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Article(id=1192149557367681182, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1192149543010582589, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250246, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1742918400000, receivedDateStr=2025-03-26, revisedDate=null, revisedDateStr=null, acceptedDate=1747584000000, acceptedDateStr=2025-05-19, onlineDate=1762160203544, onlineDateStr=2025-11-03, pubDate=1756915200000, pubDateStr=2025-09-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762160203544, onlineIssueDateStr=2025-11-03, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762160203544, creator=13701087609, updateTime=1762160203544, updator=13701087609, issue=Issue{id=1192149543010582589, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='10', pageStart='4241', pageEnd='4713', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1762160200113, creator=13701087609, updateTime=1762160638682, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1192151382586175735, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1192149543010582589, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1192151382586175736, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1192149543010582589, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4653, endPage=4666, ext={EN=ArticleExt(id=1192149557560619170, articleId=1192149557367681182, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Effects of calcium-free conditions on the functional phenotypes and gene expression of Vibrio parahaemolyticus, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To investigate the effects of calcium ion-free (Ca2+-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca2+ in culture media to create Ca2+-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca2+-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca2+-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca2+-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca2+-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca2+-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

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【目的】 分析无钙离子(Ca2+)环境对副溶血弧菌生物膜形成、毒力及基因表达的影响。 【方法】 向培养基中添加乙二醇双(2-氨基乙基醚)四乙酸(ethylene glycol-tetraacetic acid, EGTA)以螯合Ca2+，从而构建无Ca2+环境。采用结晶紫染色实验、泳动及群集运动实验检测副溶血弧菌的生物膜形成能力和运动能力；通过神奈川现象检测、HeLa细胞黏附及细胞毒性实验分析副溶血弧菌的毒力表型；利用RNA-seq技术分析无Ca2+环境对副溶血弧菌基因表达的影响。 【结果】 无Ca2+环境可抑制副溶血弧菌的生长，显著降低其生物膜形成能力、胞内c-di-GMP水平、泳动及群集运动能力。同时，该环境还能抑制副溶血弧菌的溶血活性及细胞黏附活性，但增强其细胞毒性。转录组分析显示，无Ca2+环境下有359个基因呈现显著差异表达(differentially expressed genes, DEGs)，这些基因涉及生物膜形成、运动、毒力及调控子相关基因；其中，参与侧鞭毛和极鞭毛合成的基因表达下调，多数与毒力相关的基因表达上调，推定的调控子基因表达下调。 【结论】 无Ca2+环境对副溶血弧菌的生物膜形成、运动能力、毒力以及基因表达均具有显著影响。

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作者贡献声明

李雪：提出概念、数据分析、撰写文章；张苗苗：数据收集与监管；罗茜：验证；张义全：监督管理、审阅文章；陆仁飞：获取基金。

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FEMS Microbiology Reviews, 2021, 45(5): fuab020., articleTitle=An update of the unceasingly growing and diverse AraC/XylS family of transcriptional activators, refAbstract=null), Reference(id=1192170582360866824, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, doi=null, pmid=null, pmcid=null, year=2020, volume=86, issue=10, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[31], rfOrder=31, authorNames=GREGORY GJ, MORREALE DP, BOYD EF, journalName=Applied and Environmental Microbiology, refType=null, unstructuredReference= GREGORY GJ, MORREALE DP, BOYD EF. CosR is a global regulator of the osmotic stress response with widespread distribution among bacteria[J]. Applied and Environmental Microbiology, 2020, 86(10): e00120-20., articleTitle=CosR is a global regulator of the osmotic stress response with widespread distribution among bacteria, refAbstract=null)], funds=[Fund(id=1192170579093505000, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, awardId=MS2023069, language=EN, fundingSource=the Research Projects of Nantong Health Commission(MS2023069), fundOrder=null, country=null), Fund(id=1192170580108526569, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, awardId=MS2023069, language=CN, fundingSource=南通市卫生健康委员会科研课题项目(MS2023069), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1192170575733867440, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, xref=null, ext=[AuthorCompanyExt(id=1192170575742256049, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, companyId=1192170575733867440, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Department of Clinical Laboratory, Nantong Third People’s Hospital, Affiliated Nantong Hospital 3 of Nantong University, Nantong, Jiangsu, China), AuthorCompanyExt(id=1192170575750644658, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, companyId=1192170575733867440, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=南通市第三人民医院/南通大学附属南通第三医院检验科，江苏 南通)])], figs=[ArticleFig(id=1192170577763910614, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 1, caption=Growth curves analysis. The OD 600 values were monitored at 1 h intervals.**: P<0.01., figureFileSmall=hFzk2Df/8trwb/m85nOdow==, figureFileBig=1kyhQckVXANWOEoTaxM2Tg==, tableContent=null), ArticleFig(id=1192170577818436567, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图1, caption=生长曲线分析, figureFileSmall=hFzk2Df/8trwb/m85nOdow==, figureFileBig=1kyhQckVXANWOEoTaxM2Tg==, tableContent=null), ArticleFig(id=1192170577914905560, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 2, caption=Biofilm formation assay by crystal violet staining. **: P<0.01; ****: P<0.000 1; ns: P>0.05., figureFileSmall=23b/8xxiS3BmXMc3ZikY1A==, figureFileBig=e/Cot2opZQ5s09gUzrFeNw==, tableContent=null), ArticleFig(id=1192170577969431513, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图2, caption=结晶紫染色定量分析, figureFileSmall=23b/8xxiS3BmXMc3ZikY1A==, figureFileBig=e/Cot2opZQ5s09gUzrFeNw==, tableContent=null), ArticleFig(id=1192170578028151770, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 3, caption=Relative intracellular c-di-GMP concentration. *: P<0.05., figureFileSmall=jrl0HQhRaQ4WmcidFOykzA==, figureFileBig=uovZ956hBqHxyIA9PJrb5Q==, tableContent=null), ArticleFig(id=1192170578086872027, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图3, caption=胞内c-di-GMP相对浓度, figureFileSmall=jrl0HQhRaQ4WmcidFOykzA==, figureFileBig=uovZ956hBqHxyIA9PJrb5Q==, tableContent=null), ArticleFig(id=1192170578145592284, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 4, caption=Motility phenotype analysis. Swimming (A) or swarming (B) capacity of Vibrio parahaemolyticus was measured by detection of the diameters of swimming or swarming areas in a semi-solid swimming or on swarming agar. The images depict swimming at 7 hours (A) and swarming at 72 hours (B). *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.000 1; ns: P>0.05., figureFileSmall=W4X4jKD63c0AAjCtoosfng==, figureFileBig=8Sn8wqNrWNP9vvP+A6BT4A==, tableContent=null), ArticleFig(id=1192170578208506845, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图4, caption=动力表型分析。A：游动能力(swimming)，纵坐标表示对应时间内细菌在半固体培养基中的游动直径，图片为7 h拍摄结果；B：群集运动能力(swarming)，纵坐标表示对应时间内细菌在平板表面的群集运动直径，图片为72 h拍摄结果。, figureFileSmall=W4X4jKD63c0AAjCtoosfng==, figureFileBig=8Sn8wqNrWNP9vvP+A6BT4A==, tableContent=null), ArticleFig(id=1192170578267227102, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 5, caption=Virulence phenotype analysis. A: Kanagawa phenomenon detection (The left plate is Wagatsuma agar supplemented with 4 mmol/L EGTA, while the right plate is without EGTA); B: Adherence against HeLa cells (HeLa cells were infected with Vibrio parahaemolyticus at a MOI of 10 for 90 minutes[14]. Adhesion rate is expressed as the number of adherent bacteria per initial bacterial inoculum); C: Cytotoxicity against HeLa cells (HeLa cells were infected with Vibrio parahaemolyticus at a MOI of 2.5 for 3 h. The cytotoxicity of Vibrio parahaemolyticus against HeLa cells was evaluated in terms of the release of LDH). **: P<0.01., figureFileSmall=ZgA6HRB/1puLdSAnSkgagw==, figureFileBig=jIKjfLtqXde00mgEfyU/QQ==, tableContent=null), ArticleFig(id=1192170578338530271, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图5, caption=毒力表型分析。A：神奈川现象检测(左为添加了4 mmol/L EGTA的我妻氏平板，右为无添加的我妻氏平板)；B：细胞黏附活性(副溶血弧菌按10倍MIO感染HeLa细胞90 min后[14]，黏附率表示为细菌黏附数量/初始菌量)；C：细胞毒性(副溶血弧菌按2.5倍MIO感染HeLa细胞3 h后，测定每组细胞LDH的释放量，纵坐标表示为每组LDH释放量/完全释放量)。, figureFileSmall=ZgA6HRB/1puLdSAnSkgagw==, figureFileBig=jIKjfLtqXde00mgEfyU/QQ==, tableContent=null), ArticleFig(id=1192170578405639136, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 6, caption=Transcriptome differential expression analysis. A: Volcano plot (HI-EGTA was used as the control group. Red, blue and grey points represent the up-regulated, down-regulated and no-significant regulated genes, respectively); B: The enrichment of gene ontology (GO) term (Green, red and blue bars represent molecular function, cellular component and biological process, respectively. The number on the top of each bar indicates the number of enriched genes); C: Pathways of differentially expressed genes were enriched by Kyoto encyclopedia of genes and genomes (KEGG) (The vertical axis represents KEGG classification, while the horizontal axis represents the number of DEGs); D: Cluster of orthologous groups of proteins (COG) (The vertical axis represents COG classification, whereas the horizontal axis represents the number of DEGs. Red and blue bars represent up-regulated and down-regulated genes, respectively)., figureFileSmall=7ced+9oUF9EyD+2WSdsIHQ==, figureFileBig=1mL7UbchXc+NcCG0EzbcAg==, tableContent=null), ArticleFig(id=1192170578468553697, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图6, caption=转录组差异分析。A：火山图(以HI-EGTA为对照组，红点、蓝点、灰色分别代表上调、下调、无显著差异)；B：DEGs的GO富集(绿色、红色和蓝色条分别代表分子功能、细胞成分和生物过程，每个条形图顶部的数字表示富集基因的数量)；C：DEGs的KEGG分类(纵轴表示功能类别，横轴表示基因数量)；D：DEGs的COG富集(纵轴表示COG类型，横轴表示基因数量；红色和蓝色分别表示上调基因和下调基因)。, figureFileSmall=7ced+9oUF9EyD+2WSdsIHQ==, figureFileBig=1mL7UbchXc+NcCG0EzbcAg==, tableContent=null), ArticleFig(id=1192170578523079650, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 7, caption=Validation of DEGs by RT-qPCR. A total of 16 genes from various pathways listed in Table 1 were selected for RT-qPCR analysis, with the 16S rRNA gene used as the internal control., figureFileSmall=rI8zhHpWfLCMCCJv4sW/xA==, figureFileBig=C8FImoVmBZGpn4QrtV4/Xg==, tableContent=null), ArticleFig(id=1192170578585994211, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图7, caption= RT-qPCR验证差异表达基因。选取了表1中各通路的共16个基因，RT-qPCR以16S rRNA基因作为对照。, figureFileSmall=rI8zhHpWfLCMCCJv4sW/xA==, figureFileBig=C8FImoVmBZGpn4QrtV4/Xg==, tableContent=null), ArticleFig(id=1192170578716017636, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Figure 8, caption=Regulatory effects of Ca2+-free conditions on functional phenotypes of Vibrio parahaemolyticus. Red upward arrows indicate positive regulation, red downward arrows indicate negative regulation, and question marks indicate unclear mechanisms of action., figureFileSmall=kSIju5I+cbO2QXU5DDdIPg==, figureFileBig=RZFC2XqjQb0pEue03EboOg==, tableContent=null), ArticleFig(id=1192170578778932197, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=图8, caption=无Ca2+ 环境对副溶血弧菌功能表型的调控作用模式图。红色向上箭头表示正调控，红色向下箭头表示负调控，问号表示作用方式尚不清晰。, figureFileSmall=kSIju5I+cbO2QXU5DDdIPg==, figureFileBig=RZFC2XqjQb0pEue03EboOg==, tableContent=null), ArticleFig(id=1192170578850235366, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=EN, label=Table 1, caption=

Potential biofilm/motility/virulence-related genes in differentially expressed genes

, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Name Fold change* Functional annotation/domains
Regulator
VP0067 2.1 LysR family transcriptional regulator
VP0358 2.9 DeoR family transcriptional regulator
VP1212 2.6 DNA-binding response regulator
VP1277 7.1 Histidine utilization repressor
VP1676 2.4 Transcriptional regulator
VP1763 2.3 MarR family transcriptional regulator
VP1889 2.6 Cold shock transcriptional regulator CspA
VP2406 2.9 LysR family transcriptional regulator
VP2520 pdhR 0.5 Transcriptional regulator PdhR
VP2603 0.0 Iron-regulated virulence regulatory protein
VP2927 nusG 0.4 Transcription antitermination protein NusG
VPA0148 0.2 Transcriptional regulator CpxR
VPA0149 0.1 Two-component system sensor kinase
VPA0497 2.0 ArsR family transcriptional regulator
VPA0663 0.0 AraC family transcriptional regulator
VPA0678 0.1 Response regulator
VPA0961 2.4 Transcriptional regulator
VPA1114 2.0 Transcriptional regulator BetI
VPA1124 5.0 Transcriptional regulator
VPA1423 8.3 Transcriptional regulator
c-di-GMP metabolism
VP1881 0.4 EAL-only
VP2888 2.2 GGDEF-only
Type IV pili
VP2697 mshA 2.1 MSHA pilin protein MshA
T3SS1
VP1676 2.4 Transcriptional regulator
VP1686 vopS 0.5 Adenosine monophosphate-protein transferase
VP1696 vscC 0.5 Type III secretion protein YscC
VPaI-7 and T3SS2
VPA1314 tdh2 0.5 TDH
T6SS1
VP1393 hcp1 0.3 BfdA protein
VP1401 0.5 Hypothetical protein
VP1402 vipA1 0.5 Hypothetical protein
VP1403 vipB1 0.5 Hypothetical protein
VP1410 VP1410 0.5 Hypothetical protein
Lateral/polar flagella
VP0780 flgF 4.3 Flagellar basal body rod protein FlgF
VP0781 flgG 3.7 Flagellar basal body rod protein FlgG
VP0782 flgH 3.1 Flagellar basal body L-ring protein
VP0783 flgI 2.2 P-ring biosynthesis protein FlgA
VP0784 flgJ 2.1 Flagellar rod assembly protein FlgJ
VP0785 flgK 5.9 Flagellar hook-associated protein FlgK
VP0786 flgL 3.2 Flagellar hook-associated protein FlgL
VP0788 flaC 4.1 Flagellin
VP0790 flaD 5.3 Flagellin
VP0791 flaE 3.9 Flagellin
VP2111 motY 2.4 Sodium-type flagellar protein MotY
VP2254 fliS 3.6 Flagellar protein FliS
VP2256 fliD 2.4 Flagellar capping protein
VP2258 flaA 2.2 Flagellin
VP2259 flaB 6.4 Flagellin
VP2261 flaF 3.4 Flagellin
VP2811 motX 2.2 Sodium-type polar flagellar protein MotX
VP1088 2.1 Chemotaxis transducer
VP2827 3.0 Methyl-accepting chemotaxis protein
VPA1449 2.3 Methyl-accepting chemotaxis protein
), ArticleFig(id=1192170578934121447, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1192149557367681182, language=CN, label=表1, caption=

差异表达基因中可能与生物膜、动力及毒力有关的基因

, figureFileSmall=null, figureFileBig=null, tableContent=
Gene Name Fold change* Functional annotation/domains
Regulator
VP0067 2.1 LysR family transcriptional regulator
VP0358 2.9 DeoR family transcriptional regulator
VP1212 2.6 DNA-binding response regulator
VP1277 7.1 Histidine utilization repressor
VP1676 2.4 Transcriptional regulator
VP1763 2.3 MarR family transcriptional regulator
VP1889 2.6 Cold shock transcriptional regulator CspA
VP2406 2.9 LysR family transcriptional regulator
VP2520 pdhR 0.5 Transcriptional regulator PdhR
VP2603 0.0 Iron-regulated virulence regulatory protein
VP2927 nusG 0.4 Transcription antitermination protein NusG
VPA0148 0.2 Transcriptional regulator CpxR
VPA0149 0.1 Two-component system sensor kinase
VPA0497 2.0 ArsR family transcriptional regulator
VPA0663 0.0 AraC family transcriptional regulator
VPA0678 0.1 Response regulator
VPA0961 2.4 Transcriptional regulator
VPA1114 2.0 Transcriptional regulator BetI
VPA1124 5.0 Transcriptional regulator
VPA1423 8.3 Transcriptional regulator
c-di-GMP metabolism
VP1881 0.4 EAL-only
VP2888 2.2 GGDEF-only
Type IV pili
VP2697 mshA 2.1 MSHA pilin protein MshA
T3SS1
VP1676 2.4 Transcriptional regulator
VP1686 vopS 0.5 Adenosine monophosphate-protein transferase
VP1696 vscC 0.5 Type III secretion protein YscC
VPaI-7 and T3SS2
VPA1314 tdh2 0.5 TDH
T6SS1
VP1393 hcp1 0.3 BfdA protein
VP1401 0.5 Hypothetical protein
VP1402 vipA1 0.5 Hypothetical protein
VP1403 vipB1 0.5 Hypothetical protein
VP1410 VP1410 0.5 Hypothetical protein
Lateral/polar flagella
VP0780 flgF 4.3 Flagellar basal body rod protein FlgF
VP0781 flgG 3.7 Flagellar basal body rod protein FlgG
VP0782 flgH 3.1 Flagellar basal body L-ring protein
VP0783 flgI 2.2 P-ring biosynthesis protein FlgA
VP0784 flgJ 2.1 Flagellar rod assembly protein FlgJ
VP0785 flgK 5.9 Flagellar hook-associated protein FlgK
VP0786 flgL 3.2 Flagellar hook-associated protein FlgL
VP0788 flaC 4.1 Flagellin
VP0790 flaD 5.3 Flagellin
VP0791 flaE 3.9 Flagellin
VP2111 motY 2.4 Sodium-type flagellar protein MotY
VP2254 fliS 3.6 Flagellar protein FliS
VP2256 fliD 2.4 Flagellar capping protein
VP2258 flaA 2.2 Flagellin
VP2259 flaB 6.4 Flagellin
VP2261 flaF 3.4 Flagellin
VP2811 motX 2.2 Sodium-type polar flagellar protein MotX
VP1088 2.1 Chemotaxis transducer
VP2827 3.0 Methyl-accepting chemotaxis protein
VPA1449 2.3 Methyl-accepting chemotaxis protein
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无钙环境对副溶血弧菌功能表型及基因表达的影响
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李雪 , 张苗苗 , 罗茜 , 张义全 , 陆仁飞
微生物学报 | 研究报告 2025,65(10): 4653-4666
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微生物学报 | 研究报告 2025, 65(10): 4653-4666
无钙环境对副溶血弧菌功能表型及基因表达的影响
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李雪, 张苗苗, 罗茜, 张义全, 陆仁飞
作者信息
  • 南通市第三人民医院/南通大学附属南通第三医院检验科，江苏 南通
Effects of calcium-free conditions on the functional phenotypes and gene expression of Vibrio parahaemolyticus
Xue LI, Miaomiao ZHANG, Xi LUO, Yiquan ZHANG, Renfei LU
Affiliations
  • Department of Clinical Laboratory, Nantong Third People’s Hospital, Affiliated Nantong Hospital 3 of Nantong University, Nantong, Jiangsu, China
出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250246
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摘要
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【目的】 分析无钙离子(Ca2+)环境对副溶血弧菌生物膜形成、毒力及基因表达的影响。 【方法】 向培养基中添加乙二醇双(2-氨基乙基醚)四乙酸(ethylene glycol-tetraacetic acid, EGTA)以螯合Ca2+，从而构建无Ca2+环境。采用结晶紫染色实验、泳动及群集运动实验检测副溶血弧菌的生物膜形成能力和运动能力；通过神奈川现象检测、HeLa细胞黏附及细胞毒性实验分析副溶血弧菌的毒力表型；利用RNA-seq技术分析无Ca2+环境对副溶血弧菌基因表达的影响。 【结果】 无Ca2+环境可抑制副溶血弧菌的生长，显著降低其生物膜形成能力、胞内c-di-GMP水平、泳动及群集运动能力。同时，该环境还能抑制副溶血弧菌的溶血活性及细胞黏附活性，但增强其细胞毒性。转录组分析显示，无Ca2+环境下有359个基因呈现显著差异表达(differentially expressed genes, DEGs)，这些基因涉及生物膜形成、运动、毒力及调控子相关基因；其中，参与侧鞭毛和极鞭毛合成的基因表达下调，多数与毒力相关的基因表达上调，推定的调控子基因表达下调。 【结论】 无Ca2+环境对副溶血弧菌的生物膜形成、运动能力、毒力以及基因表达均具有显著影响。

副溶血弧菌  /  钙离子  /  生物膜  /  毒力  /  转录组分析

[Objective] To investigate the effects of calcium ion-free (Ca2+-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca2+ in culture media to create Ca2+-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca2+-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca2+-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca2+-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca2+-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca2+-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

Vibrio parahaemolyticus  /  calcium ion  /  biofilm  /  virulence  /  transcriptome analysis
李雪, 张苗苗, 罗茜, 张义全, 陆仁飞. 无钙环境对副溶血弧菌功能表型及基因表达的影响. 微生物学报, 2025 , 65 (10) : 4653 -4666 . DOI: 10.13343/j.cnki.wsxb.20250246
Xue LI, Miaomiao ZHANG, Xi LUO, Yiquan ZHANG, Renfei LU. Effects of calcium-free conditions on the functional phenotypes and gene expression of Vibrio parahaemolyticus[J]. Acta Microbiologica Sinica, 2025 , 65 (10) : 4653 -4666 . DOI: 10.13343/j.cnki.wsxb.20250246
正文
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副溶血弧菌(Vibrio parahaemolyticus)是一种革兰氏阴性嗜盐弧菌，广泛分布于海洋及河口环境，是全球范围内食源性疾病的主要病原体之一[1]。自1996年首次报道以来，O3:K6及其变异血清型的大流行菌株在全球范围内快速传播，引发多起食物中毒事件暴发；尤其在东亚、东南亚沿海地区，其感染率持续攀升，已成为我国细菌性食源性疾病的重要病原菌[2-3]。流行病学数据显示，副溶血弧菌可通过摄入未煮熟的海产品引发急性胃肠炎，免疫功能低下的患者可出现败血症甚至死亡，对公共卫生安全构成重大威胁[4]。此外，该菌还可感染鱼、虾等水产养殖动物，引起急性肝胰腺坏死[5]，造成严重经济损失。
副溶血弧菌的致病性与其复杂的毒力因子网络密切相关，主要包括热稳定性直接溶血素(thermostable direct hemolysin, TDH)、TDH相关溶血素(TDH-related hemolysin, TRH)、III型分泌系统(type III secretion system, T3SS)和VI型分泌系统(type VI secretion system, T6SS)[4]。其中，TDH、TRH是副溶血弧菌的主要致病因子，TDH可直接作用于宿主红细胞膜，通过形成跨膜孔道引发β-溶血现象，同时具有肠毒性、心脏毒性和细胞毒性，是导致宿主肠道炎症及组织损伤的核心效应蛋白[6]。TRH与TDH具有约68%的氨基酸一致性[6-7]，其溶血活性较TDH稍弱，但同样可通过破坏宿主细胞膜完整性及调控离子通道功能，参与细菌的侵袭和免疫逃逸过程。T3SS1通过介导宿主细胞毒性参与系统性感染，而T3SS2主要介导肠道炎症反应[8-9]；T6SS1通过抗菌活性增强细菌的环境适应性，T6SS2则介导细菌对宿主细胞的黏附[10]。此外，副溶血弧菌可通过形成生物膜增强环境耐受性[11-12]，并利用鞭毛驱动的运动介导其在环境中的趋化性及宿主体内定殖[13]。值得注意的是，其表型变化和基因表达受多种环境因素调控，如营养成分(几丁质、脂肪酸、金属离子等)和物理因素(温度、pH、渗透压等)等[14-18]。
钙离子(Ca2+)作为关键的环境信号分子，在介导细菌环境适应和毒力调控中发挥重要作用[14,19-20]。已有报道显示，在副溶血弧菌BB22中，高浓度的Ca2+通过激活侧鞭毛基因的表达介导其在固体表面的群集运动，并诱导T3SS1基因的表达增强其细胞毒活性[20]。最近的研究发现，Ca2+浓度增加显著诱导副溶血弧菌RIMD2210633株的生物膜形成、环二鸟苷酸(c-di-GMP)合成以及游动能力，同时对毒力表型和基因表达也存在显著影响[14]。这些研究揭示了副溶血弧菌能通过调整生理状态和基因表达来响应高Ca2+环境，但无Ca2+环境对其表型和基因表达的影响仍缺乏系统性研究。此外，人体肠道中Ca2+的水平存在显著波动(0.5-4.5 mmol/L)[20]，这种梯度变化可能对副溶血弧菌的致病过程产生关键影响：高Ca2+环境可能促进其在肠道的初始定殖，而低Ca2+环境可能触发毒力因子的差异化表达。因此，解析无Ca2+环境下副溶血弧菌的生理重塑机制，不仅有助于理解其从自然环境到宿主肠道的适应策略，也为靶向钙稳态的抗菌干预提供了新思路。
本研究系统分析了无Ca2+环境对副溶血弧菌生长、生物膜形成、胞内c-di-GMP水平、运动能力、溶血活性、细胞黏附活性和细胞毒性的影响，并通过转录组学研究了其基因表达的变化，重点关注与生物膜形成、毒力因子和调控子相关的基因，旨在为理解副溶血弧菌在不同环境条件下的适应机制提供重要依据，并为开发针对钙稳态的抗菌策略提供理论支持。
1 材料与方法
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1.1 菌株和主要试剂
副溶血弧菌RIMD2210633株、HeLa细胞均由本实验室保存。Bacto heart infusion肉汤、Difco noble琼脂，碧迪医疗器械(上海)有限公司；EGTA，上海阿拉丁生化科技股份有限公司；BCA蛋白定量试剂盒，赛默飞世尔科技公司；微生物c-di-GMP ELISA试剂盒，武汉默沙克生物科技有限公司；我妻氏培养基，青岛高科技工业园海博生物技术有限公司；乳酸脱氢酶(lactate dehydrogenase, LDH)检测试剂盒，上海碧云天生物技术股份有限公司。
1.2 副溶血弧菌的预培养
取10 μL甘油菌种接种于5 mL的HI (2.5% Bacto heart infusion)肉汤中，37 ℃、200 r/min培养过夜；将过夜培养物按1:50稀释后接种至5 mL新鲜的HI肉汤中，37 ℃、200 r/min培养至对数中期(OD 600=1.4)。
1.3 生长曲线测定
将预培养物按1:1 000稀释后接种至15 mL新鲜HI肉汤或添加4 mmol/L EGTA的HI肉汤(HI-EGTA)中[20]，37 ℃、200 r/min培养，每隔1 h测定OD 600，连续培养至平台期。
1.4 结晶紫染色
采用24孔板培养法，将预培养物按1:1 000稀释后接种至1.5 mL的HI或HI-EGTA肉汤中，30 ℃、100 r/min分别培养6、12、24、48 h。吸去液体培养物，并测量菌液浊度(OD 600)；菌膜用去离子水洗涤3次，干燥后加入2 mL的0.1%结晶紫溶液，常温染色30 min，去离子水洗涤3次，干燥后加入2 mL的15%冰乙酸溶解结晶紫，并测定570 nm处的吸光度值(OD 570)。各菌株的生物膜相对生成量用OD 570/OD 600表示。
1.5 c-di-GMP测定
培养方式同结晶紫染色。收集1 mL菌体，用预冷的PBS洗涤并重悬，将菌液置于冰水浴条件下进行超声裂菌，裂解产物经4 ℃、12 000 r/min离心5 min。用微生物c-di-GMP的ELISA试剂盒按说明书操作，测定上清中的c-di-GMP含量；并用BCA蛋白定量试剂盒按说明书操作，测定上清中的总蛋白含量。c-di-GMP相对浓度用pmol/mg蛋白表示。
1.6 泳动实验
取2 μL预培养物，穿刺接种于半固体培养基(HI+0.3% Difco noble琼脂)中，37 ℃静置培养，每隔1 h测量1次菌苔直径。
1.7 群集运动实验
取2 μL预培养物，点种于平板(HI+2% Difco noble琼脂)表面，37 ℃静置培养，每隔24 h测量1次菌苔直径。
1.8 神奈川现象检测(溶血实验)
取2 μL预培养物，点种于我妻氏固体培养基表面，37 ℃下静置培养48 h，测定溶血环直径并拍照。
1.9 HeLa细胞黏附实验
收集生物膜培养12 h的产物，用DMEM洗涤并重悬，稀释菌液至106 CFU/mL。将1 mL/孔细菌悬液加入HeLa细胞预培养物中，置于37 ℃、5% CO2培养箱中孵育90 min，用PBS洗涤细胞3次，加入400 μL 0.5%的曲拉通作用10 min，产物经室温、4 000 r/min离心5 min，沉淀经PBS稀释后涂板计数，记录黏附于HeLa细胞表面的细菌数。
1.10 HeLa细胞毒试验
收集生物膜培养12 h的产物，用无酚红的DMEM洗涤并重悬，稀释菌液至106 CFU/mL，按2.5倍的感染复数(multiplicity of infection, MOI)感染HeLa细胞[14]，置于37 ℃、5% CO2培养箱中孵育3 h。采用LDH检测试剂盒测定感染后细胞的LDH释放量。
1.11 RNA提取及测序(RNA-seq)
将预培养物按1:1 000稀释后接种至10 mL新鲜HI肉汤或HI-EGTA中，37 ℃、200 r/min培养至OD 600=1.4，收集菌体，采用TRIzol法进行细菌RNA提取。RNA测序及数据分析委托苏州安升达生物技术有限公司进行。
1.12 统计方法
每个实验至少重复3次，每次至少设置3个生物学重复，所有数据均以均值±标准差(mean±SD)表示。采用学生配对或不配对t检验或双向方差分析与Tukey的事后校正进行多重比较来计算统计学显著性，P<0.05的值被认为具有统计学意义。
2 结果与分析
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2.1 无Ca2+环境抑制副溶血弧菌的生长
Ca2+作为细胞生理活动中重要的信号分子，参与众多基础代谢过程[18]。为解析无Ca2+环境对副溶血弧菌生存适应力的全局影响，本研究通过动态监测无Ca2+ (HI-EGTA)与含Ca2+ (HI)肉汤中细菌的生长曲线，评估其生长速率的差异。如图1所示，在HI-EGTA肉汤中培养的副溶血弧菌仍可生长，但进入对数期后生长速率明显低于HI组(P<0.01)，表明无Ca2+环境显著抑制副溶血弧菌的生长。
2.2 无Ca2+环境抑制副溶血弧菌的生物膜形成
副溶血弧菌通过形成生物膜来抵抗外界不利环境，其生物膜形成过程受众多信号分子和调控系统的协同作用[12,21]。Ca2+作为环境感知信号，可能通过影响相关调控通路的信号传递，参与生物膜形成的早期调控；也可能参与生物膜基质的构建，进而影响生物膜的成熟。为探究无Ca2+环境对生物膜形成的影响，本研究采用结晶紫染色法定量分析无Ca2+ (HI-EGTA)与含Ca2+ (HI)培养基中副溶血弧菌生物膜形成的动态变化。
结果显示，HI组生物膜量在6 h达到峰值，随后因成熟生物膜部分解离而逐渐下降；而HI-EGTA组生物膜量始终低于HI组，其12 h累积量仅为HI组的30%左右(P<0.05) (图2)。48 h后2组生物膜量无显著差异(P>0.05)，这可能是HI组的生物膜后期已基本解离所致。上述结果表明，无Ca2+环境明显降低副溶血弧菌的生物膜形成能力，尤其是在早期形成阶段。
2.3 无Ca2+环境抑制副溶血弧菌胞内c-di-GMP的合成
c-di-GMP作为生物膜形成过程中的调控枢纽，介导细菌在浮游-生物膜状态之间的转换，其浓度变化与生物膜形成密切相关[13]；而Ca2+可能通过影响c-di-GMP代谢酶基因表达或酶活性参与这一过程。为揭示Ca2+缺乏对c-di-GMP代谢的影响，本研究利用ELISA定量分析了无Ca2+ (HI-EGTA)与含Ca2+ (HI)条件下副溶血弧菌生物膜形成过程中胞内c-di-GMP的浓度变化。
结果显示，在生物膜形成早期(6-12 h) HI-EGTA组的c-di-GMP浓度较HI组明显降低(P<0.05) (图3)，表明Ca2+缺失显著抑制生物膜启动阶段的c-di-GMP合成；但在成熟期(24 h)，2组c-di-GMP水平趋于一致(P>0.05)。这一阶段性差异提示，Ca2+可能通过激活早期鸟苷酸环化酶(diguanylate cyclases, DGCs)或抑制磷酸二酯酶(phosphodiesterases, PDEs)活性发挥作用，而Ca2+缺失则抑制DGCs或激活PDEs，从而抑制生物膜初始定殖所需的c-di-GMP积累。综上所述，Ca2+的缺失通过抑制c-di-GMP代谢进而影响副溶血弧菌生物膜形成的启动效率。
2.4 无Ca2+环境降低副溶血弧菌的运动能力
副溶血弧菌的运动性是其适应海水环境动态变化及宿主感染的核心能力，其中极鞭毛驱动的游动(swimming)介导液体环境中的趋化行为，而侧鞭毛主导的群集运动(swarming)则促进表面迁移和组织扩散[22]。Ca2+作为重要的第二信使，可能通过调控鞭毛基因表达或蛋白活性影响运动表型。为明确Ca2+浓度变化对运动能力的影响，本研究通过点种法比较了无Ca2+ (HI-EGTA)与含Ca2+ (HI)环境下副溶血弧菌在游动平板和群集平板中的迁移能力。
结果显示，HI-EGTA组的游动能力在所有时间点均显著弱于HI组(P<0.05)，表现为游动环直径缩减20%-30% (图4A)；而在群集运动中，Ca2+缺失仅于48 h和72 h显著抑制细菌的表面迁移效率(迁移面积减少15%-20%，P<0.05) (图4B)。这表明Ca2+可能通过差异调控极鞭毛与侧鞭毛的功能影响不同运动模式的时序性表达：游动能力依赖持续性的Ca2+信号维持，而群集运动则在长期Ca2+匮乏时出现代偿性障碍。综上所述，Ca2+缺失通过抑制副溶血弧菌的游动及群集运动能力限制其环境适应与宿主侵袭效率，提示钙稳态调控是干预该菌传播与致病的关键靶点。
2.5 无Ca2+环境对副溶血弧菌的毒力表型的影响
副溶血弧菌的致病性与其毒力因子的动态调控密切相关，这些毒力因子包括TDH、TRH以及T6SS介导的宿主细胞黏附和T3SS介导的细胞毒性[10]。Ca2+作为关键信号分子，可能通过调节毒力基因表达和宿主互作过程影响细菌的致病能力。为探究Ca2+对副溶血弧菌毒力的调控作用，本研究通过神奈川现象检测、HeLa细胞黏附及细胞毒性实验，系统评估了无Ca2+ (HI-EGTA)和含Ca2+ (HI)环境下细菌的毒力表型变化。
神奈川实验结果显示，在添加了EGTA的我妻氏血琼脂平板上培养的副溶血弧菌，其β-溶血环直径较对照组(未添加EGTA)显著缩小(P<0.05) (图5A)，表明Ca2+缺失抑制了TDH/TRH介导的溶血活性。HeLa细胞感染试验结果显示，HI-EGTA组细菌的黏附率较HI组显著降低(P<0.05) (图5B)，提示Ca2+可能参与调控细菌表面黏附过程；然而与此矛盾的是，HI-EGTA组细菌的细胞毒性却显著增强(P<0.05) (图5C)，暗示Ca2+缺失可能激活了T3SS效应蛋白或毒素的释放，导致宿主细胞损伤加剧。综上所述，无Ca2+环境对副溶血弧菌的毒力表型具有双重调控作用：一方面通过抑制溶血活性和细胞黏附削弱其定殖能力，另一方面却通过增强细胞毒性加剧宿主组织损伤。这一发现揭示了Ca2+信号在细菌致病机制中的复杂角色，为靶向离子稳态的抗菌策略提供了新思路。
2.6 无Ca2+环境对副溶血弧菌基因表达的影响
为系统解析无Ca2+环境对副溶血弧菌基因表达谱的影响，本研究通过RNA-seq技术比较了无Ca2+ (HI-EGTA)和含Ca2+ (HI)条件下的转录组差异。与HI-EGTA组相比，HI组有359个基因的表达水平存在显著性差异，包括174个上调基因和185个下调基因(P<0.05) (图6A)。GO和KEGG富集显示，这些DEGs广泛参与代谢调控、环境信号感知及毒力因子表达。其中，12个DEGs属于生物体系统，122个DEGs参与细胞代谢，18个DEGs与人类疾病相关，48个DEGs涉及环境信息处理，14个DEGs在细胞过程中发挥作用(图6B、6C)。COG富集将DEGs分为至少20个功能类别，包括功能未知、仅进行一般功能预测、氨基酸转运和代谢、转录以及能量产生和转换等(图6D)。其中，可能与生物膜形成及毒力有关的基因有52个(变化倍数、P值和功能描述见表1)，并通
过RT-qPCR对部分DEGs进行验证，其表达差异与RNA-seq分析一致(图7)。生物膜及运动相关基因显著受抑制：2个c-di-GMP代谢酶基因(tpdA、VP2888)、IV型菌毛基因(VP2697)及多个鞭毛组装基因(如flaA、flaB等)显著下调，与前期生物膜形成及运动能力减弱表型一致(图2‒4)。毒力相关基因呈现差异化调控：2个T3SS1效应基因(vopS、vscC)表达上调，与细胞毒性增强现象(图5C)直接关联；而溶血素基因tdh2及T6SS1基因(如VP1393、VP1401)表达量上调，这与溶血活性和细胞黏附活性的抑制表型似乎并不一致(图5A、5B)，有待进一步讨论。此外，20个全局调控因子(如VP0067、VP0358等)的广泛下调进一步提示Ca2+可能通过转录级联协调毒力与代谢的分子机制(表1)。
3 讨论
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本研究探讨了无Ca2+环境对副溶血弧菌的功能表型和基因表达的影响，发现Ca2+缺失抑制了副溶血弧菌的生长、生物膜形成、胞内c-di-GMP水平及运动能力，同时降低了其溶血活性和细胞黏附活性，但增强了其细胞毒性(图1‒5)，Ca2+缺失还导致副溶血弧菌中359个基因的表达发生显著变化(图6、表1)。
3.1 无钙环境抑制副溶血弧菌生长
在生长特性方面，4 mmol/L Ca2+的添加似乎并不影响副溶血弧菌的生长[14]，但在无Ca2+环境中培养时副溶血弧菌的生长速率却被明显抑制(图1)。其机制可能涉及多个层次的生理调控失衡：(1) 外膜稳定性破坏：Ca2+通过中和外膜脂多糖层负电荷维持膜的低通透性和机械稳定性[23]，EGTA螯合Ca2+后膜流动性增加可能导致质子梯度紊乱，抑制ATP合成；(2) 代谢酶功能抑制：Ca2+是多种关键代谢酶的辅因子，如参与细胞壁合成的转肽酶和ATP依赖的跨膜转运蛋白[23]，其缺失可能干扰细胞壁合成与能量代谢，从而延缓生长代谢进程。
3.2 无钙环境通过调控c-di-GMP代谢抑制生物膜形成
在生物膜形成方面，无Ca2+环境显著抑制了副溶血弧菌生物膜的形成(图2)及胞内c-di-GMP水平(图3)，这与高Ca2+条件下c-di-GMP积累促进生物膜的表型形成鲜明对比[14]。尽管转录组数据中仅2个c-di-GMP代谢酶基因(tpdA和VP2888)的表达受Ca2+缺失调控(表1)，但其功能可能主导代谢平衡。其中，tpdA (编码磷酸二酯酶)在无Ca2+环境下表达上调(图7)，可能通过加速c-di-GMP降解抑制生物膜启动[24]；而VP2888 (推定的鸟苷酸环化酶)的下调(图7)则进一步减少c-di-GMP合成。结合前期研究[14]，推测Ca2+可能通过直接或间接调控TpdA和VP2888的活性或表达，动态平衡c-di-GMP代谢池。二者可能含有潜在的钙响应元件(如CalR结合位点[25])，Ca2+缺失解除其活性抑制/激活，从而减少c-di-GMP合成促进降解；但未来还需通过蛋白互作实验(如Pull-down或EMSA)进一步验证。
3.3 无钙环境通过调控鞭毛基因表达抑制运动性
在运动性方面，Ca2+缺失同时抑制群集运动和游动能力，但转录组分析仅显示1个侧鞭毛基因(VPA1535)下调，考虑到副溶血弧菌基因组中有39个侧鞭毛基因，仅1个基因的表达变化不太可能影响侧鞭毛的组装。相比之下，Ca2+缺失对极鞭毛的调控作用更加明显，有11个极鞭毛基因(表1、图7)表达下调。类似霍乱弧菌机制[26]，MotX/MotY形成Na⁺通道驱动鞭毛旋转，而motX和motY在无Ca2+环境下表达下调(表1)，可能导致鞭毛驱动力减弱。同时，钙结合蛋白CalR已被证实直接抑制鞭毛基因的转录(flaA、flaB等)[25]，Ca2+缺失可能增强CalR的DNA结合活性，从而抑制鞭毛组装。此外，侧鞭毛介导的群集运动和极鞭毛介导的游动在生物膜形成的初始黏附和成熟阶段都发挥重要作用，提示无Ca2+环境对鞭毛的抑制也是影响生物膜形成的重要原因。
3.4 无钙环境对毒力表型的双重调控：抑制溶血与黏附活性但增强细胞毒性
在毒力方面，环境Ca2+浓度对副溶血弧菌毒力的影响似乎更加复杂。一方面，Ca2+作为激活剂通过抑制CalR解除其对T3SS1基因簇和LafK的阻遏，增强黏附活性和细胞毒性[14,19-20]。另一方面，Ca2+的缺失是诱导副溶血弧菌T3SS基因表达和效应蛋白分泌的关键因素[20]。本研究中，Ca2+缺失增强副溶血弧菌的细胞毒性(图5C)，并同步上调T3SS1毒力基因簇中关键效应蛋白基因vopS和结构蛋白基因vscC的表达(表1、图7)，这与Gode-Potratz等[20]研究一致，但需要指出的是EGTA并非钙特异性，Fe2+的缺失同样会增强T3SS1转录和细胞毒力[20]。因此，EGTA对T3SS1的调控是否直接与Ca2+的缺失直接相关还需进一步确证。无Ca2+环境抑制副溶血弧菌溶血活性及黏附活性(图5A、5B)，但tdh2在无Ca2+环境中却被上调(表1、图7)，表型变化与RNA-seq数据间似乎存在一定矛盾，可能源于以下原因：一是TDH蛋白的溶血活性可能依赖Ca2+介导的构象变化[19]，无Ca2+环境下TDH虽表达量增加，但溶血活性受损[6]；二是神奈川实验与RNA-seq的培养条件不同，可能导致基因表达存在差异。相比之下，极鞭毛基因和IV型菌毛的下调(表1、图7)导致细菌与宿主细胞的物理接触减少，可能是黏附率降低的主要原因。
3.5 无钙环境下多家族调控子协同介导全局调控网络
在基因表达方面，转录组分析进一步揭示了Ca2+缺失对全局调控网络的潜在影响。除上述表型相关基因外，RNA-seq数据还显示，20个推测的调控子基因在Ca2+存在与缺失条件下表达差异显著，涵盖多种功能家族：LysR家族(VP0067、VP2406、VPA0961)、DeoR家族(VP0358)、ArsR家族(VPA0497)、AraC家族(VPA0663、VPA1423)以及MarR家族(VP1763)等；提示多家族协同作用可能构成层级化调控网络。具体而言，LysR家族成员VPA0961最近已被证实能通过激活副溶血弧菌侧鞭毛基因和极鞭毛的转录正调控游动和群集运动[27]，但其是否参与钙信号转导仍需进一步验证。DeoR家族成员VP0358的表达上调可能通过调控碳代谢途径间接影响细菌的能量供应和运动能力[28]。ArsR家族大多参与金属离子稳态调控[29]，VPA0497的上调可能与EGTA处理导致的金属离子失衡有关。AraC家族成员VPA0663和VPA1423的显著差异表达尤为关键，这类调控蛋白在多种细胞过程(如生物膜形成、T3SS等)中发挥作用[30]，其变化可能直接介导细胞毒性与生物膜表型的改变。此外，MarR家族的VP1763则可能通过调控渗透压反应基因的表达来应对环境压力[31]。值得注意的是，虽然LysR家族调控因子CalR是已知的钙结合调控因子[25]，但在转录组分析中并无显著性差异，推测Ca2+可能通过直接结合CalR蛋白(而非调控其转录)改变其构象或DNA结合能力，从而影响其对下游基因的调控。综上所述，副溶血弧菌可能通过调控这些转录因子的活性或表达，协调其对无Ca2+环境的适应性响应。
4 结论
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本研究系统揭示了无Ca2+环境对副溶血弧菌RIMD2210633株的多维度影响：抑制生长、生物膜形成、运动能力、溶血活性和黏附活性，同时通过“毒力代偿”策略增强T3SS依赖性细胞毒性。这种表型变化与Ca2+信号调控的转录密切相关，包括c-di-GMP代谢抑制、鞭毛基因下调、菌毛基因下调及T3SS1基因激活(图8)。这些发现不仅深化了对副溶血弧菌环境适应机制的理解，也为靶向钙稳态的抗菌策略提供了理论依据。然而，本研究也存在一定的局限性：首先，EGTA的非特异性效应会影响其他金属离子的浓度，仍需通过Ca2+特异性螯合剂或金属离子回补实验进一步验证；其次，差异表达的调控子(如VP0067、VPA0961)与钙信号通路的关联尚不明确，未来的研究应着重于解析这些调控因子的具体靶基因及其相互作用机制，以更全面地理解副溶血弧菌的环境适应策略；最后，实验室条件难以完全模拟宿主肠道内复杂的理化环境，后续需构建小鼠或斑马鱼感染模型，评估无Ca2+环境对副溶血弧菌体内定殖和致病力的实际影响；这些改进将有助于更全面地揭示钙信号在病原菌-宿主互作中的动态调控机制。
基金
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  • 南通市卫生健康委员会科研课题项目(MS2023069)
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2025年第65卷第10期
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doi: 10.13343/j.cnki.wsxb.20250246
  • 接收时间：2025-03-26
  • 首发时间：2025-11-03
  • 出版时间：2025-09-04
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  • 收稿日期：2025-03-26
  • 录用日期：2025-05-19
基金
the Research Projects of Nantong Health Commission(MS2023069)
南通市卫生健康委员会科研课题项目(MS2023069)
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    南通市第三人民医院/南通大学附属南通第三医院检验科，江苏 南通
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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