrate VgrG4 colocalises with the ER-mitochondria tethering proteinsMdm34andMfn2 in yeast and epithelial cells, respectively. Truncation experiments established that VgrG4 RTD containing domain is responsible for the interaction with Mfn2 whereas the VgrG domain mediates the interaction with Mdm34. The lack of structural homology between Mfn2 and Mdm34 explains why the same domain of VgrG4 cannot be responsible for the binding to both proteins. To the best of our knowledge, there are no reports of any T6SS effector showing ER colocalisation, and of any bacterial effector interacting with Mfn2. The key molecular mechanism of VgrG4 action is the distribution of Ca2+ from the ER to the mitochondria. This Ca2+ accumulation acti- vates Drp1, which is a critical regulator of mitochondrial fission within cells. Interestingly, Ca2+ is one of the signalsmodulatingDrp1-mediated mitochondria fission in homeostasis62. Therefore, our results are con- sistent with the notion that VgrG4 affects a pathway governing mito- chondria dynamics. Interestingly, Drp1 is emerging as target of bacterial pathogens asHelicobacter pylori and Legionella pneumophila- inducedmitochondria fragmentation is Drp1-dependent63,64. However, this is not always the case because Listeria monocytogenes-induced fragmentation is Drp1-independent65,66. The fragmentation of the mitochondria is not associated to a perturbation of the mitochondria physiology or cell death in stark contrast to what it is observed in other pathogens63–65. The only phe- notype we found is the release of mtDNA to the cytosol that activates STING signalling to produce type I IFN and ISGs. We have recently uncovered the importance of type I IFN signalling in host defence against K. pneumoniae67. Nonetheless, the possibility exists that other STING controlled responses may be beneficial for the pathogen sur- vival. Future studies are warranted to characterize the role of STING signalling in K. pneumoniae infection biology. The accumulation of Ca2+ in the mitochondria also results in the activation of NLRX1 to produce mtROS. NLRX1 remains an enigmatic innate receptor. Only recently, the localisation of NLRX1 within the matrix of the mitochondria has been conclusively established49. There is conflicting data concerning NLRX1 function as a negative or positive regulator of inflammation68–71.Moreover, the ligandoractivating signal of NLRX1 upon infection remains unknown. There is only consensus on its role inROSproduction following infection69,72. Our evidence reveals a role for NLRX1 limiting inflammation following K. pneumoniae infection. Our results establish that the accumulation of Ca2+ in the mitochondria is sufficient to induce NLRX1-controlled mtROS. These findings leadus topropose thatNLRX1has evolved todetect the action of pathogens on mitochondria using the non-homeostatic accumula- tion of Ca2+as a signal. Future studies are warranted to confirm this hypothesis following the infection with other bacteria and viruses. Another novel finding of this work is that VgrG4-induced acti- vation of NLRX1 decreases the NEDDylation of Cul-1 in a ROS- dependent manner to limit the activation of NF-κB. There are no reports of T6SS effectors affecting theposttranslationalmodification of proteins, being VgrG4 one of the fewbacterial effectors doing so73. The NEDDylation of Cul-1 is essential for the function of the ubiquitin ligase complex E3-SCFβ-TrCP that controls the levels of proteins involved in host-pathogen interactions, making Cul-1 an attractive target to control cell biology. Further suggesting that Cul-1 is a prime Klebsiella target, we have recently demonstrated that K. pneumoniae induces the expression of the deneddylase CSN5 to limit the mod- ification of Cul-1 with NEDD874. Targeting the ubiquitin ligase com- plex E3-SCFβ-TrCP via Cul-1 is an efficient strategy to interfere simultaneously with a number of signalling pathways instead of tar- geting directly key proteins of each pathway as it has been described for pathogens such as Listeria, Salmonella, Legionella, E. coli or Shi- gella. This may explain the need for these pathogens to encode a portfolio of effectors to manipulate cell biology, being the extreme case Legionella encoding more than 300 effectors. In this work, we have only focused on the NF-κB signalling cascade given its crucial importance in defence against K. pneumoniae infections25,51–53. How- ever, we hypothesize a number of other pathways modulated by E3- SCFβ-TrCP will be out of balanced in VgrG4-intoxicated cells. Providing initial support, we have shown the β-catenin pathway, whose acti- vation is dependent on the E3-SCFβ-TrCP-controlled levels of β-catenin, is upregulated in Klebsiella-infected cells. Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 13 The manipulation of mitochondria to subvert inflammation via NLRX1 activation in a ROS-dependent manner is an underappreciated anti-immunology strategy. Previous work from our laboratory demonstrated that Klebsiella also exploits two other innate receptors, NOD1, and TLR4, to control cell responses52,53,74. Therefore, the theme taking shape is that a signatureofK. pneumoniae infection biology is to hijack the activation of innate immune receptors to counteract the activation of host antimicrobial pathways. This strategy is radically different to those employed by other pathogens such as Listeria, Sal- monella, Legionella and Shigella, who deploy bacterial proteins to prevent the activation of signalling upon sensing the infection. Host-directed therapeutics aiming to interfere with host factors required by pathogens to counter the immune system are emerging as untapped opportunities that are urgently needed in the face of the global pandemic of antibiotic resistant infections. Our results suggest that limiting Klebsiella-governed mitochondria manipulation should affect decisively the host-Klebsiella interface contributing to the clearance of the infection. Mitochondrial dysfunction also contributes to the pathology of many common disorders, including neurodegen- eration, metabolic disease, heart failure, and ischaemia–reperfusion injury. There are efforts to develop pharmacological approaches aimed at therapeutically restoring mitochondrial function, and a number of agents have entered clinical trials75. We propose that these drugs shall show a beneficial effect to treat K. pneumoniae infections alone or as a synergistic add-on to antibiotic treatment. Future studies shall confirm whether this is the case. Methods Saccharomyces cerevisiae strains and growth conditions The S. cerevisiae strains used in this study were YPH499 (MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1), diploid SLY001 for general purposes, andVHY87 (MATα leu2-3, 112 ura3-52 his4 canR TRP1::DsRed-HDEL), a gift of M. Cyert (Stanford University, CA, USA), for the visualization of the ER. The SLY001 diploid strain was obtained by crossing strains of opposite mating type BY4741 trp1Δ (MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 trp1Δ::kanMX4) and BY4742 trp1Δ (MATa his3Δ1 leu2Δ0 met15Δ0 trp1Δ ura3Δ0) (Supplementary Table 1). Strains deleted for DNM1 and FIS1 were obtained from the yeast whole genome deletion (WGD) collection (Euroscarf) on the BY4741 background. Plasmid YEplac112-Ilv6-mCherry for the visuali- zation of mitochondria was previously described76. YPD (1% yeast extract, 2% peptone and 2% glucose) broth or agar was the non-selective medium used for growing yeast. For plasmid selection and maintenance was used selective synthetic dextrose media (SD) (0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulphate, 2% glucose, 0.12% drop-out supplements mix- ture andwas supplementwith the appropriate amino acids and nucleic acid bases). SR and SG media were SD with 1.5% (w/v) raffinose or 2% (w/v) galactose insteadof glucose. All galactose induction experiments were performed by growing transformants in SR liquid medium for 18 h at 30 °C to log-phase and then suspended in SGmedium during 4- 6 hours at 30 °C. Molecular techniques and construction of yeast expression plasmids Transformation of yeast by the lithium acetate and E. coli associated molecular techniques were performed by standard procedures. Invitrogen Gateway Cloning (Thermofisher) was used to fuse the hemagglutinin (HA) tag protein or red fluorescent protein (DsRed) to ERMES proteins Mdm34 and Mmm1. MDM34 and MMM1 entry clones in pDONR221 (a gift from Elba del Val and Julia María Coronas- Serna) were subcloned into a pAG413GPD-ccdB-HA or pAG424-GAL- ccdB-DsRed destination vectors by LR recombination reaction (Addgene kit #1000000011) to obtain the expression clone pAG413- GPD-Mdm34-HA, or the plasmids pAG424-GAL-Mdm34-DsRed and pAG424-GAL-Mmm1-DsRed. pEG(KG)-VgrG4 expressing GST fusions of VgrG4 and truncated versions were described before12. pEG(KG)- GST-VgrG4ΔRTD was constructed by overlap extension PCR, amplify- ing separately the vgrG4N-terminal-coding part and the one encoding the 837-899 extension. The fused amplicon bearing XbaI sites intro- duced in the 5’ region of the primers was cloned into the XbaI-cleaved pEG(KG) expression vector in frame with GST. Primers used for amplifying the N-terminal extension are UP-1-157XbaI and LO 1-517overlap838. The ones used for amplifying the 837-899 extension are UP-838overlap517 and LO-838-899XbaI (listed in Supplementary Table 2). pYES2-GFP-VgrG4 and pYES2-GFP-VgrG4(518-C) were con- structed by PCR amplification of vgrG4 or the region starting in codon 518, respectively, by using primers bearing BglII sites in 5’ and ligating the amplicons into BamHI-cleaved pYES2-GFP77 Primers used were UP- VgrG4-FL or UP-VgrG4-518, respectively, and LO-VgrG4 (Supplemen- tary Table 2). pYES2-GFP-VgrG4(1-517) was constructed by substituting the codon 518 in the vgrG4 sequenceby a stop codonusingDpnI-based site directedmutagenesis with the pYES2-GFP-VgrG4as a template and primers (1-517)−1 and (1-517) (Supplementary Table 2). Evaluation of oxidative damage in yeast cells ROS generation was tested by flow cytometry. Cells were cultured in SR medium lacking uracil and leucine overnight. Then, cultures were suspended in SG medium during 16 hours at 30 °C. As a positive con- trol, oxidative damage was induced by adding 125mM acetic acid during 4 hours after 12 hours of galactose induction at one of the control samples. After that, 1mL of each culture was treated with 2,5μg/mLof dihydroethidiumduring 5minutes at 30 °Cunder stirring. The samples were diluted 1:10 in PBS. Cells were analysed using a 419 FACScan (Becton Dickinson) flow cytometer through a 488 nm exci- tation laser and a 585/42 BP emission filter (FL2). At least 10,000 cells were analyzed for each experiment. Data was processed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Microscopy in yeast cells For fluorescence microscopy to visualise GFP- and DsRed-expressing cells, transformants were cultured in SRmedium for 18 hours at 30 °C, then, cells were suspended into SG medium and incubated for 4- 6 hours for GAL1 promoter induction. Finally, cells were harvested by centrifugation and observed directly. Cells were examined under Eclipse TE2000Umicroscope (Nikon, Tokyo, Japan) and digital images were acquired with an Orca C4742-95-12ER charge-coupled-device camera (Hamamatsu Photonics, Hamamatsu City, Japan) and pro- cessed by HC Image (Hamamatsu). GST pull-down in yeast cells Yeast cell cultures were grown in SR medium lacking uracil, leucine and histidine for 18 hours at 30 °C, then the appropriate number of cells was suspended in 100mL of SG medium for 5-6 h to reach exponential phase and GAL1 promoter induction. Standard proce- dures were used for collection and breakage. Cells were spun and lysed in 300 µL of ice-cold lysis buffer [10% v/v glycerol, 50mMTris/ HCl pH 7.5, 0.1% NP40, 150mM NaCl, 5 mM EDTA pH 8, 50mM NaF, 5mM sodium pyrophosphate, 50mM beta-glycerol phosphate, 1 mM sodium vanadate supplemented with PMSF up to 1mM and protease inhibitor cocktail (Roche/Sigma-Aldrich)]. Then, the extracts were diluted if necessary to balance protein concentration, and 10 µL were preserved as “inputs” at −80 °C overnight. To equi- librate the beads, three washes were performed with lysis buffer, and then 50 µL of glutathione sepharose 4B beads (GE Healthcare Life Sciences) diluted at 50% were added to 200 µL of each extract. After an overnight incubation with the beads at 4 °C, samples were centrifuged, the supernatant was discarded, and beadswerewashed 6 times with lysis buffer. Finally, they were suspended in 25 µL of SDS‐PAGE loading buffer (1 M Tris/HCl pH 6.8, 10% SDS, glycerol, Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 14 1 M DTT and bromophenol blue). 10 µL of the original lysates were preserved as “inputs” at -80 °C overnight and then suspended in 10 µL of the same SDS-PAGE loading buffer. Cells were boiled at 99 °C during 5min. In the gels, 10 µL of each input and 20 µL of the samples after affinity purification were loaded. Standard proce- dures were used for protein separation by SDS-PAGE, and transfer to nitrocellulose membranes. Anti-HAantibody (Anti-HAHighAffinity, Ratmonoclonal antibody (clone 3F10), Sigma-Aldrich) diluted 1:1000 was used to detect both HA-tagged ERMES proteins Mdm34. GST fusion proteins were detec- ted with anti-GST antibody (Anti-GST rabbit polyclonal Antibody (Z-5): sc-459 (Santa Cruz Biotechnology) diluted 1:5000. Primary antibodies were detected using IRDye800 anti-rat (Li-Cor Biosciences) or Alexa- 680 anti-mouse (Invitrogen) antibodies with an Odyssey Infrared Imaging System (Li-Cor Biosciences). Cell culture Lung epithelial A549 cells (ATCC CCL-185) were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco 21875) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10mM HEPES (Sigma), 100 U mL-1 penicillin, and 0.1 mg mL-1 streptomycin (Gibco) at 37 °C in a humidified 5% CO2 incubator. Human airway epithelial cells NuLi-1 (ATCC CRL-4011) were grown in PromoCell Small Airway Epithelial Cell Growth Medium (Promocell, C-21070) at 37 °C in a humidified 5% CO2 incubator. Flasks and plates were coated with collagen type IV from placenta (Sigma C-7521). Cells were routinely tested for Mycoplasma contamination. Bacterial strains and infection conditions Bacterial strains and plasmids used in this study are shown in Sup- plementary Table 2. Kp52145 is a clinical isolate, serotype O1:K2, belonging to the virulent CC65 clonal complex. When appropriate media were supplemented with antibiotics at the following con- centrations: chloramphenicol (Cm) 25μg/mL, carbenicillin (Cb) 50μg/mL, spectinomycin (spec) 50μg/mL. K. pneumoniae strainswere grown in5mLLB at 37 °C, harvested at mid exponential phase (2500 × g, 20min) and adjusted to an optical density of 1.0 at 600nm in PBS (5 × 108 CFU/mL). All the strains con- taining a pBAD30 plasmidwere induced for 30min with 0.05% (v/v) of arabinose prior to harvesting. Klebsiella infections were performed using a multiplicity of infection (MOI) of 100 bacteria per cell. Y. enterocolitica strains were grown overnight in 5mL LB at 21 °C, refreshed 1:10 (v/v) in Tryptic Soy Broth (TSB) media supplemented with 20mMmagnesium chloride and 20mM sodium oxalate for 3h30 at 21 °C followed by 30min at 37 °C. Infections were performed using a MOI of 50 bacteria per cell andmedia was replaced at 30minutes post infection for media containing gentamicin (100 µg/mL) to kill any extracellular bacteria. Infectionswere performed in the samemedia,without antibiotics, used tomaintain the cell line and incubated at 37 °C in a humidified 5% CO2 incubator. Infections of A549 epithelial cells were performed two days after seeding including a serum starvation step 16 h before infection using RPMI 1640 Medium (Gibco 21875) supplemented only with 10mMHEPES. Infections of NuLi-1 cells were performed two days after seeding. Construction of VgrG4 tagged with GSK3β To allow the study of vgrG4 translocation by Kp52145 into epithelial cells, the vgrG4 gene was fused with a GSK3β tag that can only be phosphorylated by mammalian cells. The tag was generated by hybridisation of the primers GSK3b_F1 and GSK3b_R1 (Supplemen- tary Table 2). The product of this PCR was cloned into SmaI site of pBAD30 and transformed into E. coli β2163, and then mobilized into K. pneumoniae by conjugation. Construction of a Y. enterocolitica strain delivering VgrG4 in a T3SS-dependent manner To allow the study of vgrG4 translocation into cells we used a Y. enterocolitica strain thatdelivers VgrG4 via the YscT3SSbecause vgrG4 is fused to the N-terminal secretion/translocation domain of YopE (the first 53 amino acids of this protein)32. This strain carries a mini- virulence plasmid (pT3SS), encoding the genes for a functional T3SS, the gene for the Yersinia adhesin (YadA) and a second plasmid con- taining the T3SS substrate YopE and the cognate chaperone SycE32. This strains does not encode any other T3SS effector32. The coding regionof vgrG4was amplifiedusingprimersKpn52_vgrG4_VSVG_F1 and Kpn52_vgrG4_VSVG_R1 and digested with BglII and XhoI. Fragments were gel purified and subcloned into the vector pYOPE53 previously digestedwith BamHI and SalI. The plasmidwas transformed into E. coli β2163, and then mobilized into Y. enterocolitica by conjugation. VgrG4 truncated versions were generated using the primers Kpn52_VgrG4_VSVG_F1 and Kpn52_VgrG4_VSVG_R2 for the N-terminal version (1-517) or Kpn52_VgrG4_518-C and Kpn52_VgrG4_VSVG_R1 for the C-terminal (518-C). Primer sequences are listed in Supplementary table 1. Transfection conditions Transfection of A549 cells with siRNAs or plasmids was carried out using Lipofectamine 2000 (Invitrogen) lipofection reagent according to manufacturer’s instructions. For transfection of siRNAs, 1.2 × 105 cells (12-well plate) were transfected in suspension with 20 nM siRNA using 2μL of Lipofectamine 2000 in a final volume of 1mL. For transfection of the MFN2-YFP plasmid, 3.0 × 105 cells (6-well plate for immunoprecipitation) or 5.0 × 104 cells (24-well plate for microscopy) were transfected the day after seeding with 2 µg (6-well) or 0.5 µg (24- well) using 6μL or 1 µL of Lipofectamine 2000 in a final volume of 2mL or 0.5mL, respectively. MFN2-YFP was a gift from Richard Youle (Addgene plasmid # 2801078). Infections were carried 48 h post transfection. All siRNA duplexes used for in vitro studies were chemically syn- thesised by Dharmacon (Horizon Discovery Group). The following siRNAs sense sequences were used: hMCU (ON-TARGETplus Human MCU, Dharmacon, J-015519-18), hDrp1 (5′-ACAAGUCGGCGGUGGAG UA-3′), hNLRX1 (ON-TARGETplus Human NLRX1 SMARTPool, Dhar- macon, L-012926-01), hVAPB (5’-UGUUACAGCCUUUCGAUUAdTd T-3’), hMFN2 (5’- GACUAUAAGCUGCGAAUUAdTdT-3’). An AllStars (AS) Negative Control scrambled siRNA (Qiagen) with no homology to any knownmammalian gene was used as a negative control. Efficiency of transfection was confirmed by qPCR analysis of duplicate samples from three independent transfections by normalising to the glycer- aldehyde 3-phosphate dehydrogenase (hGAPDH) gene and comparing gene expression in the knockdown samplewith the luciferase negative control. Primers used are listed in Supplementary Table 1. The per- centage of transcript remaining after transfection is presented in (Supplementary Fig. 2). Immunoblot analysis A549 were seeded in twelve-well plates (1.2 × 105 cells per well) and grown for 48 h prior to infection. Cells were infected with K. pneu- moniae or Y. enterocolitica strains for different time points as indicated in the figure legends. Cells were then washed in 1mL of ice-cold PBS and lysed in 80μL of 2 x SDS sample buffer (1x SDS Sample Buffer, 62.5mMTris-HCl pH 6.8, 2% w/v SDS, 10% glycerol, 50mMDTT, 0.01% w/v bromophenol blue). The cell lysates were sonicated for 10 seconds at 10% amplitude (Branson Sonifier), boiled at 95 °C for 5minutes and centrifuged at 12,000 × g for 1min. 20% of the cell lysates were resolved by standard 8 or 12% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 4% bovine serum albumine (w/v) in TBST and protein bands were detected with Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 15 specific antibodies using chemiluminescence reagents and a G:BOX Chemi XRQ chemiluminescence imager (Syngene). The following antibodies were used: anti-VSVG (1:1000, Sigma V4888), anti-phospho-GSK3β (1:1000, Cell Signaling Technology (CST) 5558), anti-GSK3β tag (1:1000, CST 9325), anti-GSK3β (1:1000, Santa Cruz Biotechnology sc-67075), anti-phospho-DRP1 (Ser616) (1:1000, CST 4494), anti Drp1 (1:1000 CST 8570), anti-Cullin-1 (1:200, Santa Cruz Biotechnology sc-12761), anti-NEDD8 (1:1000, Invitrogen 34- 1400), anti-Ubc12 (1:1000, Santa Cruz Biotechnology sc-366017), anti- β-catenin (1:1000, Santa Cruz Biotechnology sc-7199), anti-K48 Poly- ubiquitin (1:1000, CST 4289), anti-IκBα (1:1000, CST 4814), anti- phospho-Iκκαβ (1:1000, CST 2697), anti-phospho-IκBα (1:1000, Santa Cruz Biotechnology sc-7977), anti-GFP (1:5000, Proteintech 66002), anti-Tom20 (1:500, Santa Cruz Biotechnology F-10 sc-17764), anti- calnexin (1:1000, Santa Cruz Biotechnology AF-18 sc-23954), anti-E.coli RNA polymerase α (1:5000, BioLegend 663102). Immunoreactive bands were visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins (1:5000, BioRad 170-6515), goat anti-mouse immunoglobulins (1:5000, BioRad 170-6516) or mouse anti-goat immunoglobulins (1:5000, Santa Cruz Biotechnology sc-2354). To detect multiple proteins, membranes were reprobed after stripping of previously used antibodies using a pH 2.2 glycine-HCl/SDS buffer. To ensure that equal amounts of proteins were loaded, blots were reprobed withmouse anti-human tubulin (1:3000, Sigma T5168). Densitometry analysis Drp1 blots were quantified using Image Studio Lite version 5.2 (Li-cor) and normalized to the loading control. Graphs represent fold change compared to non-infected cells set to 100. Co-immunoprecipitation analysis Cells were seeded (3 × 105 cells per well) in six-well plates and twowells were used per sample. At the designated time points, cells were washed with pre-chilled PBS (1mL) and then lysed with 500μL of pre- chilled RIPA buffer (1% [w/v] Triton X-100, 1% [w/v] Sodium Deox- ycholate, 0.1% [w/v] SDS, 0.15M NaCl, 50mM Tris-HCl, pH 7.2, 1mM PMSF and Halt Protease Inhibitor Cocktail (Thermo Scientific)) for 30min on ice. Lysateswere centrifuged at 12,000 × g for 15min at 4 °C. Supernatants were transferred to pre-chilled tubes and incubated for 2 h with the appropriate antibody (1μg) at 4 °C with rocking. Normal mouse IgG (Santa Cruz Biotechnology, sc-2025) was used as the anti- body binding control. This was followed by the addition of protein A/G agarose beads (20μL per sample; Santa Cruz Biotechnology, sc-2003) and incubation overnight at 4 °C with rocking. Immunoprecipitates were collected by centrifugation at 1000 × g for 2min at 4 °C and the beads were then washed two times with RIPA buffer (500μL) lacking the protease inhibitor mixture. The beads were resuspended in 2x sample buffer (40 μL) and boiled for 5min at 95 °C. Samples were centrifuged at 12,000× g for 1min and subjected to immunoblotting. The immunoreactive bands were detected using HRP conjugated TidyBlot (BioRad, 1:1000). Cell fractionation Epithelial cells were seeded in a 10 cm dish, at a cell density of 1 × 106 cells per dish, and two dishes were used per sample. Cells were infected as described previously for 90min with Y. enterocolitica strains. To isolatemitochondria fromendoplasmic reticulum fractions a protocol was adapted from Wieckowski and colleagues79. Briefly, cells were washed with PBS, scrapped and homogenised in 2mL of IB buffer [225mM mannitol, 75mM sucrose, 0.1mM EDTA and 30mM Tris-HCl (pH 7.4)] using a pestle. Lysates were centrifuged at 600× g for 5min at 4 °C. Supernatants were transferred to pre-chilled tubes and centrifuged at 7000 × g for 10min at 4 °C. The resulting pellets were resuspended in 1mLof buffer IBwithout EDTAand centrifuged at 7000× g for 10min at 4 °C (resulting pellet is the mitochondrial frac- tion). The supernatants were centrifuged at 20,000 × g for 60min at 4 °C to isolate the ER fraction (resulting pellet). Fluorescence dye-based detection of ROS Epithelial cells were seeded in a 96-well black clear bottom plate, at a cell density of 2 × 104 cells per well. Cells were infected as described previously for 5 h with K. pneumoniae strains or for 90min with Y. enterocolitica strains. Cells were treated with 50 µM 2′,7′-Dichloro- fluorescein (Sigma, 35848) for 30min, washed twice with pre-warmed Hanks’ Balanced Salt Solution (HBSS) and fluorescence was measured in a POLARStar Omega BMG LabTech plate reader at Excitation/ Emission of 485/520nm. All the experiments were carried out in tri- plicate wells in three independent occasions. ATP determination A549 cells were seeded in 12 well plates at a density of 1 × 105/well two days prior to infection. Cells were starved for 16 h using RPMI 1640 Medium supplemented only with 10mMHEPES and changed tomedia without antibiotics prior to infection. Cells were infected with K. pneumoniae for 5 h, and with Y. enterocolitica strains for 2 h. Cells were lysed in0.5% (w/v) saponin in PBS and20%of the cell lysatewasused to determine ATP concentration using an ATP Determination Kit (Invi- trogen, A22066) following manufacturer’s instructions and measured in a Promega Glomax with an integration time of 0.1 seconds. Mitochondrial membrane potential determination Epithelial cells were seeded in a 96-well black clear bottom plate, at a cell density of 2 × 104 cells per well. Cells were infected as described previously for 3 h with K. pneumoniae strains or 1h30 with Y. enter- ocolitica strains. Mitochondrial Membrane Potential was assessed with a TMRM assay kit (Abcam, ab228569) following manufacturer’s instructions. Briefly TMRM was added to the culture media and incu- bated for 30minutes afterwhichcellswerewashed in0.2% (w/v) BSA in PBS, bathed in LiveCell ImagingBuffer andfluorescencewasmeasured in a POLARStar Omega BMG LabTech plate reader at an Excitation/ Emission of 544/590nm. Seahorse analysis A549 cells were seeded in Seahorse XF Cell Culture Microplates at a density of 2 × 104 cells per well two days prior to infection. Cells were starved for 16 h using RPMI 1640 Medium supplemented only with 10mM HEPES and changed to media without antibiotics prior to infection. Seahorse cartridge was hydrated overnight in Seahorse XF Calibrant solution at 37 °C overnight in a non-CO2 incubator. Infection was performed at a 100:1M.O.I. and after 3 h contact, cells were washed and media replaced with XF culture media (pH 7.4 supple- mented with 1mM pyruvate, 2mM glutamine, 10mM glucose and 30 µg/mL gentamicin to remove extracellular bacteria). Metabolic activity of cells was assessed using the Seahorse Mito Stress Test Kit (Ref: 103015-100, Agilent) according to manufacturer’s instructions and experiment analysed with the Seahorse XFe96 analyser. 8 wells were used per condition across 3 independent experiments. Determination of cytosolic mitochondrial DNA A549 or NuLi-1 cells were grown as monolayers in 10 cm dishes (1.0 × 106 cells), infected with Y. enterocolitica strains for 2 hours after which cells were washed in ice-cold PBS, scraped from culture dishes on ice using a plastic cell scraper and collected in micro-centrifuge tubes in 1mL of ice-cold PBS. After centrifugation at maximum speed for 10 seconds, cell pelletswere resuspended in900μLof ice-cold0.1% NP40 in PBS to lyse the cells. One third of this cytosolic fraction was then submitted to a PCR clean up kit (Qiagen) and the resulting DNA was submitted to qPCR for mitochondrial cytochrome oxidase 1 (mtCO1) using ashousekeeping gene humanDNApolymeraseβ (Polβ). Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 16 Mitochondria calcium detection Epithelial cells were seeded in a 96-well black clear bottom plate, at a cell density of 2 × 104 cells per well two days prior to infection. Cells were starved for 16 h using RPMI 1640 Medium supplemented only with 10mM HEPES and changed to media without antibiotics prior to infection. Cells were infected for 30minutes with Y. enterocolitica strains after which media was replaced with HBSS supplemented with 100 µg/mL gentamicin to remove any extracellular bacteria and with 50 µMRhod2-AM (Sigma, catalogue number 35848). Fluorescence was measured in a POLARStar Omega BMG LabTech plate reader at an Excitation/Emission of 544/590nm at 20minutes interval for 2 hours. All the experiments were carried out in triplicate wells in three inde- pendent occasions. Mitochondrial ROS detection Epithelial cells were seeded in a 96-well black clear bottom plate, at a cell density of 2 × 104 cells per well. Cells were treated with 50 µM MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, M36008) for 30minutes and were replenished with fresh antibiotic free media. Cells were then infected as described previously for 5 h with K. pneumoniae strains or for 1h30 with Y. enterocolitica strains. After infection cells were washed twice with pre-warmed Hanks’ Balanced Salt Solution (HBSS) and fluorescence was measured in a POLARStar Omega BMG LabTech plate reader at Excitation/Emission of 544/590 nm. All the experiments were carried out in triplicate wells in three independent occasions. Cell viability The thiazolyl blue tetrazolium bromide (MTT) assay was used to determine cell viability. Cells were seeded at a concentration of 1 × 105 cells/mL in 96-well plates and infected under normal conditions. After 3 h, 10μL ofMTT (5mg/mL in PBS) was added and the plates incubated at 37 oC in a humidified incubator for 3 hours.Media was removed from all wells and replaced with 100 µL DMSO to dissolve any dye crystals, and incubated for 30minutes at 37 oC in a humidified incubator. Stainingwas thenquantifiedbydetermining theOD570 in aplate reader. Generation of the A549 mCherryER cell line To generate a stable cell line with fluorescently-labelled ER, the mCherry gene including KDEL ER-targeting sequence and Calreticulin ER-retention signal was amplified by PCR (5’-CTAGGGATCCGCC ACCATGCTGCTATCCG-3’; 5’-TCTACTCGAGTTACAGCTCATCCTTCTT GTACAGCTCGTCCATGC-3’) from pmCherryER/ pICC158480 and inserted into the retroviral expression vector pMXs-IRES-Puro using standard restriction enzyme (BamHI, XhoI) cloning. Attenuated retro- virus for transduction was produced as described previously (PMID: 26216420). Briefly, Hek293E cells were transfected with pMXs-IP and the pCMV-VSV-G envelope and pCMV-MMLV-gag-pol packaging plas- mids, the medium replaced with fresh growth medium after 24 h and cell supernatant containing virions harvested after another 24 h of incubation. A549 cells were infected for 24 h, washed and growth medium containing 1.5 µg/mL puromycin added. After 7 days of selection including media exchange to remove dead cells and visual confirmation of mCherry fluorescence throughout the culture, the attached cells were harvested, and stocks frozen and used for experi- ments without further selection. Microscopy A549 orNuLi-1 cells were seeded (5 × 104 cells perwell) in 24-well plates with sterile round glass cover slips, grown for 24 h to 70-80% con- fluence and starved for 18 h. Cells were pre-incubated with 50 µM Mitotracker Red FM (Invitrogen, M22425) for 30minutes and media was replaced to antibiotic free medium prior to infection. After incu- bation with either bacterial strains or PBS (control) for the indicated timepoints in figure legends,mediumwas removed, and the cells were gently washed twice with PBS. Cells were then fixed by the addition of 4% (w/v) paraformaldehyde for 20min. Cells were washed twice with PBS and kept at 4 °C in PBS supplemented with 1mM NH4Cl until staining. Nuclei were stained with Hoechst 33342 (1.5μg/mL; Sigma) for 30minutes, washed with PBS and were mounted with ProLong Gold Antifade Mountant (Molecular Probes Inc). Fluores- cence images were captured using the ×100 objective lens on a Leica SP8 laser scanning confocal microscope equipped with the appropriate filter sets. Acquired images were analysed using the LAS imaging software (version 3.7, Leica). Quantitative analysis of mito- chondrial morphology was performed using the plugin for ImageJ Mitochondria Analyzer in 20 images for each condition from at least three independent experiments (https://github.com/AhsenChaudhry/ Mitochondria-Analyzer)31. Analysis of VSV-G co-localisation was performed using anti-VSVG (1:400, Sigma V4888) or anti-Flag (in the case of Kp52145 co-localisa- tion) (1:5000, Sigma F3165) stained for 1 hour followed by Alexa Fluor 647 (1:400, Life-tech A21244), Alexa Fluor 488 (1:400, Life-tech A21206) or Alexa Fluor 488 (1:400, Abcam ab150117). Analysis of p65 translocation was determined in cells fixed as above and stainedwith p65 antibody (1:400, SantaCruz Biotechnology sc-372) using Alexa Fluor 488 (1:400, Life-tech A21206) with fluores- cence images acquired in a Leica DM5500 microscope using the ×64 objective lens. Transmission electron microscope A549 cells were seeded (3 × 105 cells per well) in 6-well plates, grown for 24 h to 70–80% confluence and starved for 18 h. Media was replaced to antibiotic free medium prior to infection and after incubation with either Kp52145 or PBS (non-infected control) for 3 hours, medium was removed, and the cells were gently washed twice with PBS. Cells were then fixed by the addition of 2.5% (v/v) glutaraldehyde + 1.5% (v/v) paraformaldehyde in PHEM buffer (Elec- tron Microscopy Sciences, catalogue number 11165) for 1 hour at room temperature. Cells were them scraped and collected into a microcentrifuge tube. Cell suspension was pelleted at 800 g for 15minutes and embedded in agarose (2%, w/v, in PHEM buffer). Cells were then post fixed with 2% osmium tetroxide (v/v in PHEM) and immersed in Spurr replacement (Low viscosity resin kit, TAAB, T262) and polymerised at 60 °C for 48 hours. The block was trimmed, thin sectioned (90–100 nm thick) and then applied to copper grids and air dried. The grid was loaded in a JEOL1400 transmission electron microscopy (Jeol). At least 10 randomly selected electron micro- graphs from each sample were examined. RNA extraction and quantitative real‐time PCR analysis Cells were washed twice in PBS and RNA was extracted using TRIzol Reagent (Ambion) according to the manufacturer’s instructions. Duplicate cDNA preparations from each sample were generated from 1μg of RNA using Moloney murine leukaemia virus (M‐MLV) reverse transcriptase (Sigma‐Aldrich) according to the manufacturer’s instructions. Quantitative real‐time PCR analysis of gene expression was undertaken using the KAPA SYBR FAST qPCRKit and a Rotor-Gene Q real-time PCR cycler System (Qiagen). Thermal cycling conditions were as follows: 95 °C for 3min for enzyme activation, 40 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 20 s. Primers used in qPCR reactions are listed in Supplementary Table 1. cDNA samples were tested in duplicate and relative mRNA quantity was determined by the comparative threshold cycle (ΔΔCT) method, using glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) gene normalisation. Quantification of cytokines Infectionswereperformed in twelve-well plates (1.2 × 105 cells perwell) using Kp52145 at a MOI of 100:1. After 3 hours of infection media was Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 17 https://github.com/AhsenChaudhry/Mitochondria-Analyzer https://github.com/AhsenChaudhry/Mitochondria-Analyzer replaced by media containing gentamicin to clear any extracellular bacteria and supernatants from infected cells were collected after 16 h and spun down at 12,000 × g for 5min to remove any debris. IL-8 in the supernatants was determined using a Human IL-8 Standard TMB ELISA Development Kit (PeproTech, catalogue number 900-T18), according to manufacturer’s instructions. Experiments were run in duplicate and repeated at least three times. Statistical analysis Statistical analyses were performed using ANOVA, or when the requirements were not met, by unpaired t‐test. Multiple comparison wasperformedusingHolm-Sidak’smultiple comparisons test. P‐values of <0.05 were considered statistically significant. 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Research at the V.J.C and M.M. lab was funded by Grant PID2019-105342GB-I00 from Ministerio de Ciencia e Innova- ción (Spain) and by Grant S2017/BMD-3691-InGEMICS-CM, funded by Comunidad de Madrid and European Structural and Investment Funds. S.L-M. was recipient of predoctoral contracts by Universidad Complu- tense de Madrid (UCM) (Spain) and PEJD-2019-PRE/BMD-16928 by Comunidad de Madrid. This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC, BB/T001976/1, BB/ P020194/1, BB/N00700X/1, BB/V007939/1) to J.A.B. Author contributions Conceptualization, J.S-P., M.M., V.J.C. and J.A.B.; Methodology, J.S-P., S.L.-M., K.P., G.N.S., P.B., T.C. and I. R.-E.; Investigation, J.S-P., S.L.-M., K.P., H.M., A.O., and P.B.; Resources, G.N.S.; Writing-Original draft, J.S- P., S.L.-M., G.N.S., M.M., V.J.C. and J.A.B.; Writing-Review & Editing, J.S- P., G.N.S., M.M., V.J.C., T.C. and J.A.B.; Supervision, M.M., V.J.C. and J.A.B; Funding acquisition, M.M., V.J.C. and J.A.B. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-36629-3. Correspondence and requests for materials should be addressed to José A. Bengoechea. Peer review information Nature Communications thanks the anon- ymous reviewers for their contribution to the peer review of this work. Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2023 Article https://doi.org/10.1038/s41467-023-36629-3 Nature Communications | (2023) 14:871 20 https://doi.org/10.1038/s41467-023-36629-3 http://www.nature.com/reprints http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection Results K. pneumoniae VgrG4 RTD domain induces oxidative stress and mitochondrial condensation in Saccharomyces cerevisiae VgrG4 colocalises with the yeast ERMES component Mdm34 through its VgrG domain VgrG4 is translocated into human lung epithelial cells and induces mitochondria fragmentation VgrG4-induced mitochondrial fragmentation is Drp1-dependent VgrG4-induced mitochondrial fragmentation does not impair mitochondria physiology VgrG4 colocalises with the ER protein mitofusin 2 VgrG4 triggers the transfer of Ca2+ to the mitochondria from the ER VgrG4-induced Ca2+ accumulation in the mitochondria mediates the fragmentation of the mitochondria network VgrG4 induces mitochondria ROS upon activation of the innate receptor NLRX1 NLRX1 elicited ROS mediates cullin 1 deNEDDylation to modulate epithelial signalling Discussion Methods Saccharomyces cerevisiae strains and growth conditions Molecular techniques and construction of yeast expression plasmids Evaluation of oxidative damage in yeast cells Microscopy in yeast cells GST pull-down in yeast cells Cell culture Bacterial strains and infection conditions Construction of VgrG4 tagged with GSK3β Construction of a Y. enterocolitica strain delivering VgrG4 in a T3SS-dependent manner Transfection conditions Immunoblot analysis Densitometry analysis Co-immunoprecipitation analysis Cell fractionation Fluorescence dye-based detection of ROS ATP determination Mitochondrial membrane potential determination Seahorse analysis Determination of cytosolic mitochondrial DNA Mitochondria calcium detection Mitochondrial ROS detection Cell viability Generation of the A549 mCherryER cell line Microscopy Transmission electron microscope RNA extraction and quantitative real‐time PCR analysis Quantification of cytokines Statistical analysis Reporting summary Data availability References Acknowledgements Author contributions Competing interests Additional information