SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力
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浙江省自然科学基金(LY21C010004);浙江省大学生科技创新活动计划(新苗人才计划)(2020R413040);温州市基础性科研项目(Y20210082)


Cyclic di-GMP produced by SadC regulates swimming motility of Pseudomonas aeruginosa via PilZ and FlgZ
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    摘要:

    【目的】探究铜绿假单胞菌(Pseudomonas aeruginosa)鸟苷酸环化酶(diguanylate cyclase,DGC) SadC合成的环二鸟苷酸(cyclic di-GMP,c-di-GMP)信号与PilZ结构域受体间的信号传递关系,分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析;SadC过表达背景下,PilZ结构域受体突变各菌株的泳动表型分析和筛选;基因敲除和过表达解析筛选出的PilZ结构域受体功能;定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力;PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制;功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强,而过表达PilZ或FlgZ则抑制了泳动能力;定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要,多序列比对分析表明这些位点是其保守的c-di-GMP结合位点。【结论】SadC合成的c-di-GMP信号通过PilZ和FlgZ调控铜绿假单胞菌的泳动能力。

    Abstract:

    [Objective] To identify the PilZ domain-containing receptor(s) that sense the second messenger cyclic di-GMP (c-di-GMP) produced by the diguanylate cyclase SadC in Pseudomonas aeruginosa and investigate the functions and regulatory mechanisms of the identified receptor(s). [Methods] We constructed the strains in which sadC gene was deleted or overexpressed and tested their ability to swim by using a plate-based approach. We then added sadC in multicopy in each deletion mutant of the eight PilZ domain-containing receptors and screened for the mutants with alleviated swimming repression compared to the wild-type PA14 overexpressing SadC. For the mutations screened out, single gene knockout and overexpression strategies were used to explore the function of the identified receptor(s). Furthermore, site-directed mutagenesis and genetic complementation were employed to test whether the identified receptor's role in SadC-mediated swimming repression requires its c-di-GMP-binding motif. [Results] The SadC-mediated repression of swimming motility was associated with flagellar malfunction rather than flagellum formation. Two PilZ domain-containing receptors, PilZ and FlgZ, were identified to be involved in SadC-mediated swimming repression. The deletion of gene pilZ or flgZ increased the swimming motility, while overexpression of them significantly impaired swimming. A R10A substitution in the conserved c-di-GMP-binding motif of PilZ, or a R140A substitution in FlgZ, resulted in a variant that was no longer able to repress swimming in ΔpilZ or ΔflgZ overexpressing SadC, indicating that the conserved residue required for c-di-GMP binding is critical for PilZ or FlgZ to repress swimming in response to SadC-derived c-di-GMP. [Conclusion] PilZ and FlgZ are the effector relay proteins that respond to SadC c-di-GMP signaling to mediate swimming repression in P. aeruginosa.

    参考文献
    [1] JENAL U, REINDERS A, LORI C. Cyclic di-GMP:second messenger extraordinaire[J]. Nature Reviews Microbiology, 2017, 15(5):271-284.
    [2] HALL CL, LEE VT. Cyclic-di-GMP regulation of virulence in bacterial pathogens[J]. Wiley Interdisciplinary Reviews RNA, 2018, 9(1):10.1002/wrna.1454.
    [3] PETCHIAPPAN A, NAIK SY, CHATTERJI D. Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria[J]. Biophysical Reviews, 2020, 12(3):719-730.
    [4] 管文静, 吴茂森, 何晨阳. c-di-GMP信号途径对细菌致病性的调控作用[J]. 微生物学通报, 2009, 36(3):427-431. GUAN WJ, WU MS, HE CY. Cyclic diguanylate signalling in regulation of bacterial pathogenesis[J]. Microbiology, 2009, 36(3):427-431(in Chinese).
    [5] CHOU SH, GALPERIN MY. Diversity of cyclic di-GMP-binding proteins and mechanisms[J]. Journal of Bacteriology, 2016, 198(1):32-46.
    [6] HENGGE R. High-specificity local and global c-di-GMP signaling[J]. Trends in Microbiology, 2021, 29(11):993-1003.
    [7] 杨凤环, 田芳, 陈华民, 何晨阳. 病原细菌受体介导的c-di-GMP信号传导及其调控机制[J]. 植物保护, 2017, 43(1):9-14. YANG FH, TIAN F, CHEN HM, HE CY. Cyclic di-GMP signal receptor-mediated regulation of bacterial behaviors[J]. Plant Protection, 2017, 43(1):9-14(in Chinese).
    [8] KULASAKARA H, LEE V, BRENCIC A, LIBERATI N, URBACH J, MIYATA S, LEE DG, NEELY AN, HYODO M, HAYAKAWA Y, AUSUBEL FM, LORY S. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(8):2839-2844.
    [9] MERIGHI M, LEE VT, HYODO M, HAYAKAWA Y, LORY S. The second messenger bis-(3'-5')-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa[J]. Molecular Microbiology, 2007, 65(4):876-895.
    [10] HOU LL, DEBRU A, CHEN QQ, BAO QY, LI KW. AmrZ regulates swarming motility through cyclic di-GMP-dependent motility inhibition and controlling pel polysaccharide production in Pseudomonas aeruginosa PA14[J]. Frontiers in Microbiology, 2019, 10:1847.
    [11] CHOI KH, SCHWEIZER HP. Mini-Tn7 insertion in bacteria with single attTn7 sites:example Pseudomonas aeruginosa[J]. Nature Protocols, 2006, 1(1):153-161.
    [12] HMELO LR, BORLEE BR, ALMBLAD H, LOVE ME, RANDALL TE, TSENG BS, LIN CY, IRIE Y, STOREK KM, YANG JJ, SIEHNEL RJ, HOWELL PL, SINGH PK, TOLKER-NIELSEN T, PARSEK MR, SCHWEIZER HP, HARRISON JJ. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange[J]. Nature Protocols, 2015, 10(11):1820-1841.
    [13] HA DG, KUCHMA SL, O'TOOLE GA. Plate-based assay for swimming motility in Pseudomonas aeruginosa[J]. Methods in Molecular Biology:Clifton, N J, 2014, 1149:59-65.
    [14] MERRITT JH, BROTHERS KM, KUCHMA SL, O'TOOLE GA. SadC reciprocally influences biofilm formation and swarming motility via modulation of exopolysaccharide production and flagellar function[J]. J Bacteriol, 2007, 189(22):8154-8164.
    [15] YEUNG ATY, TORFS ECW, JAMSHIDI F, BAINS M, WIEGAND I, HANCOCK REW, OVERHAGE J. Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of transcriptional regulators, including MetR[J]. Journal of Bacteriology, 2009, 191(18):5592-5602.
    [16] ORR MW, LEE VT. A PilZ domain protein for chemotaxis adds another layer to c-di-GMP-mediated regulation of flagellar motility[J]. Science Signaling, 2016, 9(450):fs16.
    [17] ALM RA, BODERO AJ, FREE PD, MATTICK JS. Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa[J]. Journal of Bacteriology, 1996, 178(1):46-53.
    [18] BENSE S, BRUCHMANN S, STEFFEN A, STRADAL TEB, HÄUSSLER S, DÜVEL J. Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility[J]. Molecular Microbiology, 2019, 111(6):1544-1557.
    [19] CHEANG QW, XIN LY, CHEA RYF, LIANG ZX. Emerging paradigms for PilZ domain-mediated C-di-GMP signaling[J]. Biochemical Society Transactions, 2019, 47(1):381-388.
    [20] BAKER AE, DIEPOLD A, KUCHMA SL, SCOTT JE, HA DG, ORAZI G, ARMITAGE JP, O'TOOLE GA. PilZ domain protein FlgZ mediates cyclic di-GMP-dependent swarming motility control in Pseudomonas aeruginosa[J]. Journal of Bacteriology, 2016, 198(13):1837-1846.
    [21] DAHLSTROM KM, O'TOOLE GA. A symphony of cyclases:specificity in diguanylate cyclase signaling[J]. Annual Review of Microbiology, 2017, 71:179-195.
    [22] O'TOOLE GA, KOLTER R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[J]. Molecular Microbiology, 1998, 30(2):295-304.
    [23] HA DG, RICHMAN ME, O'TOOLE GA. Deletion mutant library for investigation of functional outputs of cyclic diguanylate metabolism in Pseudomonas aeruginosa PA14[J]. Applied and Environmental Microbiology, 2014, 80(11):3384-3393.
    [24] BHASME P, WEI Q, XU AM, NAQVI STA, WANG D, MA LZ. Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa[J]. MicrobiologyOpen, 2020, 9(3):e975.
    [25] SCHMIDT A, HAMMERBACHER AS, BASTIAN M, NIEKEN KJ, KLOCKGETHER J, MERIGHI M, LAPOUGE K, POSCHGAN C, KÖLLE J, ACHARYA KR, ULRICH M, TÜMMLER B, UNDEN G, KAEVER V, LORY S, HAAS D, SCHWARZ S, DÖRING G. Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa[J]. Environmental Microbiology, 2016, 18(10):3390-3402.
    [26] MOSCOSO JA, JAEGER T, VALENTINI M, HUI K, JENAL U, FILLOUX A. The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa[J]. Journal of Bacteriology, 2014, 196(23):4081-4088.
    [27] ZHU B, LIU CL, LIU SH, CONG HJ, CHEN YH, GU LC, MA LZ. Membrane association of SadC enhances its diguanylate cyclase activity to control exopolysaccharides synthesis and biofilm formation in Pseudomonas aeruginosa[J]. Environmental Microbiology, 2016, 18(10):3440-3452.
    [28] MCCARTHY RR, MAZON-MOYA MJ, MOSCOSO JA, HAO YA, LAM JS, BORDI C, MOSTOWY S, FILLOUX A. Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion[J]. Nature Microbiology, 2017, 2:17027.
    [29] BAKER AE, WEBSTER SS, DIEPOLD A, KUCHMA SL, BORDELEAU E, ARMITAGE JP, O'TOOLE GA. Flagellar stators stimulate c-di-GMP production by Pseudomonas aeruginosa[J]. Journal of Bacteriology, 2019, 201(18):e00741-e00718.
    [30] LEWIS KA, BAKER AE, CHEN AI, HARTY CE, KUCHMA SL, O'TOOLE GA, HOGAN DA. Ethanol decreases Pseudomonas aeruginosa flagellar motility through the regulation of flagellar stators[J]. Journal of Bacteriology, 2019, 201(18):e00285-e00219.
    [31] AMIKAM D, GALPERIN MY. PilZ domain is part of the bacterial c-di-GMP binding protein[J]. Bioinformatics:Oxford, England, 2006, 22(1):3-6.
    [32] MARTÍNEZ-GRANERO F, NAVAZO A, BARAHONA E, REDONDO-NIETO M, GONZÁLEZ de HEREDIA E, BAENA I, MARTÍN-MARTÍN I, RIVILLA R, MARTÍN M. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas[J]. PLoS One, 2014, 9(2):e87608.
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董友明,林心铭,付春情,王倩琪,吴玲娟,卢冰倩,林羲,包其郁,李科伟. SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力[J]. 微生物学报, 2023, 63(3): 1115-1127

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  • 收稿日期:2022-07-17
  • 录用日期:2022-09-22
  • 在线发布日期: 2023-03-08
  • 出版日期: 2023-03-04
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