Effects of cbpD gene on virulence and related biological characteristics of Vibrio alginolyticus
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摘要: 为研究几丁质结合蛋白 (Chitin-binding protein D, CbpD) 对溶藻弧菌 (Vibrio alginolyticus) 毒力和相关生物学特性的影响，通过同源重组技术构建溶藻弧菌ZJ-T的cbpD基因缺失突变株ZJ-T-ΔcbpD，比较突变株与野生株对斑马鱼 (Danio rerio) 的毒性，以及毒力相关生理过程，包括生长能力、运动性、胞外蛋白酶分泌活性、溶血活性、抗生素敏感性、生物膜形成、过氧化氢 (H2O2) 和铜离子 (Cu2+) 抗性以及铁离子 (Fe3+) 吸收能力的差异。研究发现，cbpD缺失后，溶藻弧菌对斑马鱼的毒力显著减弱，且细菌游动能力、涌动能力和胞外蛋白酶活性均降低，突变株对 Cu2+的抗性提高；cbpD的突变不影响溶藻弧菌在富营养培养基中的生长、生物膜的形成、溶血活性、对大部分抗生素敏感性、对H2O2的抗性和Fe3+的获取能力。结果表明，cbpD可能通过正调控溶藻弧菌的运动能力和胞外蛋白酶分泌活性，而促进溶藻弧菌的毒力。Abstract: In order to study the effect of the chitin binding protein D (CbpD) on the virulence and related biological characteristics of Vibrio alginolyticus, we constructed the cbpD gene deletion mutant strain of V. alginolyticus ZJ-T by homologous recombination technique. Then we compared the virulence to Danio rerio and other related physiological processes between the wild type strain and the cbpD gene mutant strain, including the growth ability, motility, extracellular protease secretion activity, hemolytic activity and antibiotic sensitivity, biofilm formation, hydrogen peroxide and copper ion resistance as well as ion absorption capacity. The results show that due to the absence of cbpD gene, the swimming motility, swarming motility and extracellular protease activity all reduced, while the resistance to copper ion of the mutant strain strengthened, and its virulence toD. rerio was weakened. However, the deletion of cbpD gene did not affect the growth of the strain in nutrient-rich medium, biofilm formation, hemolytic activity, stress response to H2O2, uptake and utilization of iron and sensitivity to most antibiotics. In conclusion, the results indicate that cbpD may promote the virulence by positively regulating the motility, extracellular protease secretion activity of V. alginolyticus.
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Key words:
- Vibrio alginolyticus /
- cbpD gene /
- Gene knockout /
- Virulence regulation /
- Biological characteristics
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图 1 缺失株ZJ-T-ΔcbpD的构建
注：a. 质粒 pSW7848 线性化条带；b. cbpD 上下游片段，泳道 2 为 cbpD 上游同源臂，泳道 3 为 cbpD 下游同源臂；c. 重组自杀质粒 PCR 鉴定扩增片段；d. cbpD 缺失株的鉴定，泳道 5 为野生株扩增结果，泳道 6 为潜在突变株扩增结果。
Figure 1. Construction of deletion strain ZJ-T-ΔcbpD
Note: a. Plasmid pSW7848 linearization band; b. cbpD upstream and downstream fragments, and Lane 2 is the upstream, while Lane 3 is the downstream; c. PCR to identify segment of recombinant suicide plasmid; d. Identification of cbpD deletion strain, Lane 5 is the amplification result of the wild strain, and Lane 6 is the amplification result of the potential mutant strain.
图 3 溶藻弧菌ZJ-T和突变株ZJ-T-ΔcbpD的运动性
Figure 3. Mobility of V. alginolyticus ZJ-T and mutant strain ZJ-T-ΔcbpD
图 4 运动性统计分析
注：*. P<0.05；**. P<0.01；图5-a、8-b同此。
Figure 4. Motility statistical analysis
Note: *. P<0.05; **. P<0.01; The same case in Fig. 5-a and 8-b.
图 5 蛋白酶分泌能力
注： a. 蛋白质透明分解圈直径与菌落直径的比值；b. 脱脂牛奶平板图。
Figure 5. Secretion of extracellular protease capacity
Note: a. The ratio of protein transparent decomposition circle diameter to colony diameter; b. Picture of skim milk plate.
图 6 溶藻弧菌ZJ-T和ZJ-T-ΔcbpD的溶血性
Figure 6. Hemolytic activity of V. alginolyticus ZJ-T and ZJ-T-ΔcbpD
图 7 溶藻弧菌ZJ-T和ZJ-T-ΔcbpD对过氧化氢、硫酸铜以及铁离子螯合剂2-2'-联吡啶的敏感性
注：a. LBS 琼脂平板；b. 0.003% H2O2-LBS 平板；c. 4.5 mmol·L−1 CuSO4-LBS 平板；d. 150 μmol·L−1 DIP-LBS 平板。
Figure 7. Sensitivity to H2O2, CuSO4 and DIP of wild type and cbpD mutant on LBS plate
Note: a. LBS agar plate; b. 0.003% H2O2-LBS agar plate; c. 4.5 mmol·L−1 CuSO4-LBS agar plate; d. 150 μmol·L−1 DIP-LBS agar plate.
图 9 溶藻弧菌ZJ-T和突变株ZJ-T-ΔcbpD感染斑马鱼后成活率
Figure 9. Survival rate of V. alginolyticus ZJ-T and mutant strain ZJ-T-ΔcbpD after infection of D. rerio
表 1 本研究所用菌株及质粒
Table 1. Strains and plasmids used in this study
菌株或质粒
Strain or plasmid相关特征
Relevant characteristics来源
Source菌株 Strain Vibrio alginolyticus ZJ-T 氨苄抗性；野生菌株ZJ-51半透明/光滑变体；分离自中国南部沿海患病石斑鱼 [15] ZJ-T-ΔcbpD 缺失cbpD基因的ZJ-T突变株 本文构建 E. coliⅡ3813 lacIQ，thi1，supE44，endA1，recA1，hsdR17，gyrA462，zei298::Tn10[Tc]，ΔthyA::erm-pir116；自杀质粒pSW7848的中间宿主 [16] E. coli GEB883 大肠杆菌野生株K12 ΔdapA::ermpir RP4-2 ΔrecAgyrA462，zei298::Tn10；接合作用供体菌 [17] 质粒 Plasmid pSW7848 氯霉素抗性；具有R6K起点的自杀质粒，需要pir蛋白进行复制，带ccdB毒性基因 [18] pSW7848-cbpDup-cbpD down 氯霉素抗性；包括cbpD上下游同源臂的pSW7848质粒 本文构建 
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表 2 本研究所用引物
Table 2. Primers used in this study
引物
Primer引物序列 (5'—3')
Primer sequence (5'–3')用途
ApplicationpSW7848_fwd GTCTGATTCGTTACCAATTATGACAAC 扩增pSW7848片段 pSW7848_rev GAATTCGATATCAAGCTTATCGATAC cbpD-U-F ataagcttgatatcgaattcCAACATATGTGGGATAGTGCGT 扩增cbpD上游同源臂 cbpD-U-R ggtccattctttaccCTGAAACACAAGCGGTTTAAGC cbpD-D-F aaccgcttgtgtttcagGGTAAAGAATGGACCGCTC 扩增cbpD下游同源臂 cbpD-D-R taattggtaacgaatcagacGTCTTGGTTCTTAAAGGAGCTG Del-check-pSW7848-F TCACTGTCCCTTATTCGCACC 验证cbpD上下游片段是否与
质粒pSW7848重组成功Del-check-pSW7848-R CTGCTTTTGAGCACTACCCG Del-cbpD-check-F GTCACAACATTACGGGATCTAAC 检验cbpD基因是否成功敲除 Del-cbpD-check-R GCGAATAACGATATGTGCTCTG
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表 3 药敏试验结果
Table 3. Drug sensitivity tests results of ZJ-T and ZJ-T-ΔcbpD
药物
Drug每片含量
Content of each tablet/(μg·片−1)ZJ-T ZJ-T-ΔcbpD 抑菌圈直径
Size of inhibition zone/mm敏感性
Sensitivity抑菌圈直径
Size of inhibition zone/mm敏感性
Sensitivity克拉霉素 Claricid 15 18.24±1.10 S 15.59±0.81 I 利福平 Rifampin 5 15.35±0.35 R 13.66±0.33 R 阿莫西林 Amoxicillin 20 7.67±0.45 R 0 R 多西环素 Doxycycline 30 12.79±0.32 I 13.08±0.19 I 磺胺异噁唑 Sulfafurazole 300 0 R 0 R 麦迪霉素 Medimycin 30 9.63±0.64 R 8.52±0.12 R 头孢氨苄 Cephalexin 30 8.06±0.62 R 7.40±0.24 R 氟苯尼考 Florfenicol 30 22.15±1.31 S 20.96±0.78 S 妥布霉素 Tobramycin 10 15.07±1.32 S 15.25±2.01 S 环丙沙星 Ciprofloxacin 5 16.37±1.07 I 16.98±1.56 I 恩诺沙星 Enrofloxacin 10 15.04±0.87 R 14.91±0.44 R 呋喃唑酮 Furazolidone 100 11.77±1.08 R 10.81±0.43 R 复方新诺明 SMZ/TMP 23.75/1.25 0 R 0 R 庆大霉素 Gentamicin 10 13.43±0.47 I 12.38±0.17 I 链霉素 Streptomycin 10 8.12±0.16 R 8.08±0.12 R 头孢唑啉 Cefazolin 30 10.01±0.78 R 9.73±0.04 R 四环素 Tetracycline 30 12.89±0.73 R 12.90±1.15 R 红霉素 Erythromycin 15 16.86±0.91 I 15.04±0.68 I 氯霉素 Chloramphenicol 30 20.93±1.08 S 21.19±0.45 S 氨苄西林 Ampicillin 10 0 R 0 R 氧氟沙星 Ofloxacin 5 15.72±0.73 I 15.88±0.28 I 诺氟沙星 Norfloxacin 10 14.24±0.62 I 14.75±0.84 I 万古霉素 Vancomycin 30 0 R 0 R 注：R. 耐药；I. 中度敏感；S. 高度敏感。 Note: R. Resistance; I. Moderately sensitivity; S. Highly sensitivity.
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