H3-T6SS of Pseudomonas aeruginosa PA14 contributes to environmental adaptation via secretion of a biofilm-promoting effector

Abstract

Microbial species often occur in complex communities and exhibit intricate synergistic and antagonistic interactions. To avoid predation and compete for favorable niches, bacteria have evolved specialized protein secretion systems. The type VI secretion system (T6SS) is a versatile secretion system widely distributed among Gram-negative bacteria that translocates effectors into target cells or the extracellular milieu via various physiological processes. Pseudomonas aeruginosa is an opportunistic pathogen responsible for many diseases, and it has three independent T6SSs (H1-, H2-, and H3-T6SS). In this study, we found that the H3-T6SS of highly virulent P. aeruginosa PA14 is negatively regulated by OxyR and OmpR, which are global regulatory proteins of bacterial oxidative and acid stress. In addition, we identified a H3-T6SS effector PA14_33970, which is located upstream of VgrG3. PA14_33970 interacted directly with VgrG3 and translocated into host cells. Moreover, we found that H3-T6SS and PA14_33970 play crucial roles in oxidative, acid, and osmotic stress resistance, as well as in motility and biofilm formation. PA14_33970 was identified as a new T6SS effector promoting biofilm formation and thus named TepB. Furthermore, we found that TepB contributes to the virulence of P. aeruginosa PA14 toward Caenorhabditis elegans. Overall, our study indicates that H3-T6SS and its biofilm-promoting effector TepB are regulated by OxyR and OmpR, both of which are important for adaptation of P. aeruginosa PA14 to multiple stressors, providing insights into the regulatory mechanisms and roles of T6SSs in P. aeruginosa.

Keywords

P. aeruginosa PA14 / Regulation / H3-T6SS / TepB / Stress resistance / Virulence

References

[1] AllsoppLP, WoodTE, HowardSA, MaggiorelliF, NolanLM, WettstadtS, FillouxA. RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A, 2017, 114: 7707-7712

[2] Al-WrafyF, BrzozowskaE, GorskaS, GamianA. Pathogenic factors of Pseudomonas aeruginosa - the role of biofilm in pathogenicity and as a target for phage therapy. Postepy Hig Med Dosw (Online), 2017, 71: 78-91

[3] BalasubramanianD, SchneperL, KumariH, MatheeK. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res, 2013, 41: 1-20

[4] BieleckiP, JensenV, SchulzeW, GodekeJ, StrehmelJ, EckweilerD, et al.. Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa. Nucleic Acids Res, 2015, 43: 6413-6425

[5] BlevesS, ViarreV, SalachaR, MichelGP, FillouxA, VoulhouxR. Protein secretion systems in Pseudomonas aeruginosa: a wealth of pathogenic weapons. Int J Med Microbiol, 2010, 300: 534-543

[6] BondageDD, LinJS, MaLS, KuoCH, LaiEM. VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex. Proc Natl Acad Sci U S A, 2016, 113: E3931-E3940

[7] BonemannG, PietrosiukA, DiemandA, ZentgrafH, MogkA. Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J, 2009, 28: 315-325

[8] BoulantT, BoudehenYM, FillouxA, PlesiatP, NaasT, DortetL. Higher prevalence of PldA, a Pseudomonas aeruginosa trans-kingdom H2-type VI secretion system effector, in clinical isolates responsible for acute infections and in multidrug resistant strains. Front Microbiol, 2018, 9: 2578

[9] BrencicA, LoryS. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Mol Microbiol, 2009, 72: 612-632

[10] BurrowsLL. Pseudomonas aeruginosa twitching motility: type IV pili in action. Annu Rev Microbiol, 2012, 66: 493-520

[11] ChenL, ZouY, KronflAA, WuY. Type VI secretion system of Pseudomonas aeruginosa is associated with biofilm formation but not environmental adaptation. Microbiologyopen, 2020, 9: e991

[12] ChenL, ZouY, SheP, WuY. Composition, function, and regulation of T6SS in Pseudomonas aeruginosa. Microbiol Res, 2015, 172: 19-25

[13] Cherny KE, Sauer K (2019) Pseudomonas aeruginosa requires the DNA-specific endonuclease EndA to degrade extracellular genomic DNA to disperse from the biofilm. J Bacteriol 201. https://doi.org/10.1128/JB.00059-19

[14] CianfanelliFR, MonlezunL, CoulthurstSJ. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol, 2016, 24: 51-62

[15] CoulthurstS. The type VI secretion system: a versatile bacterial weapon. Microbiology (Reading), 2019, 165: 503-515

[16] da Cruz Nizer WS, Inkovskiy V, Versey Z, Strempel N, Cassol E, Overhage J (2021) Oxidative stress response in Pseudomonas aeruginosa. Pathogens 10. https://doi.org/10.3390/pathogens10091187

[17] DarbyC, HsuJW, GhoriN, FalkowS. Caenorhabditis elegans: plague bacteria biofilm blocks food intake. Nature, 2002, 417: 243-244

[18] DurandE, CambillauC, CascalesE, JournetL. VgrG, Tae, Tle, and beyond: the versatile arsenal of type VI secretion effectors. Trends Microbiol, 2014, 22: 498-507

[19] Fei N, Ji W, Yang L, Yu C, Qiao P, Yan J et al (2022) Hcp of the type VI secretion system (T6SS) in Acidovorax citrulli group II strain Aac5 has a dual role as a core structural protein and an effector protein in colonization, growth ability, competition, biofilm formation, and ferric Iron absorption. Int J Mol Sci 23. https://doi.org/10.3390/ijms23179632

[20] FillouxA. The type VI secretion system: a tubular story. EMBO J, 2009, 28: 309-310

[21] Flemming H-C, van Hullebusch ED, Neu TR, Nielsen PH, Seviour T, Stoodley P et al (2022) The biofilm matrix: multitasking in a shared space. Nat Rev Microbiol. https://doi.org/10.1038/s41579-022-00791-0

[22] Gerken H, Vuong P, Soparkar K, Misra R (2020) Roles of the EnvZ/OmpR two-component system and porins in iron acquisition in Escherichia coli. mBio 11. https://doi.org/10.1128/mBio.01192-20

[23] GueguenE, DurandE, ZhangXY, d'AmalricQ, JournetL, CascalesE. Expression of a Yersinia pseudotuberculosis type VI secretion system is responsive to envelope stresses through the OmpR transcriptional activator. PLoS One, 2013, 8: e66615

[24] HachaniA, AllsoppLP, OdukoY, FillouxA. The VgrG proteins are “à la Carte” delivery systems for bacterial type VI effectors. J Biol Chem, 2014, 289: 17872-17884

[25] HanY, WangT, ChenG, PuQ, LiuQ, ZhangY, et al.. A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition. PLoS Pathog, 2019, 15: e1008198

[26] HoBT, FuY, DongTG, MekalanosJJ. Vibrio cholerae type 6 secretion system effector trafficking in target bacterial cells. Proc Natl Acad Sci U S A, 2017, 114: 9427-9432

[27] HoangTT, KutchmaAJ, BecherA, SchweizerHP. Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid, 2000, 43: 59-72

[28] InoueT, ShingakiR, FukuiK. Inhibition of swarming motility of Pseudomonas aeruginosa by branched-chain fatty acids. FEMS Microbiol Lett, 2008, 281: 81-86

[29] JiangF, WaterfieldNR, YangJ, YangG, JinQ. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe, 2014, 15: 600-610

[30] KasettyS, Katharios-LanwermeyerS, O’TooleGA, NadellCD. Differential surface competition and biofilm invasion strategies of Pseudomonas aeruginosa PA14 and PAO1. J Bacteriol, 2021, 203: e0026521

[31] KatoJ, ChakrabartyAM. Purification of the regulatory protein AlgR1 and its binding in the far upstream region of the algD promoter in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A, 1991, 88: 1760-1764

[32] Kenney LJ, Anand GS (2020) EnvZ/OmpR two-component signaling: an archetype system that can function noncanonically. EcoSal Plus 9. https://doi.org/10.1128/ecosalplus.ESP-0001-2019

[33] Kim S, Li XH, Hwang HJ, Lee JH (2020) Thermoregulation of Pseudomonas aeruginosa biofilm formation. Appl Environ Microbiol 86. https://doi.org/10.1128/AEM.01584-20

[34] KumarA, AlamA, RaniM, EhteshamNZ, HasnainSE. Biofilms: survival and defense strategy for pathogens. Int J Med Microbiol, 2017, 307: 481-489

[35] LeimanPG, BaslerM, RamagopalUA, BonannoJB, SauderJM, PukatzkiS, et al.. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci U S A, 2009, 106: 4154-4159

[36] LesicB, StarkeyM, HeJ, HazanR, RahmeLG. Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology (Reading), 2009, 155: 2845-2855

[37] Li C, Zhu L, Pan D, Li S, Xiao H, Zhang Z et al (2019) Siderophore-mediated Iron acquisition enhances resistance to oxidative and aromatic compound stress in Cupriavidus necator JMP134. Appl Environ Microbiol 85. https://doi.org/10.1128/AEM.01938-18

[38] Li C, Zhu L, Wang D, Wei Z, Hao X, Wang Z et al (2021) T6SS secretes an LPS-binding effector to recruit OMVs for exploitative competition and horizontal gene transfer. ISME J. https://doi.org/10.1038/s41396-021-01093-8

[39] LinJ, ChengJ, ChenK, GuoC, ZhangW, YangX, et al.. The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1. Front Cell Infect Microbiol, 2015, 5: 70

[40] Lin J, Xu L, Yang J, Wang Z, Shen X (2021) Beyond dueling: roles of the type VI secretion system in microbiome modulation, pathogenesis and stress resistance. Stress Biol 1. https://doi.org/10.1007/s44154-021-00008-z

[41] LinJ, ZhangW, ChengJ, YangX, ZhuK, WangY, et al.. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition. Nat Commun, 2017, 8: 14888

[42] LiuL, HaoS, LanR, WangG, XiaoD, SunH, XuJ. The type VI secretion system modulates flagellar gene expression and secretion in Citrobacter freundii and contributes to adhesion and cytotoxicity to host cells. Infect Immun, 2015, 83: 2596-2604

[43] Martinez-GarciaS, Rodriguez-MartinezS, Cancino-DiazME, Cancino-DiazJC. Extracellular proteases of Staphylococcus epidermidis: roles as virulence factors and their participation in biofilm. APMIS, 2018, 126: 177-185

[44] MauraD, HazanR, KitaoT, BallokAE, RahmeLG. Evidence for direct control of virulence and defense gene circuits by the Pseudomonas aeruginosa quorum sensing regulator, MvfR. Sci Rep, 2016, 6: 34083

[45] MelstromKA Jr, KozlowskiR, HassettDJ, SuzukiH, BatesDM, GamelliRL, ShankarR. Cytotoxicity of Pseudomonas secreted exotoxins requires OxyR expression. J Surg Res, 2007, 143: 50-57

[46] Montenegro BenavidesNA, AlvarezBA, Arrieta-OrtizML, RodriguezRL, BoteroD, TabimaJF, et al.. The type VI secretion system of Xanthomonas phaseoli pv. manihotis is involved in virulence and in vitro motility. BMC Microbiol, 2021, 21: 14

[47] MoscosoJA, MikkelsenH, HeebS, WilliamsP, FillouxA. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ Microbiol, 2011, 13: 3128-3138

[48] MougousJD, CuffME, RaunserS, ShenA, ZhouM, GiffordCA, et al.. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science, 2006, 312: 1526-1530

[49] NanB, ZusmanDR. Novel mechanisms power bacterial gliding motility. Mol Microbiol, 2016, 101: 186-193

[50] O’TooleGA, KolterR. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol, 1998, 30: 295-304

[51] PanP, WangX, ChenY, ChenQ, YangY, WeiC, et al.. Effect of Hcp iron ion regulation on the interaction between Acinetobacter baumannii with human pulmonary alveolar epithelial cells and biofilm formation. Front Cell Infect Microbiol, 2022, 12: 761604

[52] PanmaneeW, CharoenlapN, AtichartpongkulS, MahavihakanontA, WhitesideMD, WinsorG, et al.. The OxyR-regulated phnW gene encoding 2-aminoethylphosphonate:pyruvate aminotransferase helps protect Pseudomonas aeruginosa from tert-butyl hydroperoxide. PLoS One, 2017, 12: e0189066

[53] RashidMH, KornbergA. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A, 2000, 97: 4885-4890

[54] RecordsAR. The type VI secretion system: a multipurpose delivery system with a phage-like machinery. Mol Plant-Microbe Interact, 2011, 24: 751-757

[55] RussellAB, PetersonSB, MougousJD. Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol, 2014, 12: 137-148

[56] SanaTG, BerniB, BlevesS. The T6SSs of Pseudomonas aeruginosa strain PAO1 and their effectors: beyond bacterial-cell targeting. Front Cell Infect Microbiol, 2016, 6: 61

[57] SanaTG, HachaniA, BuciorI, SosciaC, GarvisS, TermineE, et al.. The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells. J Biol Chem, 2012, 287: 27095-27105

[58] SanaTG, SosciaC, TongletCM, GarvisS, BlevesS. Divergent control of two type VI secretion systems by RpoN in Pseudomonas aeruginosa. PLoS One, 2013, 8: e76030

[59] ShaJ, RosenzweigJA, KozlovaEV, WangS, ErovaTE, KirtleyML, et al.. Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis. Microbiology (Reading), 2013, 159: 1120-1135

[60] ShenX, BangaS, LiuY, XuL, GaoP, ShamovskyI, et al.. Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response. Cell Microbiol, 2009, 11: 911-926

[61] SiM, WangY, ZhangB, ZhaoC, KangY, BaiH, et al.. The type VI secretion system engages a redox-regulated dual-functional heme transporter for zinc acquisition. Cell Rep, 2017, 20: 949-959

[62] SiM, ZhaoC, BurkinshawB, ZhangB, WeiD, WangY, et al.. Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis. Proc Natl Acad Sci U S A, 2017, 114: E2233-E2242

[63] SongL, PanJ, YangY, ZhangZ, CuiR, JiaS, et al.. Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties. Nat Commun, 2021, 12: 423

[64] SongY, XiaoX, LiC, WangT, ZhaoR, ZhangW, et al.. The dual transcriptional regulator RovM regulates the expression of AR3- and T6SS4-dependent acid survival systems in response to nutritional status in Yersinia pseudotuberculosis. Environ Microbiol, 2015, 17: 4631-4645

[65] TanMW, Mahajan-MiklosS, AusubelFM. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl Acad Sci U S A, 1999, 96: 715-720

[66] Tolker-NielsenT. Biofilm development. Microbiol Spectr, 2015, 3: MB-0001-2014

[67] VinckxT, WeiQ, MatthijsS, CornelisP. The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin. Microbiology (Reading), 2010, 156: 678-686

[68] Wadhwa N, Berg HC (2021) Bacterial motility: machinery and mechanisms. Nat Rev Microbiol. https://doi.org/10.1038/s41579-021-00626-4

[69] WangT, SiM, SongY, ZhuW, GaoF, WangY, et al.. Type VI secretion system transports Zn²⁺ to combat multiple stresses and host immunity. PLoS Pathog, 2015, 11: e1005020

[70] WeberB, HasicM, ChenC, WaiSN, MiltonDL. Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum. Environ Microbiol, 2009, 11: 3018-3028

[71] WeiQ, MinhPN, DotschA, HildebrandF, PanmaneeW, ElfarashA, et al.. Global regulation of gene expression by OxyR in an important human opportunistic pathogen. Nucleic Acids Res, 2012, 40: 4320-4333

[72] WettstadtS. Should I kill or should I go: T6SS regulation networks in Vibrio. Environ Microbiol, 2020, 22: 1-4

[73] WhiteleyM, BangeraMG, BumgarnerRE, ParsekMR, TeitzelGM, LoryS, GreenbergEP. Gene expression in Pseudomonas aeruginosa biofilms. Nature, 2001, 413: 860-864

[74] WuCF, LienYW, BondageD, LinJS, PilhoferM, ShihYL, et al.. Effector loading onto the VgrG carrier activates type VI secretion system assembly. EMBO Rep, 2020, 21: e47961

[75] XuH, YangJ, GaoW, LiL, LiP, ZhangL, et al.. Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome. Nature, 2014, 513: 237-241

[76] XuL, ShenX, BryanA, BangaS, SwansonMS, LuoZQ. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog, 2010, 6: e1000822

[77] Yang X, Liu H, Zhang Y, Shen X (2021) Roles of type VI secretion system in transport of metal ions. Front Microbiol 12. https://doi.org/10.3389/fmicb.2021.756136

[78] YangX, PanJ, WangY, ShenX. Type VI secretion systems present new insights on pathogenic Yersinia. Front Cell Infect Microbiol, 2018, 8: 260

[79] YuH, HeX, XieW, XiongJ, ShengH, GuoS, et al.. Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipid-mediated regulation. Can J Microbiol, 2014, 60: 227-235

[80] Yu KW, Xue P, Fu Y, Yang L (2021) T6SS mediated stress responses for bacterial environmental survival and host adaptation. Int J Mol Sci 22. https://doi.org/10.3390/ijms22020478

[81] ZhangL, LiS, LiuX, WangZ, JiangM, WangR, et al.. Sensing of autoinducer-2 by functionally distinct receptors in prokaryotes. Nat Commun, 2020, 11: 5371

[82] ZhangW, WangY, SongY, WangT, XuS, PengZ, et al.. A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environ Microbiol, 2013, 15: 557-569

[83] ZhengY, KahntJ, KwonIH, MackieRI, ThauerRK. Hydrogen formation and its regulation in Ruminococcus albus: involvement of an electron-bifurcating [FeFe]-hydrogenase, of a non-electron-bifurcating [FeFe]-hydrogenase, and of a putative hydrogen-sensing [FeFe]-hydrogenase. J Bacteriol, 2014, 196: 3840-3852

[84] Zhu L, Xu L, Wang C, Li C, Li M, Liu Q et al (2021) T6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese. Proc Natl Acad Sci U S A 118. https://doi.org/10.1073/pnas.2103526118

[85] ZouedA, BrunetYR, DurandE, AschtgenMS, LoggerL, DouziB, et al.. Architecture and assembly of the type VI secretion system. Biochim Biophys Acta, 2014, 1843: 1664-1673

Funding

Key Technologies Research and Development Program(2021YFA0909600); Young Scientists Fund (CN)(32100034); Young Scientists Fund(32100149); National Natural Science Foundation of China(31970114)