Orf1/SpcS Chaperones ExoS for Type Three Secretion by Pseudomonas aeruginosa

Objective Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type Ⅲ secretion system （TTSS） to inject effector proteins directly into the cytosol of target cells to subvert the host cell＇s functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P aeruginosa, we analyzed the role of a postulated chaperone termed Orfl. Methods By allelic exchange, we constructed the mutant with the deletion of gene Orfl. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orfl, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF The role of Orfl in the expression of exoS was evaluated by gene reporter analysis. Results Pull-down assay showed that Orfl binds to ExoS and ExoT. Secretion profile analysis showed that Orfl was necessary for the optimal secretion of ExoS and ExoT. However, Orfl had no effect on the expression of exoS. Conclusion Orfl is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orfl as SpcS for ＂specific Pseudomonas chaperone for ExoS＂.

Molecular Cloning,Bioinformatics Analysis and Expression Analysis of Type III Secretion System(T3SS)Injectisome Gene vscX from Vibrio alginolyticus

In order to enrich the research about type III secretion system （T3SS） injectisome of Vibrio alginolyticus, vscX gene was cloned from E algianlyticus strain HY9901 for bioinformatics analysis and expression analysis. Specific primers were designed according to the full-length geanme sequence of V. alginolyticus in GenBank. vscX gene （C, enBank accession number： FR780679） contained a 378 bp open reading frame （ORF）, encoding a putative protein of 125 amino acids. The theoretical molecular weight was 14.209 2 kD and theoretical pI was 5.75. By using Signal 4.1 Server and TMHMM Server 2.0, it was predicted that VscX protein had no transmembrane domain or signal peptide. The results of prediction using SoftBerry-Psite software showed that VscX protein contained three casein ki- nase lI phosphorylation sites, one N-myristoylation site and three C-terminal targeting signal sites. Subcellular localization revealed that VscX might be located in cytoplasm with the possibility of 56.5%. According to SMART prediction, VscX had one Pfam （ 1 - 125 aa） domain. Phylogenetic analysis revealed that VscX from V. alginolyticus and VscX from E parahemolyticus were clustered into the same group. Network interaction analysis showed that vscX was adjacent to vseY, vopB and sycN. By real-time fluorescent quantitative PCR technique and 2-△△△ method, the differences in expression levels of VscX mRNA in V. alginolyticus strain HY9901, T3SS deletion strain AvscO and complementary strain C-vscO at different growth stages were analyzed. The results showed that the expression levels of VscX mRNA in V. algianlyticus strain HY9901 and C-vscO were significantly up-regulated at stable growth stage （ P 〈0.01 ） ; the expression levels of VscX mRNA in deletion strain △vscO were significantly up-regulated at late growth stage （ P 〈0.01 ）. This study provided the basis for revealing the transport mechanism of T3SS injectisome of E alginolyticus.

T4SP: A Novel Tool and Database for Type IV Secretion Systems in Bacterial Genomes

Secretion systems, macromolecules to pass which can mediate the across cellular membranes, are essential for virulent and genetic material exchange among bacterial species[1]. Type IV secretion system （T4SS） is one of the secretion systems and it usually consists of 12 genes： VirB1, VirB2 ...VirB11, and VirD4[2]. The structure and molecular mechanisms of these genes have been well analyzed in Gram-negative strains[3] and Gram-positive strains were once believed to be lack of T4SS. However, some recent studies revealed that one or more virB/D genes also exist in some kinds of Gram-positive bacteria and play similar role, and form a T4SS-like system[3]. The VirBl-like, VirB4, VirB6, and VirD4 genes were identified in the chromosome of Gram-positive bacterium Streptococcus suis in our previous studies and their role as important mobile elements for horizontal transfer to recipients in an 89 K pathogenicity island （PAl） was demonstrated[45]. However, their structure and molecular mechanisms in other strains, especially in Gram-positive strains, are remained unclear.

Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran:A descriptive-analytical study

Objective:To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection.Methods:In this study,100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational-therapeutic hospitals and were identified using standard microbiological tests.Then,the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method.The bacterial DNAs were extracted by the alkaline lysis method.Finally,the presence of exoU and exoY genes was evaluated by the PCR test.Results:In this study,47%,72%,29%,39%,40%,and 44%of the isolates were non-susceptible to piperacillin,aztreonam,ceftazidime,imipenem,tobramycin,and ciprofloxacin,respectively.In addition,95%and 93%of the clinical isolates carried the exoU and exoY genes.Blood and fecal isolates had both virulence genes,while only one wound isolate had neither genes.Meanwhile,all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene.Also,88 isolates simultaneously had both exoU and exoY genes.Conclusions:High prevalence of exoU and exoY genes in this region indicates a significant role of typeⅢsecretion system in pathogenesis of Pseudomonas aeruginosa.The typeⅢsecretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Mogroside IIE,an in vivo metabolite of sweet agent,alleviates acute lung injury via Pla2g2a-EGFR inhibition

In the face of increasingly serious environmental pollution,the health of human lung tissues is also facing serious threats.Mogroside IIE(M2E)is the main metabolite of sweetening agents mogrosides from the anti-tussive Chinese herbal Siraitia grosvenori.The study elucidated the anti-inflammatory action and molecular mechanism of M2E against acute lung injury(ALI).A lipopolysaccharide(LPS)-induced ALI model was established in mice and MH-S cells were employed to explore the protective mechanism of M2E through the western blotting,co-immunoprecipitation,and quantitative real time-PCR analysis.The results indicated that M2E alleviated LPS-induced lung injury through restraining the activation of secreted phospholipase A2 type IIA(Pla2g2a)-epidermal growth factor receptor(EGFR).The interaction of Pla2g2a and EGFR was identified by co-immunoprecipitation.In addition,M2E protected ALI induced with LPS against inflammatory and damage which were significantly dependent upon the downregulation of AKT and m TOR via the inhibition of Pla2g2a-EGFR.Pla2g2a may represent a potential target for M2E in the improvement of LPS-induced lung injury,which may represent a promising strategy to treat ALI.

SecReT6 update:a comprehensive resource of bacterial TypeⅥSecretion Systems

TypeⅥSecretion System(T6SS)plays significant roles in microbial activities via injecting effectors into adjacent cells or environments.T6SS increasingly gained attention due to its important influence on pathogenesis,microbial competition,etc.T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource.The SecReT6 version3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors.To assist T6SS researches like microbial competition and regulatory mechanisms,SecReT6 v3developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk.We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3.17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected,analyzed and incorporated into the database.The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/.

Type Ⅳ secretion system in Helicobacter pylori:a new insight into pathogenicity

Objective To review the research progress on Type Ⅳ secretion system （T4SS） in HelicobacterpylorL Data sources The data used in this review were identified by searching of PUBMED （1995-2007） online resources using the key terms ＇Type Ⅳ secretion system＇ and ＇Helicobacter pylon. Study selection Mainly original articles and critical reviews written by major pioneer investigators of this field were selected. Results The research progress on T4SS in Helicobacter pylori was summarized. The structure and function was discussed. Conclusions T4SS is not only involved in toxin secretion and injection of virulence factors into eukaryotic host target cells, but also involved in horizontal DNA transfer to other bacteria and eukaryotic cells, through DNA uptake from or release into the extracellular milieu. It provides a new insight into the pathogenicity of Helicobacter pylori and a novel target for antimicrobials development. However, many challenges remain for us in understanding the biological role of T4SS in Helicobacter pylori.

Towards a better understanding of antimicrobial resistance dissemination:what can be learnt from studying model conjugative plasmids?

Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.

Molecular Cloning,Bioinformatics Analysis and Transcriptional Expression of Virulence-related Gene(exsA)of Vibrio alginolyticus

[Objectives]The purpose was to investigate the function and mechanism of exsA gene in type III secretion system of Vibrio alginolyticus.[Methods]The full length of exsA was cloned using molecular biology techniques to analyze its biological information.Fluorescence quantitative PCR technology was used to analyze the expression of exsA after different media stress.[Results]The exsA gene contains an open reading frame(ORF)of 861 bp,encoding 286 amino acids.The physico-chemical analysis shows that the molecular structural formula is C1442H2267N393O441S12,the theoretical molecular weight is 32.549 kD,the theoretical pI value is 6.0,and the protein is non-hydrophilic and unstable.The gene does not contain a transmembrane region,and there is no obvious signal peptide.The prediction result of protein subcellular localization shows that the protein is inside the cell.The deduced amino acid sequence and constructed phylogenetic tree show that V.alginolyticus has a close relationship with Vibrio antiquarius.The qPCR results show that the expression level of exsA in different media is different,highest in TSB medium containing bile salts,followed by DMEM medium,and lowest in ordinary TSB medium.[Conclusions]The gene sequence,molecular structure and isoelectric point of exsA,as well as its expression in three different media were obtained.

Beyond dueling: roles of the type VI secretion system in microbiome modulation, pathogenesis and stress resistance

Bacteria inhabit diverse and dynamic environments,where nutrients may be limited and toxic chemicals can be prevalent.To adapt to these stressful conditions,bacteria have evolved specialized protein secretion systems,such as the type VI secretion system(T6SS)to facilitate their survival.As a molecular syringe,the T6SS expels various effectors into neighboring bacterial cells,eukaryotic cells,or the extracellular environment.These effectors improve the competitive fitness and environmental adaption of bacterial cells.Although primarily recognized as antibacterial weapons,recent studies have demonstrated that T6SSs have functions beyond interspecies competition.Here,we summarize recent research on the role of T6SSs in microbiome modulation,pathogenesis,and stress resistance.

Molecular Cloning and Bioinformatics Analysis of T3SS Effector HopPmaJ from Vibrio alginolyticus

In this paper, the hopPmaJ gene, which encodes type III effector HopPmaJ of Vibrio alginolyticus, was cloned, sequenced and bioinformatically predicted. The results showed that hopPmaJ （GenBank accession number： KX245315） is 345 bp in length and encodes 114 amino acids, with a theoretical molecular mass of 12. 774 kD, and a pI of 4.45. The protein has multiple functional domains and binding sites, but no signal peptide. The tertiary structure of the protein is a homodimer. Based on multiple parameters, the possible dominant B cell antigenic epitopes of HopPmaJ were predicted to be at positions 13 - 16, 29 -30, 40 -42, 45 -49 and 84 -91.

Diverse interactions of five core type Ⅲ effectors from Ralstonia solanacearum with plants

Ralstonia solanacearum is a widespread plant bacterial pathogen that can launch a range of type Ⅲ effectors(T3Es)to cause disease.In this study,we isolate a pathogenic R.solanacearum strain named P380 from tomato rhizosphere.Five out of 12 core T3Es of strain P380 are introduced into Pseudomonas syringae DC3000D36E separately to determine their functions in interacting with plants.DC3000D36E that harbors each effector suppresses FliC-triggered Pti5 and ACRE31 expression,ROS burst,and callose deposition.RipAE,RipU,and RipW elicit cell death as well as upregulate the MAPK cascades in Nicotiana benthamiana.The derivatives RipC1^(△DDXDX(T/V))and RipW^(△DDKXXQ)but not RipAE^(K310R) fail to suppress ROS burst.Moreover,RipAE^(K310R) and RipW^(△DDKXXQ) retain the cell death elicitation ability.RipAE and RipW are associated with salicylic acid and jasmonic acid pathways,respectively.RipAE and RipAQ significantly promote the propagation of DC3000D36E in plants.The five core T3Es localize in diverse subcellular organelles of nucleus,plasma membrane,endoplasmic reticulum,and Golgi network.The suppressor of G2 allele of Skp1 is required for RipAE but not RipU-triggered cell death in N.benthamiana.These results indicate that the core T3Es in R.solanacearum play diverse roles in plantpathogen interactions.

The putative propeptide of MycP1 in mycobacterial type VII secretion system does not inhibit protease activity but improves protein stability

Mycosin-1 protease(MycP1)is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis,and is essential in virulence factor secretion through the ESX-1 type VII secretion system(T7SS).Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence,primarily through cleavage of its secretion substrate EspB.MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser,classic hallmarks of a sub-tilase family serine protease.The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation.In this study,we have deter-mined crystal structures of MycP1 with(MycP124-422)and without(MycP1^(63-422))the propeptide,and conducted EspB cleavage assays using the two proteins.Very high struc-tural similarity was observed in the two crystal structures.Interestingly,protease assays demonstrated positive EspB cleavage for both proteins,indicating that the putative propeptide does not inhibit protease activity.Molecu-lar dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP124-422 than in MycP1^(63-422),suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.

Chemical constituents and theiranti-infective activity of Paeonia suffruticosa

Objective:Research the chemical constituents of Paeonia suffruticosa that are responsible for its anti-infective properties.Methods:Several column chromatographic methods were used to purify the chemical constituents from P.suffruticosa,including medium pressure liquid chromatography,Sephadex LH-20,and normal silica gel.A disc diffusion method was used to screen for antibacterial activity,and their anti-virulence activity was assessed on the type III secretion system(T3SS)of Salmonella pathogenicity island 1(SPI-1)in Salmonella enterica serovar Typhimurium UK-1 g 8956 by SDS-PAGE and western blots.Results:Twenty-one compounds were identified.Compounds 7,8 and 17 showed moderate activity against S.aureus ATCC25923,compounds 8,9 and 10 showed weak activities against B.subtilis ACCC11060.Meanwhile,phenols(14-18)and flavonoids(20 and 21)inhibited T3SS protein secretion of S.typhimurium without affecting bacterial growth.Furthermore,a strong inhibitory effect was observed for 17 and 20 on SPI-1 mediated invasion of HeLa cells.Additionally,no toxicity was observed for these compounds.Conclusion:P.suffruticosa has anti-infective properties due in part to the fact that phenols and flavonoids can block the secretion of T3SS-associated protein effectors.

A Fur-regulated type Ⅵ secretion system contributes to oxidative stress resistance and virulence in Yersinia pseudotuberculosis

The type Ⅵ secretion system(T6SS)is a widespread protein secretion apparatus deployed by many Gram-negative bacterial species to interact with competitor bacteria,host organisms,and the environment.Yersinia pseudotuber-culosis T6SS4 was recently reported to be involved in manganese acquisition;however,the underlying regulatory mechanism still remains unclear.In this study,we discovered that T6SS4 is regulated by ferric uptake regulator(Fur)in response to manganese ions(Mn^(2+)),and this negative regulation of Fur was proceeded by specifically recogniz-ing the promoter region of T6SS4 in Y.pseudotuberculosis.Furthermore,T6SS4 is induced by low Mn^(2+)and oxidative stress conditions via Fur,acting as a Mn^(2+)-responsive transcriptional regulator to maintain intracellular manganese homeostasis,which plays important role in the transport of Mn^(2+)for survival under oxidative stress.Our results pro-vide evidence that T6SS4 can enhance the oxidative stress resistance and virulence for Y.pseudotuberculosis.This study provides new insights into the regulation of T6SS4 via the Mn^(2+)-dependent transcriptional regulator Fur,and expands our knowledge of the regulatory mechanisms and functions of T6SS from Y.pseudotuberculosis.

WEDeepT3: predicting type Ⅲ secreted effectors based on word embedding and deep learning

Background:The type Ⅲ secreted effectors(T3SEs)are one of the indispensable proteins in the growth and reproduction of Gram-negative bacteria.In particular,the pathogenesis of Gram-negative bacteria depends on the type Ⅲ secreted effectors,and by injecting T3SEs into a host cell,the host cell's immunity can be destroyed.The high diversity of T3SE sequences and the lack of defined secretion signals make it difficult to identify and predict.Moreover,the related study of the pathological system associated with T3SE remains a hot topic in bioinformatics.Some computational tools have been developed to meet the growing demand for the recognition of T3SEs and the studies of type Ⅲ secretion systems(T3SS).Although these tools can help biological experiments in certain procedures,there is still room for improvement,even for the current best model,as the existing methods adopt handdesigned feature and traditional machine learning methods.Methods:In this study,we propose a powerful predictor based on deep learning methods,called WEDeepT3.Our work consists mainly of three key steps.First,we train word embedding vectors for protein sequences in a large-scale amino acid sequence database.Second,we combine the word vectors with traditional features extracted from protein sequences,like PSSM,to construct a more comprehensive feature representation.Finally,we construct a deep neural network model in the prediction of type Ⅲ secreted effectors.Results:The feature representation of WEDeepT3 consists of both word embedding and position-specific features.Working together with convolutional neural networks,the new model achieves superior performance to the state-ofthe-art methods,demonstrating the effectiveness of the new feature representation and the powerful learning ability of deep models.Conclusion:WEDeepT3 exploits both semantic information of Ar-mer fragments and evolutional information of protein sequences to accurately difYerentiate between T3SEs and non-T3SEs.WEDeepT3 is available at bcmi.sjtu.edu.cn/~yangyang/WEDeepT3.html.

Emergence of hypervirulent Pseudomonas aeruginosa pathotypically armed with co-expressed T3SS effectors ExoS and ExoU

Pseudomonas aeruginosa is a significant pathogen mainly causing healthcare-associated infections(HAIs).Newly emerging high-risk clones of P.aeruginosa with elevated virulence profiles furtherly cause severe community-acquired infections(CAIs).Usually,it is not common for P.aeruginosa to co-carry exoU and exoS genes,encoding two type III secretion system(T3SS)effectors.The pathogenicity mechanism of exoS+/exoU+strains of P.aeruginosa remains unclear.Here,we provide detailed evidence for a subset of hypervirulent P.aeruginosa strains,which abundantly co-express and secrete the T3SS effectors ExoS and ExoU.The exoS+/exoU+P.aeruginosa strains were available to cause both HAIs and CAIs.The CAI-associated strains could elicit severe inflammation and hemorrhage,leading to higher death rates in a murine acute pneumonia model,and had great virulence potential in establishing chronic infections,demonstrating hypervirulence when compared to PAO1(exoS+/exoU-)and PA14(exoS-/exoU+).Both ExoS and ExoU were co-expressed and co-secreted in abundance in exoS+/exoU+strains.Their abundant protein secretion could boost exoS+/exoU+strains’potentials for cytotoxicity in vitro and pathogenicity in vivo.Genomic evidence indicates that exoU acquisition is likely mediated by horizontal gene transfer(HGT)of the pathogenicity island PAPI-2,while deletion of exoU was sufficient to mitigate virulence in the exoS+/exoU+strains.Furthermore,bioinformatics analysis showed that such exoS+/exoU+P.aeruginosa strains turned out to be widely distributed across the globe.Overall,the research provide detailed evidence for the high virulence and epidemicity of exoS+/exoU+strains of P.aeruginosa,highlighting an urgent need for surveillance against these high-risk hypervirulent strains.

A plasma membrane nucleotide-binding leucinerich repeat receptor mediates the recognition of the Ralstonia pseudosolanacearum effector RipY in Nicotiana benthamiana

Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops.Only a few functional resistance genes against bacterial wilt have been cloned to date.Here,we showthat the broadly conserved typeⅢsecreted effector RipY is recognized by the Nicotiana benthamiana immune system,leading to cell death induction,induction of defense-related gene expression,and restriction of bacterial pathogen growth.Using a multiplexed virus-induced gene-silencing-based N.benthamiana nucleotide-binding and leucine-rich repeat receptor(NbNLR)library,we identified a coiled-coil(CC)nucleotide-binding and leucine-rich repeat receptor(CNL)required for recognition of RipY,which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY(RRS-Y).Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death andRipY-induced immunity to Ralstonia pseudosolanacearum.RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1,ACTIVATED DISEASE RESISTANCE 1,and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2,-3,and-4 in N.benthamiana.We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition.RRSY also broadly recognizes RipY homologs across Ralstonia species.Lastly,we show that the C-terminal region of RipY is indispensable for RRS-Y activation.Together,our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.

Sensing bacterial infections by NAIP receptors in NLRC4 inflammasome activation

The inflammasome is an emerging new pathway in innate immune defense against microbial infection or endogenous danger signals.The inflammasome stimulates activation of inflammatory caspases,mainly caspase-1.Caspase-1 activation is responsible for processing and secretion of IL-1βand IL-18 as well as for inducing macrophage pyroptotic death.Assembly of the large cytoplasmic inflammasome complex is thought to be mediated by members of NOD-like receptor(NLR)family.While functions of most of the NLR proteins remain to be defined,several NLR proteins including NLRC4 have been shown to assemble distinct inflammasome complexes.These inflammasome pathways,particularly the NLRC4 inflammasome,play a critical role in sensing and restricting diverse types of bacterial infections.Here we review recent advances in defining the exact bacterial ligands and the ligand-binding receptors involved in NLRC4 inflammasome activation.Implications of the discovery of the NAIP family of inflammasome receptors for bacterial flagellin and type III secretion apparatus on future inflammasome and bacterial infection studies are also discussed.

Legionella pneumophila temporally regulates the activity of ADP/ATP translocases by reversible ADP-ribosylation

The mitochondrion is an important signaling hub that governs diverse cellular functions,including metabolism,energy production,and immunity.Among the hundreds of effectors translocated into host cells by the Dot/Icm system of Legionella pneumophila,several are targeted to mitochondria but the function of most of them remains elusive.Our recent study found that the effector Ceg3 inhibits the activity of ADP/ATP translocases(ANTs)by ADP-ribosylation(ADPR).Here,we show that the effect of Ceg3 is antagonized by Larg1,an effector encoded by lpg0081,a gene that is situated next to ceg3.Larg1 functions to reverse Ceg3-mediated ADPR of ANTs by cleaving the N-glycosidic bond between the ADPR moiety and the modified arginine residues in ANTs,leading to restoration of their activity in ADP/ATP exchange.Structural analysis of Larg1 and its complex with ADPR reveals that this ADPR glycohydrolase harbors a unique macrodomain that catalyzes the removal of ADPR modification on ANTs.Our results also demonstrate that together with Ceg3,Larg1 imposes temporal regulation of the activity of ANTs by reversible ADPR during L.pneumophila infection.