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

Abstract

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 pseudotuberculosis 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 (Mn²⁺), and this negative regulation of Fur was proceeded by specifically recognizing the promoter region of T6SS4 in Y. pseudotuberculosis. Furthermore, T6SS4 is induced by low Mn²⁺ and oxidative stress conditions via Fur, acting as a Mn²⁺-responsive transcriptional regulator to maintain intracellular manganese homeostasis, which plays important role in the transport of Mn²⁺ for survival under oxidative stress. Our results provide 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 Mn²⁺-dependent transcriptional regulator Fur, and expands our knowledge of the regulatory mechanisms and functions of T6SS from Y. pseudotuberculosis.

Keywords

Type VI secretion system / Fur / Manganese / Oxidative stress / Virulence

References

[1] AguirreJD, CulottaVC. Battles with iron: manganese in oxidative stress protection. J Biol Chem, 2012, 287(17):13541-13548

[2] AndersonMC, VonaeschP, SaffarianA, MarteynBS, SansonettiPJ. Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy. Cell Host Microbe, 2017, 21(6):769-776 e763

[3] AskouraM, SarvanS, CoutureJF, StintziA. The campylobacter jejuni ferric uptake regulator promotes acid survival and cross-protection against oxidative stress. Infect Immun, 2016, 84(5):1287-1300

[4] BarneseK, GrallaEB, ValentineJS, CabelliDE. Biologically relevant mechanism for catalytic superoxide removal by simple manganese compounds. Proc Natl Acad Sci U S A, 2012, 109(18):6892-6897

[5] BaslerM, PilhoferM, HendersonGP, JensenGJ, MekalanosJJ. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature, 2012, 483(7388):182-186

[6] BeardenSW, PerryRD. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol Microbiol, 1999, 32(2):403-414

[7] BeckerKW, SkaarEP. Metal limitation and toxicity at the interface between host and pathogen. FEMS Microbiol Rev, 2014, 38(6):1235-1249

[8] Brunet YR, Bernard CS, Cascales E (2020) Fur-dam regulatory interplay at an internal promoter of the enteroaggregative Escherichia coli type VI secretion sci1 gene cluster. J Bacteriol 202(10). https://doi.org/10.1128/JB.00075-20

[9] BrunetYR, BernardCS, GavioliM, LloubesR, CascalesE. An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster. PLoS Genet, 2011, 7(7):e1002205

[10] CaiR, GaoF, PanJ, HaoX, YuZ, QuY, LiJ, WangD, WangY, ShenX, LiuX, YangY. The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis. Microbiol Res, 2021, 249: 126787

[11] ChakrabortyS, SivaramanJ, LeungKY, MokYK. Two-component PhoB-PhoR regulatory system and ferric uptake regulator sense phosphate and iron to control virulence genes in type III and VI secretion systems of Edwardsiella tarda. J Biol Chem, 2011, 286(45):39417-39430

[12] ChoJY, LiuR, MacbethJC, HsiaoA. The interface of vibrio cholerae and the gut microbiome. Gut Microbes, 2021, 13(1):1937015

[13] CianfanelliFR, MonlezunL, CoulthurstSJ. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol, 2016, 24(1):51-62

[14] Diaz-OchoaVE, LamD, LeeCS, KlausS, BehnsenJ, LiuJZ, ChimN, NuccioSP, RathiSG, MastroianniJR, EdwardsRA, JacoboCM, CerasiM, BattistoniA, OuelletteAJ, GouldingCW, ChazinWJ, SkaarEP, RaffatelluM. Salmonella mitigates oxidative stress and thrives in the inflamed gut by evading calprotectin-mediated manganese sequestration. Cell Host Microbe, 2016, 19(6):814-825

[15] 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(6):e66615

[16] 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(35):9427-9432

[17] HohleTH, O'BrianMR. Metal-specific control of gene expression mediated by Bradyrhizobium japonicum Mur and Escherichia coli Fur is determined by the cellular context. Mol Microbiol, 2016, 101(1):152-166

[18] HsiehPF, LuYR, LinTL, LaiLY, WangJT. Klebsiella pneumoniae type VI secretion system contributes to bacterial competition, cell invasion, type-1 fimbriae expression, and in vivo colonization. J Infect Dis, 2019, 219(4):637-647

[19] IshikawaT, SabharwalD, BromsJ, MiltonDL, SjostedtA, UhlinBE, WaiSN. Pathoadaptive conditional regulation of the type VI secretion system in vibrio cholerae O1 strains. Infect Immun, 2012, 80(2):575-584

[20] KapiteinN, MogkA. Deadly syringes: type VI secretion system activities in pathogenicity and interbacterial competition. Curr Opin Microbiol, 2013, 16(1):52-58

[21] Kehl-FieTE, ChitayatS, HoodMI, DamoS, RestrepoN, GarciaC, MunroKA, ChazinWJ, SkaarEP. Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus. Cell Host Microbe, 2011, 10(2):158-164

[22] KehresDG, JanakiramanA, SlauchJM, MaguireME. Regulation of Salmonella enterica serovar typhimurium mntH transcription by H₂O₂, Fe²⁺, and Mn²⁺. J Bacteriol, 2002, 184(12):3151-3158

[23] KimYH, YuMH. Overexpression of reactive cysteine-containing 2-nitrobenzoate nitroreductase (NbaA) and its mutants alters the sensitivity of Escherichia coli to reactive oxygen species by reprogramming a regulatory network of disulfide-bonded proteins. J Proteome Res, 2012, 11(6):3219-3230

[24] KitphatiW, Ngok-NgamP, SuwanmaneeratS, SukchawalitR, MongkolsukS. Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence. Appl Environ Microbiol, 2007, 73(15):4760-4768

[25] KoskiniemiS, LamoureuxJG, NikolakakisKC, t’Kint de RoodenbekeC, KaplanMD, LowDA, HayesCS. Rhs proteins from diverse bacteria mediate intercellular competition. Proc Natl Acad Sci U S A, 2013, 110(17):7032-7037

[26] LampsLW, MadhusudhanKT, HavensJM, GreensonJK, BronnerMP, ChilesMC, DeanPJ, ScottMA. Pathogenic Yersinia DNA is detected in bowel and mesenteric lymph nodes from patients with Crohn's disease. Am J Surg Pathol, 2003, 27(2):220-227

[27] LiC, PanD, LiM, WangY, SongL, YuD, ZuoY, WangK, LiuY, WeiZ, LuZ, ZhuL, ShenX. Aerobactin-mediated iron acquisition enhances biofilm formation, oxidative stress resistance, and virulence of Yersinia pseudotuberculosis. Front Microbiol, 2021, 12: 699913

[28] Li C, Zhu L, Pan D, Li S, Xiao H, Zhang Z, Shen X, Wang Y, Long M (2019) Siderophore-mediated iron acquisition enhances resistance to oxidative and aromatic compound stress in Cupriavidus necator JMP134. Appl Environ Microbiol 85(1). https://doi.org/10.1128/AEM.01938-18

[29] LiC, ZhuL, WangD, WeiZ, HaoX, WangZ, LiT, ZhangL, LuZ, LongM, WangY, WeiG, ShenX. T6SS secretes an LPS-binding effector to recruit OMVs for exploitative competition and horizontal gene transfer. ISME J, 2022, 16(2):500-510

[30] 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(1). https://doi.org/10.1007/s44154-021-00008-z

[31] LinJ, ZhangW, ChengJ, YangX, ZhuK, WangY, WeiG, QianPY, LuoZQ, ShenX. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition. Nat Commun, 2017, 8: 14888

[32] MiyataST, KitaokaM, BrooksTM, McAuleySB, PukatzkiS. Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect Immun, 2011, 79(7):2941-2949

[33] Monjaras FeriaJ, ValvanoMA. An overview of anti-eukaryotic T6SS effectors. Front Cell Infect Microbiol, 2020, 10: 584751

[34] MorrisseyJA, CockayneA, BrummellK, WilliamsP. The staphylococcal ferritins are differentially regulated in response to iron and manganese and via PerR and Fur. Infect Immun, 2004, 72(2):972-979

[35] MougousJD, CuffME, RaunserS, ShenA, ZhouM, GiffordCA, GoodmanAL, JoachimiakG, OrdonezCL, LoryS, WalzT, JoachimiakA, MekalanosJJ. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science, 2006, 312(5779):1526-1530

[36] MurphyEC, FriedmanAJ. Hydrogen peroxide and cutaneous biology: translational applications, benefits, and risks. J Am Acad Dermatol, 2019, 81(6):1379-1386

[37] Pasqua M, Visaggio D, Lo Sciuto A, Genah S, Banin E, Visca P, Imperi F (2017) Ferric uptake regulator Fur is conditionally essential in Pseudomonas aeruginosa. J Bacteriol 199(22). https://doi.org/10.1128/JB.00472-17

[38] PlateroR, PeixotoL, O'BrianMR, FabianoE. Fur is involved in manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti. Appl Environ Microbiol, 2004, 70(7):4349-4355

[39] PorcheronG, GarenauxA, ProulxJ, SabriM, DozoisCM. Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence. Front Cell Infect Microbiol, 2013, 3: 90

[40] Radin JN, Zhu J, Brazel EB, McDevitt CA, Kehl-Fie TE (2019) Synergy between nutritional immunity and independent host defenses contributes to the importance of the MntABC manganese transporter during Staphylococcus aureus infection. Infect Immun 87(1). https://doi.org/10.1128/IAI.00642-18

[41] RiderCV, ChanP, HerbertRA, KisslingGE, FombyLM, HejtmancikMR, WittKL, WaidyanathaS, TravlosGS, KadiiskaMB. Dermal exposure to cumene hydroperoxide: assessing its toxic relevance and oxidant potential. Toxicol Pathol, 2016, 44(5):749-762

[42] Ruiz-LagunaJ, Prieto-AlamoMJ, PueyoC. Oxidative mutagenesis in Escherichia coli strains lacking ROS-scavenging enzymes and/or 8-oxoguanine defenses. Environ Mol Mutagen, 2000, 35(1):22-30 https://doi.org/10.1002/(sici)1098-2280(2000)35:1<22::aid-em4>3.0.co;2-x

[43] RussellAB, HoodRD, BuiNK, LeRouxM, VollmerW, MougousJD. Type VI secretion delivers bacteriolytic effectors to target cells. Nature, 2011, 475(7356):343-347

[44] SanaTG, FlaugnattiN, LugoKA, LamLH, JacobsonA, BaylotV, DurandE, JournetL, CascalesE, MonackDM. Salmonella typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut. Proc Natl Acad Sci U S A, 2016, 113(34):E5044-E5051

[45] SanaTG, HachaniA, BuciorI, SosciaC, GarvisS, TermineE, EngelJ, FillouxA, BlevesS. 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(32):27095-27105

[46] SchellMA, UlrichRL, RibotWJ, BrueggemannEE, HinesHB, ChenD, LipscombL, KimHS, MrazekJ, NiermanWC, DeshazerD. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol, 2007, 64(6):1466-1485

[47] SchweerJ, KulkarniD, KochutA, PezoldtJ, PisanoF, PilsMC, GenthH, HuehnJ, DerschP. The cytotoxic necrotizing factor of Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by activation of Rho GTPases. PLoS Pathog, 2013, 9(11):e1003746

[48] SeoSW, KimD, LatifH, O'BrienEJ, SzubinR, PalssonBO. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli. Nat Commun, 2014, 5: 4910

[49] SepterAN, LyellNL, StabbEV. The iron-dependent regulator fur controls pheromone signaling systems and luminescence in the squid symbiont Vibrio fischeri ES114. Appl Environ Microbiol, 2013, 79(6):1826-1834

[50] ShneiderMM, ButhSA, HoBT, BaslerM, MekalanosJJ, LeimanPG. PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature, 2013, 500(7462):350-353

[51] SiM, WangY, ZhangB, ZhaoC, KangY, BaiH, WeiD, ZhuL, ZhangL, DongTG, ShenX. The type VI secretion system engages a redox-regulated dual-functional heme transporter for zinc acquisition. Cell Rep, 2017, 20(4):949-959

[52] SiM, ZhaoC, BurkinshawB, ZhangB, WeiD, WangY, DongTG, ShenX. Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis. Proc Natl Acad Sci U S A, 2017, 114(11):E2233-E2242

[53] SilvermanJM, BrunetYR, CascalesE, MougousJD. Structure and regulation of the type VI secretion system. Annu Rev Microbiol, 2012, 66: 453-472

[54] SongL, PanJ, YangY, ZhangZ, CuiR, JiaS, WangZ, YangC, XuL, DongTG, WangY, ShenX. Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties. Nat Commun, 2021, 12(1):423

[55] SongY, XiaoX, LiC, WangT, ZhaoR, ZhangW, ZhangL, WangY, ShenX. 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(11):4631-4645

[56] StoreyD, McNallyA, AstrandM, Sa-Pessoa Graca SantosJ, Rodriguez-EscuderoI, ElmoreB, PalaciosL, MarshallH, HobleyL, MolinaM, CidVJ, SalminenTA, BengoecheaJA. Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent. PLoS Pathog, 2020, 16(3):e1007969

[57] TownsleyL, Sison MangusMP, MehicS, YildizFH. Response of vibrio cholerae to low-temperature shifts: CspV regulation of type VI secretion, biofilm formation, and association with zooplankton. Appl Environ Microbiol, 2016, 82(14):4441-4452

[58] TroxellB, HassanHM. Transcriptional regulation by ferric uptake regulator (Fur) in pathogenic bacteria. Front Cell Infect Microbiol, 2013, 3: 59

[59] TrunkK, PeltierJ, LiuYC, DillBD, WalkerL, GowNAR, StarkMJR, QuinnJ, StrahlH, TrostM, CoulthurstSJ. The type VI secretion system deploys antifungal effectors against microbial competitors. Nat Microbiol, 2018, 3(8):920-931

[60] VazeND, ParkS, BrooksAD, FridmanA, JoshiSG. Involvement of multiple stressors induced by non-thermal plasma-charged aerosols during inactivation of airborne bacteria. PLoS One, 2017, 12(2):e0171434

[61] Wang S, Yang D, Wu X, Yi Z, Wang Y, Xin S, Wang D, Tian M, Li T, Qi J, Ding C, Yu S (2019) The ferric uptake regulator represses type VI secretion system function by binding directly to the clpV promoter in Salmonella enterica Serovar typhimurium. Infect Immun 87(10). https://doi.org/10.1128/IAI.00562-19

[62] WangT, SiM, SongY, ZhuW, GaoF, WangY, ZhangL, ZhangW, WeiG, LuoZQ, ShenX. Type VI secretion system transports Zn²⁺ to combat multiple stresses and host immunity. PLoS Pathog, 2015, 11(7):e1005020

[63] WeberJ, FinlaysonNB, MarkJB. Mesenteric lymphadenitis and terminal ileitis due to Yersinia pseudotuberculosis. N Engl J Med, 1970, 283(4):172-174

[64] XuH, YangJ, GaoW, LiL, LiP, ZhangL, GongYN, PengX, XiJJ, ChenS, WangF, ShaoF. Innate immune sensing of bacterial modifications of rho GTPases by the pyrin inflammasome. Nature, 2014, 513(7517):237-241

[65] XuS, PengZ, CuiB, WangT, SongY, ZhangL, WeiG, WangY, ShenX. FliS modulates FlgM activity by acting as a non-canonical chaperone to control late flagellar gene expression, motility and biofilm formation in Yersinia pseudotuberculosis. Environ Microbiol, 2014, 16(4):1090-1104

[66] YangX, LiuH, ZhangY, ShenX. Roles of type VI secretion system in transport of metal ions. Front Microbiol, 2021, 12: 756136

[67] 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(2). https://doi.org/10.3390/ijms22020478

[68] ZhangW, WangY, SongY, WangT, XuS, PengZ, LinX, ZhangL, ShenX. A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environ Microbiol, 2013, 15(2):557-569

[69] ZhangW, XuS, LiJ, ShenX, WangY, YuanZ. Modulation of a thermoregulated type VI secretion system by AHL-dependent quorum sensing in Yersinia pseudotuberculosis. Arch Microbiol, 2011, 193(5):351-363

[70] ZhouD, QinL, HanY, QiuJ, ChenZ, LiB, SongY, WangJ, GuoZ, ZhaiJ, DuZ, WangX, YangR. Global analysis of iron assimilation and Fur regulation in Yersinia pestis. FEMS Microbiol Lett, 2006, 258(1):9-17

[71] ZhuC, ChenJ, WangY, WangL, GuoX, ChenN, ZhengP, SunJ, MaY. Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli. Biotechnol Bioeng, 2019, 116(8):2018-2028

[72] Zhu L, Xu L, Wang C, Li C, Li M, Liu Q, Wang X, Yang W, Pan D, Hu L, Yang Y, Lu Z, Wang Y, Zhou D, Jiang Z, Shen X (2021) T6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese. Proc Natl Acad Sci U S A 118(42). https://doi.org/10.1073/pnas.2103526118

Funding

National Key R&D Program of China(2018YFA0901200); National Natural Science Foundation of China(32100149); Postdoctoral Research Foundation of China(2020M673501); Scientific Startup Foundation for Doctors of Northwest A and F University(Z1090122002); Young Talent Support Program of Shaanxi Province University(20220206)