Impacting of cAMP receptor protein on the bile tolerance of Aeromonas hydrophila in early growth
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摘要:
目的 本研究拟确定cAMP 受体蛋白 对嗜水气单胞菌胆汁耐受性的影响。 方法 构建嗜水气单胞菌的luxS、crp的缺失株及crp回补株，通过胆汁生长实验确定嗜水气单胞菌及其构建株的胆汁耐受性。 结果 嗜水气单胞菌BJ018、BJ018ΔluxS和BJ018Δcrp在LB培养基中生长速度基本一致。 10%胆汁LB中，BJ018和缺失株ΔluxS前8 h生长被抑制，后期呈对数快速生长；其缺失株Δcrp生长速度领先于野生株，早期生长未被抑制。 嗜水气单胞菌BJ018Δcrp的回补株早期生长重新被抑制，与野生株BJ018基本一致。 其他两株嗜水气单胞菌BJ017、BJ054及其缺失株Δcrp胆汁生长结果与BJ018、BJ018Δcrp一致。 这些结果提示嗜水气单胞菌CRP影响菌株对胆汁的耐受。 结论 CRP的存在影响嗜水气单胞菌在胆汁中的生长，可能在胆汁耐受调控中起到重要作用。 Abstract:Objective Bile is an important signaling molecule that regulates the intestinal microflora and environment in both directions, playing an important role in viral and bacterial infections. CRP (cAMP receptor protein) is involved in the bile tolerance of Escherichia coli and Salmonella in infections. In this study, we evaluated the effect of CRP (cAMP receptor protein) on bile tolerance of Aeromonas hydrophila. Methods The luxS-deleted, crp-deleted and crp-complemented strains of A. hydrophila were constructed, and the bile tolerance of A. hydrophila and its constructed strains was detected by bile growth experiments. Results The growth rates of A. hydrophila strains BJ018, BJ018ΔluxS and BJ018Δcrp in Luria broth (LB ) were similar. However, in 10% bile LB, the growth of BJ018 and BJ018ΔluxS was inhibited in the first 8 hours, and the growth rate was logarithmic in the later stage; while the growth rate of BJ018Δcrp was higher than that of the wild strain, and the early growth was not inhibited. The early growth of the complemented strain BJ018Δcrp-pSRK-crp was re-inhibited in the early stage, which was similar to that of the wild strain BJ018 containing plasmid pSRK. The other A. hydrophila strains BJ017, BJ054 and their crp-deleted strains had the same result with BJ018 and BJ018Δcrp. The result suggested that CRP had influence on bile tolerance of A. hydrophila. Conclusion CRP negatively regulated the growth of A. hydrophila in bile and might play an important role in bile tolerance regulation. -
Key words:
- Aeromonas hydrophila /
- Bile /
- cAMP receptor protein
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图 1 嗜水气单胞相关菌株电泳结果
注：M. Marker 2000； 2～10. 以内引物crpin扩增结果；11～21. 以外引物crp112upF/crp112dnL扩增结果；3、4、10、12、13、19. 野生株扩增结果，阳性对照；2、5～9、11、14～18. crp缺失株扩增结果；20. pRE112-crp扩增结果，阳性对照；21. 水，阴性对照
Figure 1. Agarose GEL electrophoresis of A. hydrophila strains
图 2 0%或10%胆盐浓度LB中嗜水气单胞菌BJ018及相关菌株的生长曲线
注：A、C. 无胆汁的LB培养基，B、D. 含10%胆汁的LB培养基
Figure 2. 2 Growth determination of A. hydrophila strains BJ018 and related strains in 0% or 10% bile salts LB
图 3 0%或10%胆盐浓度LB中嗜水气单胞菌BJ017、BJ054及其crp缺失株的生长曲线
注：A、C. 无胆汁的LB培养基，B、D. 含10%胆汁的LB培养基
Figure 3. Growth determination of A. hydrophila strains BJ017, J054 and their crp-deleted strains in 0% or 10% bile salts LB
表 1 嗜水气单胞菌缺失株及回补株构建相关引物
Table 1. Primers for deletion and complementation of A. hydrophila
编号 引物序列（5'～3'） 酶切位点 产物（bp） crp112upF GCtctagaGCGATCGGCGATGACGAACTGA Xba I 565 crp112upR GCACTGCCACCGGCAAGACCATAGTGGTGT crp112dnF GGTCTTGCCGGTGGCAGTGCGACAAGAACC Kpn I 553 crp112dnL GGggtaccCCTGACGCGCCTTCTCCAGGAT luxSupF GCtctagaGCAGTGGGGAACCCCGATCCAGGTG Xba I 496 luxSupR CAGCTCGTCAGTAAAACTGTCCAATAACGGCAT luxSdnF ACAGTTTTACTGACGAGCTGGCCCTGCCGGAAG Kpn I 491 luxSdnR GGggtaccCCACATAGACCGCTTCAAGAGTTTCAAC crpF TGCtctagaTCAGCGGGTGCCGAACAC Xba I 639 crpR GGAATTCcatatgGTCATTGGCAAACCGCAAAGC Nde I crpinF GCGGAGATCAGCTCCTGC 576 crpinR TCCCACCCTGGAATGGTTCT 注：下划线部分为引入的酶切位点和保护性碱基，粗体字母为反向互补序列 
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[1] Fu JY, Kuipers F. Systems genetics approach reveals cross-talk between bile acids and intestinal microbes[J]. PLoS Genet, 2019, 15(8): e1008307. DOI: 10.1371/journal.pgen.1008307. [2] Goodrich JK, Davenport ER, Waters JL, et al. Cross-species comparisons of host genetic associations with the microbiome[J]. Science, 2016, 352(6285): 532–535. DOI: 10.1126/science.aad9379. [3] Kemis JH, Linke V, Barrett KL, et al. Genetic determinants of gut microbiota composition and bile acid profiles in mice[J]. PLoS Genet, 2019, 15(8): e1008073. DOI: 10.1371/journal.pgen.1008073. [4] Wahlström A, Sayin SI, Marschall HU, et al. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism[J]. Cell Metab, 2016, 24(1): 41–50. DOI: 10.1016/j.cmet.2016.05.005. [5] Sayin SI, Wahlström A, Felin J, et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist[J]. Cell Metab, 2013, 17(2): 225–235. DOI: 10.1016/j.cmet.2013.01.003. [6] Hu JP, Wang CK, Huang XY, et al. Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling[J]. Cell Rep, 2021, 36(12): 109726. DOI: 10.1016/j.celrep.2021.109726. [7] Begley M, Gahan CGM, Hill C. The interaction between bacteria and bile[J]. FEMS Microbiol Rev, 2005, 29(4): 625–651. DOI: 10.1016/j.femsre.2004.09.003. [8] Urdaneta V, Casadesús J. Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts[J]. Front Med, 2017, 4: 163. DOI: 10.3389/fmed.2017.00163. [9] Eade CR, Hung CC, Bullard B, et al. Bile acids function synergistically to repress invasion gene expression in Salmonella by destabilizing the invasion regulator HilD[J]. Infect Immun, 2016, 84(8): 2198–2208. DOI: 10.1128/IAI.00177−16. [10] Murakami K, Tenge VR, Karandikar UC, et al. Bile acids and ceramide overcome the entry restriction for GⅡ. 3 human norovirus replication in human intestinal enteroids[J]. Proc Natl Acad Sci USA, 2020, 117(3): 1700–1710. DOI: 10.1073/pnas.1910138117. [11] Sawabe T, Oliver JD. International committee on systematics of prokaryotes subcommittee on the taxonomy of Aeromonadaceae, Vibrionaceae and related organisms Minutes of the meetings, 31 March 2016, Roscoff, France[J]. Int J Syst Evol Microbiol, 2017, 67(3): 759–760. DOI: 10.1099/ijsem.0.001675. [12] Stratev D, Odeyemi OA. Antimicrobial resistance of Aeromonas hydrophila isolated from different food sources: a mini-review[J]. J Infect Public Health, 2016, 9(5): 535–544. DOI: 10.1016/j.jiph.2015.10.006. [13] Ghenghesh KS, Rahouma A, Zorgani A, et al. Aeromonas in Arab countries: 1995−2014[J]. Comp Immunol Microbiol Infect Dis, 2015, 42: 8–14. DOI: 10.1016/j.cimid.2015.07.002. [14] Chao CM, Lai CC, Tang HJ, et al. Biliary tract infections caused by Aeromonas species[J]. Eur J Clin Microbiol Infect Dis, 2013, 32(2): 245–251. DOI: 10.1007/s10096−012−1736−1. [15] Cameron ADS, Redfield RJ. Non-canonical CRP sites control competence regulons in Escherichia coli and many other γ-proteobacteria[J]. Nucleic Acids Res, 2006, 34(20): 6001–6014. DOI: 10.1093/nar/gkl734. [16] Kidd SP, Pemberton JM. The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect[J]. J Appl Microbiol, 2002, 93(5): 787–793. DOI: 10.1046/j.1365−2672.2002.01750.x. [17] van Velkinburgh JC, Gunn JS. PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp[J]. Infect Immun, 1999, 67(4): 1614–1622. DOI: 10.1128/IAI.67.4.1614−1622.1999. [18] Yu HB, Zhang YL, Lau YL, et al. Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91[J]. Appl Environ Microbiol, 2005, 71(8): 4469–4477. DOI: 10.1128/AEM.71.8.4469−4477.2005. [19] 南征, 于礼, 杨伟民, 等. 气单胞菌不同菌种胆汁耐受特征分析[J]. 疾病监测,2021,37(4):453–456. DOI: 10.3784/jbjc.202104140209.Nan Z, Yu L, Yang WM, et al. Bile tolerance characteristics of different Aeromonas species[J]. Dis Surveill, 2021, 37(4): 453–456. DOI: 10.3784/jbjc.202104140209. [20] Wang YD, Wang H, Liang WL, et al. Quorum sensing regulatory cascades control Vibrio fluvialis pathogenesis[J]. J Bacteriol, 2013, 195(16): 3583–3589. DOI: 10.1128/JB.00508−13. [21] Kozlova EV, Khajanchi BK, Popov VL, et al. Impact of QseBC system in c-di-GMP-dependent quorum sensing regulatory network in a clinical isolate SSU of Aeromonas hydrophila[J]. Microb Pathog, 2012, 53(3/4): 115–124. DOI: 10.1016/j.micpath.2012.05.008. [22] Shimizu K. Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism[J]. ISRN Biochem, 2013, 2013: 645983. DOI: 10.1155/2013/645983. [23] Chen BL, Liang WL, Wu R, et al. Phenotype microarray screening of carbon sources used by Vibrio cholerae identifies genes regulated by the cAMP receptor protein[J]. Can J Microbiol, 2013, 59(7): 472–478. DOI: 10.1139/cjm−2013−0084. [24] Liang WL, Silva AJ, Benitez JA. The cyclic AMP receptor protein modulates colonial morphology in Vibrio cholerae[J]. Appl Environ Microbiol, 2007, 73(22): 7482–7487. DOI: 10.1128/AEM.01564−07. [25] Chu CY, Wang SY, Chen ZW, et al. Heterologous protection in pigs induced by a plasmid-cured and crp gene-deleted Salmonella choleraesuis live vaccine[J]. Vaccine, 2007, 25(41): 7031–7040. DOI: 10.1016/j.vaccine.2007.07.063. [26] Le VVH, Biggs PJ, Wheeler D, et al. Novel mechanisms of TolC-independent decreased bile-salt susceptibility in Escherichia coli[J]. FEMS Microbiol Lett, 2020, 367(10): fnaa083. DOI: 10.1093/femsle/fnaa083. [27] Villarreal JM, Hernández-Lucas I, Gil F, et al. cAMP receptor protein (CRP) positively regulates the yihU-yshA operon in Salmonella enterica serovar Typhi[J]. Microbiology (Reading) , 2011, 157(Pt 3): 636–647. DOI: 10.1099/mic.0.046045−0. [28] Chen ZW, Hsuan SL, Liao JW, et al. Mutations in the Salmonella enterica serovar choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 type III secretion system[J]. Vet Res, 2010, 41(1): 5. DOI: 10.1051/vetres/2009053. [29] Zhou YY, Yu L, Nan Z, et al. Taxonomy, virulence genes and antimicrobial resistance of Aeromonas isolated from extra-intestinal and intestinal infections[J]. BMC Infect Dis, 2019, 19(1): 158. DOI: 10.1186/s12879−019−3766−0. -
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