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Article(id=1226956559557771350, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250186, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1741190400000, receivedDateStr=2025-03-06, revisedDate=null, revisedDateStr=null, acceptedDate=1743350400000, acceptedDateStr=2025-03-31, onlineDate=1770458839549, onlineDateStr=2026-02-07, pubDate=1756915200000, pubDateStr=2025-09-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1770458839549, onlineIssueDateStr=2026-02-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1770458839549, creator=13701087609, updateTime=1770458839549, updator=13701087609, issue=Issue{id=1226956547847275311, tenantId=1146029695717560320, journalId=1192105938417971205, year='2025', volume='65', issue='9', pageStart='3821', pageEnd='4232', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1770458836757, creator=13701087609, updateTime=1770459153781, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1226957877613605816, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1226957877613605817, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1226956547847275311, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=4188, endPage=4197, ext={EN=ArticleExt(id=1226956560874782839, articleId=1226956559557771350, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Infection ability of LPXTG motif-anchored protein Lmo0130 in Listeria monocytogenes, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To investigate the role of Listeria monocytogenes LPXTG motif-anchored protein Lmo0130 in infection causing diseases, the bacterial growth, infection in cell and host among the L. monocytogenes wild-type, lmo0130-deleted and lmo0130-complementary strains were compared. [Methods] The lmo0130-deleted strain Δlmo0130 and lmo0130-complementary strain CΔlmo0130 were constructed to investigate the effects of Lmo0130 on the abilities of bacterial growth, cell surface adhesion and invasion, intracellular proliferation, intercellular migration, survival of infected mice, and bacterial load in mouse organs, ultimately demonstrated the role of L. monocytogenes Lmo0130 in cell and host infection. [Results] LPXTG motif-anchored protein Lmo0130 contributed to cell surface adhesion and invasion, intracellular proliferation, specific colonization in the liver and spleen, and pathogenicity in mice. However, it had no effect on bacterial growth or intercellular migration. [Conclusion] Lmo0130 contributes to cell and host infection of L. monocytogenes finally.

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*E-mail: CHENG Changyong,
SONG Houhui,
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【目的】 为了探究LPXTG基序锚定蛋白Lmo0130对单核增生李斯特氏菌EGD-e在感染致病中的影响，比较该菌EGD-e、lmo0130基因缺失株和回补株在生长、细胞和宿主感染等方面的差异。 【方法】 构建单核增生李斯特氏菌lmo0130基因缺失株Δlmo0130和回补株CΔlmo0130，探究Lmo0130对单核增生李斯特氏菌的生长能力、细胞表面的细菌黏附与侵袭能力、细胞内的细菌增殖能力、细胞间的迁移能力，以及感染小鼠的存活能力和细菌定殖能力等方面的影响，从而阐明Lmo0130对单核增生李斯特氏菌在细胞和宿主感染中的作用。 【结果】 LPXTG基序锚定蛋白Lmo0130有助于单核增生李斯特氏菌在细胞表面的黏附与侵袭、在细胞内的增殖、在感染小鼠组织中的定殖，并最终有助于在小鼠中的致病力，但对该菌的生长和胞间迁移能力无显著影响。 【结论】 本研究阐明了Lmo0130在单核增生李斯特氏菌感染细胞和宿主中的作用。

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作者贡献声明

邓思敏：研究构思和设计，数据收集和处理，论文撰写与修改；葛泓睿：实验操作和数据收集；单䶮：协助实验操作；毛敏杰：指导实验操作和图片绘制；徐加利：提供技术指导；夏菁：提供技术支持；宋厚辉：研究构思和设计；程昌勇：研究构思和设计，论文修改。

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M: DNA marker., figureFileSmall=VLO3AtN+4M8fbg88CRxxIg==, figureFileBig=Pos3J5xDBf5niHT5tb64Dw==, tableContent=null), ArticleFig(id=1226964056305746792, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图1, caption=单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 的菌落PCR验证, figureFileSmall=VLO3AtN+4M8fbg88CRxxIg==, figureFileBig=Pos3J5xDBf5niHT5tb64Dw==, tableContent=null), ArticleFig(id=1226964056444158835, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Figure 2, caption=The comparison of growth abilities among Listeria monocytogenes EGD-e, Δlmo0130, and CΔlmo0130 strains., figureFileSmall=YkCuHdMNU/YzwXWswhfY0g==, figureFileBig=zJEjeIedWA6FBhW5mYSQkg==, tableContent=null), ArticleFig(id=1226964056574182265, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图2, caption=比较单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 的生长水平差异, figureFileSmall=YkCuHdMNU/YzwXWswhfY0g==, figureFileBig=zJEjeIedWA6FBhW5mYSQkg==, tableContent=null), ArticleFig(id=1226964056687428480, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Figure 3, caption=The comparison of adherence (A) and invasion (B) abilities among Listeria monocytogenes EGD-e, Δlmo0130, and CΔlmo0130 in Caco-2 cells. *: 0.01<P<0.05; ns: P>0.05., figureFileSmall=gXjCyRyMMjRBhOfSH2btMA==, figureFileBig=mGzgiCRURFwQUYCJ0hBrnw==, tableContent=null), ArticleFig(id=1226964056788091783, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图3, caption=比较单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 的黏附(A)和侵袭(B)能力, figureFileSmall=gXjCyRyMMjRBhOfSH2btMA==, figureFileBig=mGzgiCRURFwQUYCJ0hBrnw==, tableContent=null), ArticleFig(id=1226964056922309519, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Figure 4, caption=The comparison of proliferation abilities among Listeria monocytogenes EGD-e, Δlmo0130, and CΔlmo0130 in RAW264.7 cells. A: Bacterial counts in cell; B: Proliferation abilities after 8 hours infection. *: 0.01<P<0.05; ***: P<0.001; ns: P>0.05., figureFileSmall=NytmjLGo8S5GPat8/1/FVw==, figureFileBig=ifQtABoEBsPRsBbseoL0Bw==, tableContent=null), ArticleFig(id=1226964057006195605, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图4, caption=比较单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 在RAW264.7中增殖能力的差异。A：胞内细菌数；B：感染8 h后的增殖率。, figureFileSmall=NytmjLGo8S5GPat8/1/FVw==, figureFileBig=ifQtABoEBsPRsBbseoL0Bw==, tableContent=null), ArticleFig(id=1226964057098470298, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Figure 5, caption=The comparison of intercellular migration abilities among Listeria monocytogenes EGD-e, Δlmo0130, and CΔlmo0130 in L929 cells. A: Cell plaque; B: Plaque size; C: Plaque number. ns: P>0.05., figureFileSmall=x1z1pxH2jHI2YqFESFQxyw==, figureFileBig=knL7nR05vxyIVmUdGjCu/A==, tableContent=null), ArticleFig(id=1226964057211716512, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图5, caption=比较单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 的胞间迁移能力差异。A：细菌蚀斑；B：蚀斑大小；C：蚀斑数目。, figureFileSmall=x1z1pxH2jHI2YqFESFQxyw==, figureFileBig=knL7nR05vxyIVmUdGjCu/A==, tableContent=null), ArticleFig(id=1226964057320768424, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Figure 6, caption=The comparison of pathogenicity among Listeria monocytogenes EGD-e, Δlmo0130 and CΔlmo0130 in infected mice. A: The survival curve of infected mice; B: The bacterial loads in liver and spleen of mice. *: 0.01<P<0.05; **: 0.001<P<0.01; ***: P<0.001; ns: P>0.05., figureFileSmall=XH5+T/H5i/KF93q9eTDvqw==, figureFileBig=j7PZtgC4sq2fcxnOKCTjrw==, tableContent=null), ArticleFig(id=1226964057459180462, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=图6, caption=比较单核增生李斯特氏菌EGD-e、Δlmo0130 和CΔlmo0130 对小鼠的致病力的差异。A：感染后小鼠的存活曲线；B：肝脏和脾脏的组织载菌量。, figureFileSmall=XH5+T/H5i/KF93q9eTDvqw==, figureFileBig=j7PZtgC4sq2fcxnOKCTjrw==, tableContent=null), ArticleFig(id=1226964057614369718, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=EN, label=Table 1, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)
lmo0130-up-FCCCAAGCTTGCTGCTTGCTACATAATGCGCC
lmo0130-up-RCAATCCAGCTGTATCAAATTTGTTCACTTTCACACTCTCCCTTTTT
lmo0130-down-FAAAGTGAACAAATTTGATACAGCTGGATTGGCAACTGTATTTG
lmo0130-down-RCCGGAATTCCACGGAATGAATGATCGTGGAATGAAGAAA
lmo0130-front-FCCGCGATAGCAAGTTCCGTTATTTC
CΔlmo0130-FGGGGATCGGAATTCGAGCTCTTCAAGAAAACTACACATGTTTTACTCGTAGCAG
CΔlmo0130-RGCAGCCCGGGGGATCCACCAGTTGTTGGTAAGGAAGTATTGG
lmo0130-in-FTTTTTTCCATCGAAATGAAAATATAGCCAAATTTTATCATTACA
lmo0130-in-RAGGGTGTTCCAGGAAGCTGG
lmo0130-out-FTGACAATGCTATCCGTGTTCAAGC
lmo0130-out-FAGGGTGTTCCAGGAAGCTGG
), ArticleFig(id=1226964057819890624, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1226956559557771350, language=CN, label=表1, caption=

引物序列信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Primers namePrimer sequences (5′→3′)
lmo0130-up-FCCCAAGCTTGCTGCTTGCTACATAATGCGCC
lmo0130-up-RCAATCCAGCTGTATCAAATTTGTTCACTTTCACACTCTCCCTTTTT
lmo0130-down-FAAAGTGAACAAATTTGATACAGCTGGATTGGCAACTGTATTTG
lmo0130-down-RCCGGAATTCCACGGAATGAATGATCGTGGAATGAAGAAA
lmo0130-front-FCCGCGATAGCAAGTTCCGTTATTTC
CΔlmo0130-FGGGGATCGGAATTCGAGCTCTTCAAGAAAACTACACATGTTTTACTCGTAGCAG
CΔlmo0130-RGCAGCCCGGGGGATCCACCAGTTGTTGGTAAGGAAGTATTGG
lmo0130-in-FTTTTTTCCATCGAAATGAAAATATAGCCAAATTTTATCATTACA
lmo0130-in-RAGGGTGTTCCAGGAAGCTGG
lmo0130-out-FTGACAATGCTATCCGTGTTCAAGC
lmo0130-out-FAGGGTGTTCCAGGAAGCTGG
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单核增生李斯特氏菌LPXTG基序锚定蛋白Lmo0130的感染生物学作用
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邓思敏 , 葛泓睿 , 单䶮 , 毛敏杰 , 徐加利 , 夏菁 , 宋厚辉 , 程昌勇
微生物学报 | 研究报告 2025,65(9): 4188-4197
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微生物学报 | 研究报告 2025, 65(9): 4188-4197
单核增生李斯特氏菌LPXTG基序锚定蛋白Lmo0130的感染生物学作用
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邓思敏, 葛泓睿, 单䶮, 毛敏杰, 徐加利, 夏菁, 宋厚辉 , 程昌勇
作者信息
  • 浙江农林大学 动物医学院，浙江省畜禽绿色生态健康养殖应用技术研究重点实验室，动物健康互联网检测技术浙江省工程研究中心，浙江省动物医学与健康管理国际科技合作基地，同一健康和食品安全“一带一路”国际联合实验室，中澳动物健康大数据分析联合实验室，浙江 杭州
Infection ability of LPXTG motif-anchored protein Lmo0130 in Listeria monocytogenes
Simin DENG, Hongrui GE, Yan SHAN, Minjie MAO, Jiali XU, Jing XIA, Houhui SONG , Changyong CHENG
Affiliations
  • Key Laboratory of Applied Technology on Green-Eco-Healthy on Animal Husbandry of Zhejiang Province, Zhejiang Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, the Belt and Road International Joint Laboratory for One Health and Food Safety, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Veterinary Medicine, Zhejiang A&F University, Hangzhou, Zhejiang, China
出版时间: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250186
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摘要
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【目的】 为了探究LPXTG基序锚定蛋白Lmo0130对单核增生李斯特氏菌EGD-e在感染致病中的影响，比较该菌EGD-e、lmo0130基因缺失株和回补株在生长、细胞和宿主感染等方面的差异。 【方法】 构建单核增生李斯特氏菌lmo0130基因缺失株Δlmo0130和回补株CΔlmo0130，探究Lmo0130对单核增生李斯特氏菌的生长能力、细胞表面的细菌黏附与侵袭能力、细胞内的细菌增殖能力、细胞间的迁移能力，以及感染小鼠的存活能力和细菌定殖能力等方面的影响，从而阐明Lmo0130对单核增生李斯特氏菌在细胞和宿主感染中的作用。 【结果】 LPXTG基序锚定蛋白Lmo0130有助于单核增生李斯特氏菌在细胞表面的黏附与侵袭、在细胞内的增殖、在感染小鼠组织中的定殖，并最终有助于在小鼠中的致病力，但对该菌的生长和胞间迁移能力无显著影响。 【结论】 本研究阐明了Lmo0130在单核增生李斯特氏菌感染细胞和宿主中的作用。

LPXTG基序锚定蛋白Lmo0130  /  感染致病  /  胞内增殖  /  单核增生李斯特氏菌

[Objective] To investigate the role of Listeria monocytogenes LPXTG motif-anchored protein Lmo0130 in infection causing diseases, the bacterial growth, infection in cell and host among the L. monocytogenes wild-type, lmo0130-deleted and lmo0130-complementary strains were compared. [Methods] The lmo0130-deleted strain Δlmo0130 and lmo0130-complementary strain CΔlmo0130 were constructed to investigate the effects of Lmo0130 on the abilities of bacterial growth, cell surface adhesion and invasion, intracellular proliferation, intercellular migration, survival of infected mice, and bacterial load in mouse organs, ultimately demonstrated the role of L. monocytogenes Lmo0130 in cell and host infection. [Results] LPXTG motif-anchored protein Lmo0130 contributed to cell surface adhesion and invasion, intracellular proliferation, specific colonization in the liver and spleen, and pathogenicity in mice. However, it had no effect on bacterial growth or intercellular migration. [Conclusion] Lmo0130 contributes to cell and host infection of L. monocytogenes finally.

LPXTG motif-anchored protein Lmo0130  /  infection causing diseases  /  intracellular proliferation  /  Listeria monocytogenes
邓思敏, 葛泓睿, 单䶮, 毛敏杰, 徐加利, 夏菁, 宋厚辉, 程昌勇. 单核增生李斯特氏菌LPXTG基序锚定蛋白Lmo0130的感染生物学作用. 微生物学报, 2025 , 65 (9) : 4188 -4197 . DOI: 10.13343/j.cnki.wsxb.20250186
Simin DENG, Hongrui GE, Yan SHAN, Minjie MAO, Jiali XU, Jing XIA, Houhui SONG, Changyong CHENG. Infection ability of LPXTG motif-anchored protein Lmo0130 in Listeria monocytogenes[J]. Acta Microbiologica Sinica, 2025 , 65 (9) : 4188 -4197 . DOI: 10.13343/j.cnki.wsxb.20250186
正文
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食源性胞内致病菌单核增生李斯特氏菌(Listeria monocytogenes)是重要的革兰氏阳性菌，也是引起人类食物中毒性死亡的第三大病原微生物[1-2]。单核增生李斯特氏菌经受污染的食物进入感染机体后，通过裂解吞噬泡进入细胞质中并大量增殖，进而在细胞间传播[3-4]，甚至突破血脑屏障或孕妇的胎盘屏障，引发孕妇流产，幼儿、老人和免疫力低下者脑膜炎和败血症等临床症状，病死率高达30%[5-6]。单核增生李斯特氏菌表达多种表面蛋白和分泌蛋白参与单核增生李斯特氏菌感染宿主的细胞表面黏附、细胞侵袭、细胞内增殖和细胞间传播等过程[7-8]。其中LPXTG基序锚定蛋白是羧基端带有LPXTG保守基序的一类蛋白质，通过SrtA分选酶将其共价结合到细胞壁肽聚糖上，并呈现在细菌表面。这类表面蛋白在感染机体过程中发挥重要的毒力作用，如重要毒力因子InlA[1,9]、Vip[10]和LapB[11]。在单核增生李斯特氏菌EGD-e的全基因组中发现41个LPXTG基序锚定蛋白[12-13]，且其中多个LPXTG基序锚定蛋白的感染生物学功能和机制未知。
本研究通过比较单核增生李斯特氏菌EGD-e、缺失株Δlmo0130和回补株CΔlmo0130在生长、细胞和宿主感染等方面的差异，明确LPXTG基序锚定蛋白Lmo0130在单核增生李斯特氏菌感染致病中的作用，旨在为进一步解析Lmo0130介导的感染机制奠定理论基础。
1 材料与方法
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1.1 菌株、质粒和引物
单核增生李斯特氏菌参考菌株EGD-e、缺失株Δlmo0130和回补株CΔlmo0130培养于牛脑心浸出液肉汤培养基(brain heart infusion, BHI)中，在37 ℃摇床或生化培养箱中振荡或静置培养。
本研究所涉及引物见表1。
1.2 Δlmo0130 缺失株和CΔlmo0130 回补株的构建和筛选
基于无痕基因敲除原理构建单核增生李斯特氏菌基因缺失株[14]。以lmo0130-up-F/R和lmo0130-down-F/R (表1)为引物，以过夜培养的单核增生李斯特氏菌EGD-e (GenBank登录号：NC_003210.1)菌液作为基因组DNA模板进行PCR反应分别扩增lmo0130基因的上、下游同源臂。PCR反应体系(50 μL)：2×KOD OneTM PCR Master Mix 25 µL，上、下游引物(10 µmol/L)各2 µL，DNA模板2 µL，ddH2O 19 µL。PCR反应条件：98 ℃预变性5 min；98 ℃变性10 s，58 ℃退火5 s，72 ℃延伸1 s，35个循环；72 ℃终延伸7 min。以lmo0130-up-F和lmo0130-down-R为引物，以lmo0130基因的上、下游同源臂扩增产物各1 µL为基因组DNA模板，参照上述PCR反应体系和条件通过重叠PCR获得上下游同源臂融合片段。该融合片段经Hind Ⅲ和EcoR Ⅰ双酶切后构建至pKSV7质粒中获得重组质粒pKSV7_lmo0130。随后将该重组质粒电转至单核增生李斯特氏菌EGD-e感受态中，通过温度和Cmr (10 μg/mL)抗性双重选择压力下进行同源重组克隆的筛选，并用引物lmo0130-front-F/lmo0130-down-R (表1)对筛选出的重组单克隆进行PCR和测序双重验证，最终获得缺失菌株Δlmo0130。
通过biocyc数据库查明单核增生李斯特氏菌lmo0130基因为单转录本，参考文献[14]构建回补株。设计引物CΔlmo0130-F/R (表1)扩增lmo0130的启动子和开放阅读框。扩增产物经Sac Ⅰ和BamH Ⅰ双酶切后构建至pIMK2质粒中获得重组质粒pIMK2_lmo0130。将该质粒电转至Δlmo0130感受态中，通过卡那霉素(50 μg/mL)抗性筛选，并对筛选出的单克隆经菌落PCR和测序验证后最终获得回补株CΔlmo0130。
通过lmo0130-in-F/R和lmo0130-out-F/R (表1)基因内外部引物对Δlmo0130和CΔlmo0130进行PCR鉴定。
1.3 生长曲线的绘制和菌落计数
参考文献[15]比较单核增生李斯特氏菌各菌株生长曲线的差异。过夜培养的菌液调整至相同OD600吸光值后转接至新鲜培养基中培养12 h，并每隔1 h用多功能酶标仪SynergyTM H1测定OD600吸光值。每个菌株设置3个重复。
1.4 细菌黏附与侵袭试验
参考文献[16]探究Lmo0130对单核增生李斯特氏菌在人肠上皮细胞Caco-2上黏附和侵袭的影响。培养到OD600为0.6的单核增生李斯特氏菌菌液经1×PBS (10 mmol/L)洗涤2次后用RPMI 1640细胞培养基稀释至2×106 CFU/mL备用。稀释后的菌液以感染复数(multiplicity of infection, MOI)=10感染人肠上皮细胞Caco-2。感染30 min后洗涤细胞3次，裂解细胞并倍比稀释后涂布于BHI平板上计数，黏附率计算如公式(1)所示。
黏附率=黏附后细菌数/感染前细菌数×100%
感染1.5 h后，加入50 µg/mL庆大霉素的作用30 min杀灭胞外菌，随后洗涤细胞3次，并裂解细胞计数，侵袭率计算如公式(2)所示。
侵袭率=侵袭后细菌数/感染前细菌数×100%
1.5 胞内增殖试验
参考文献[17]探究Lmo0130对单核增生李斯特氏菌在小鼠巨噬细胞RAW264.7中增殖能力的影响。培养到OD600为0.6的单核增生李斯特氏菌菌液，经1×PBS (10 mmol/L)洗涤2次后用DMEM细胞培养基稀释至5×106 CFU/mL。稀释后的菌液以MOI=10感染RAW264.7细胞。感染30 min后用50 μg/mL庆大霉素处理30 min。处理后的细胞洗涤3次后用含5 μg/mL庆大霉素的DMEM培养基(含10% FBS)继续培养1、4和7 h，然后裂解细胞进行倍比稀释并涂布在BHI平板上进行细菌计数。
1.6 细菌空斑试验
参考文献[14]探究Lmo0130对单核增生李斯特氏菌在小鼠成纤维细胞L929间迁移的影响。培养到OD600为0.6的单核增生李斯特氏菌菌液，经1×PBS洗涤2次后用DMEM细胞培养基稀释至2×105 CFU/mL。稀释后的菌液以MOI=0.2在6孔板中感染L929细胞。感染后的细胞继续培养1 h并在期间不时晃动细胞板使细菌分布均匀。随后洗涤细胞3次，加入50 µg/mL庆大霉素处理细胞1 h用于杀灭胞外细菌。洗涤细胞3次，然后加入3 mL含10 µg/mL庆大霉素和10% FBS的无酚红DMEM培养基与0.7%低熔点琼脂糖充分混匀后配制成的细胞覆盖琼脂，待琼脂凝固后倒置细胞板继续培养3 d直至空斑出现。将细胞经40%甲醛溶液固定和0.5%结晶紫染色10 min，再用ddH2O冲洗后测定空斑数量和大小。
1.7 细菌存活和组织定殖试验
参考文献[18]探究Lmo0130对单核增生李斯特氏菌感染小鼠存活能力和小鼠脏器中细菌定殖能力的影响。过夜培养的单核增生李斯特氏菌用1×PBS洗涤并稀释至1×107 CFU/mL备用。每只6周龄的雌性ICR小鼠腹腔注射200 µL稀释后的菌液，每组7只，共21只。感染24 h和48 h后，采集小鼠的脾脏(spleen)和肝脏(liver)进行组织匀浆，随后将组织悬液倍比稀释后涂布在BHI平板上进行细菌计数，每组去除最高值和最低值后进行数据处理。
腹腔注射200 µL稀释后的1×107 CFU/mL单核增生李斯特氏菌菌液感染雌性ICR小鼠，10只/组，共30只，感染后每隔12 h观察小鼠的存活情况并记录数据，共观察7 d。动物实验通过浙江农林大学实验动物伦理委员会审查，编号：ZAFUAC202482。
1.8 数据统计与处理
使用Graphpad Prism 8.0进行数据处理和显著性分析。除小鼠存活曲线采用LogRank以外，其余均采用T检验进行显著性分析，其中ns表示P>0.05，*表示0.01<P<0.05，**表示0.001<P<0.01，***表示P<0.001。使用Adobe Illustrator 2025进行图片排版。
2 结果与分析
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2.1 单核增生李斯特氏菌缺失株Δlmo0130 和回补株CΔlmo0130 的构建
将筛选到的缺失株和回补株利用内部引物lmo0130-in-F/R和外部引物lmo0130-out-F/R (表1)进行菌落PCR验证。如图1所示，当用外部引物进行PCR扩增时，以EGD-e为模板扩增出732 bp的PCR产物，以缺失菌株Δlmo0130和回补菌株CΔlmo0130为模板扩增产物大小仅为282 bp；当用内部引物进行PCR扩增时，以EGD-e和CΔlmo0130为模板扩增出492 bp的PCR产物，而以Δlmo0130为模板未扩增出明显条带。进一步将扩增产物进行DNA测序验证，测序结果与目的序列比对一致，表明Δlmo0130缺失株和CΔlmo0130回补株构建成功。
2.2 Lmo0130对单核增生李斯特氏菌生长的影响
测定单核增生李斯特氏菌在37 ℃ BHI肉汤中生长0-12 h的OD600吸光值，发现Lmo0130不影响单核增生李斯特氏菌的生长(图2)。
2.3 Lmo0130有助于单核增生李斯特氏菌对细胞的黏附和侵袭
利用人肠上皮细胞Caco-2探究Lmo0130对单核增生李斯特氏菌黏附和侵袭细胞的影响。与EGD-e和CΔlmo0130相比，感染0.5 h时的Δlmo0130黏附率显著下降了68.21%和54.02% (图3A)；感染1.5 h时的∆lmo0130侵袭率显著下降了77.80%和71.31% (图3B)。可见，缺失lmo0130基因显著降低单核增生李斯特氏菌的黏附和侵袭能力。因此Lmo0130有助于单核增生李斯特氏菌对细胞的黏附和侵袭。
2.4 Lmo0130有助于单核增生李斯特氏菌在小鼠巨噬细胞RAW264.7中的增殖
比较单核增生李斯特氏菌EGD-e、Δlmo0130和CΔlmo0130在RAW264.7细胞中增殖能力的差异。单核增生李斯特氏菌在感染0.5、2、5和8 h时，胞内细菌数呈明显上升趋势；在0.5-5 h之间，EGD-e、Δlmo0130和CΔlmo0130在RAW264.7中细菌数无显著差异；而在感染8 h时，RAW264.7细胞中Δlmo0130细菌数显著低于EGD-e和CΔlmo0130 (图4A)，EGD-e和CΔlmo0130的增殖率分别是Δlmo0130的1.93倍和2.61倍(图4B)。因此Lmo0130有助于单核增生李斯特氏菌在小鼠巨噬细胞RAW264.7中的增殖。
2.5 Lmo0130不影响单核增生李斯特氏菌在细胞间的迁移能力
探究Lmo0130对单核增生李斯特氏菌在小鼠成纤维细胞L929间迁移能力的影响。在单核增生李斯特氏菌EGD-e、Δlmo0130和CΔlmo0130的感染孔中均可见均匀分布的蚀斑(图5A)，且Lmo0130不影响蚀斑数目和大小(图5B、5C)，因此Lmo0130不影响该菌在小鼠成纤维细胞L929中的胞间迁移能力。
2.6 Lmo0130有助于单核增生李斯特氏菌对小鼠的致病力
通过小鼠感染模型来探究Lmo0130对单核增生李斯特氏菌致病力的影响。在小鼠存活试验中，每隔12 h观察腹腔注射感染单核增生李斯特氏菌小鼠的存活情况。结果发现(图6A)，在感染后60 h感染EGD-e、Δlmo0130和CΔlmo0130的小鼠存活率分别为70%、90%和80%；在感染80 h后，感染EGD-e和CΔlmo0130的小鼠存活率分别降低了30%和50%，而感染Δlmo0130小鼠的存活率仅降低了10%。这表明lmo0130基因缺失显著提高了感染小鼠的存活能力。小鼠组织载菌量试验结果发现，感染单核增生李斯特氏菌24 h时，各菌株在小鼠肝脏和脾脏中的定殖能力无显著差异；而在感染48 h时，与EGD-e和CΔlmo0130相比，感染Δlmo0130的小鼠肝脏载菌量分别下降了84.98%和61.60%，小鼠脾脏载菌量分别下降了95.21%和84.37% (图6B)。这表明lmo0130基因缺失后使感染小鼠脏器中单核增生李斯特氏菌的定殖能力显著降低。因此Lmo0130有助于单核增生李斯特氏菌对小鼠的致病力。
3 讨论与结论
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单核增生李斯特氏菌是食源性疾病中病死率最高的致病菌之一，食用该菌污染的食物易引起败血症、脑膜炎、脑炎、流产或死胎等临床症状[19-20]。表面蛋白对于革兰阳性菌在环境中的存活和宿主感染至关重要的，而LPXTG基序锚定蛋白是表面蛋白的重要组成部分，在黏附、侵袭、胞内增殖、逃逸吞噬体和胞间传播等宿主感染过程中发挥重要作用[21]。本研究明确了功能未知的LPXTG基序锚定蛋白Lmo0130在单核增生李斯特氏菌生长和感染致病中的作用，对解析该菌感染宿主的机制具有重要意义。
本研究通过细胞和动物感染模型发现LPXTG基序锚定蛋白Lmo0130在单核增生李斯特氏菌的细胞和宿主感染中发挥重要作用，最终影响该菌的致病力。LPXTG表面蛋白锚定到细胞壁上并介导多种革兰阳性菌的毒力，如李斯特氏菌[13]、葡萄球菌[22-23]、链球菌[24]和肠球菌[25]。SrtA对于单核增生李斯特氏菌内化素InlA等LPXTG表面蛋白的表达和在细菌表面的锚定都是必需的[26]，srtA基因缺失导致该菌在小鼠肝、脾中的定殖能力和对小鼠的致病力都严重受损[27-28]。单核增生李斯特氏菌内化素家族蛋白在该菌的宿主感染及致病中起重要作用，如InlA[29-31]、InlF[32]、InlJ[33]、InlH[34]、InlK[35]和Lmo0171[36]。其中InlA通过LRR结构域发挥黏附侵袭和细菌内化，并在跨越肠道屏障和胎盘屏障中起着关键作用[29-31]；InlF与表面波形蛋白Vimentin的结合对于单核增生李斯特氏菌在大脑中的最佳定殖是必需的[32]。黏附素InlJ在单核增生李斯特氏菌感染宿主时特异性表达，有助于该菌发挥完整毒力[33]。LPXTG表面蛋白LapB也是一种黏附素，对于单核增生李斯特氏菌侵袭哺乳动物细胞和致病力具有重要意义[11]。此外，Vip通过与宿主gp96相互作用在单核增生李斯特氏菌侵袭哺乳动物细胞和发挥毒力时起关键作用[10]。李斯特氏菌侵袭素LmiA有助于细菌与宿主黏蛋白之间的互作并介导细菌的侵袭[37]。缺失iap基因后显著降低该菌在细胞间传播能力和对小鼠致病力[38]。因此LPXTG蛋白在单核增生李斯特氏菌在感染致病中的作用和机制有待进一步探索。
本研究通过构建lmo0130的缺失株并探究其感染生物学功能，发现LPXTG基序锚定蛋白Lmo0130在单核增生李斯特氏菌细胞和宿主感染中发挥重要作用。这一发现对进一步解析Lmo0130介导宿主感染的机制及明确LPXTG基序锚定蛋白的功能奠定了重要基础。
基金
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  • 国家重点研发计划(2023YFD1801800)
  • 国家自然科学基金(32473026)
  • 国家自然科学基金(32302961)
  • 2025年浙江省大学生科技创新活动计划暨新苗人才计划资助项目(2025R412A011)
  • 2025年浙江省大学生创新训练项目
文献
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2025年第65卷第9期
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doi: 10.13343/j.cnki.wsxb.20250186
  • 接收时间：2025-03-06
  • 首发时间：2026-02-07
  • 出版时间：2025-09-04
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  • 收稿日期：2025-03-06
  • 录用日期：2025-03-31
基金
National Key Research and Development Program of China(2023YFD1801800)
国家重点研发计划(2023YFD1801800)
National Natural Science Foundation of China(32473026)
国家自然科学基金(32473026)
National Natural Science Foundation of China(32302961)
国家自然科学基金(32302961)
Zhejiang University Students’ Scientific and Technological Innovation Project in 2025(2025R412A011)
2025年浙江省大学生科技创新活动计划暨新苗人才计划资助项目(2025R412A011)
College Students’ Innovative Entrepreneurial Training Plan Program in Zhejiang Province in 2025
2025年浙江省大学生创新训练项目
作者信息
    浙江农林大学 动物医学院，浙江省畜禽绿色生态健康养殖应用技术研究重点实验室，动物健康互联网检测技术浙江省工程研究中心，浙江省动物医学与健康管理国际科技合作基地，同一健康和食品安全“一带一路”国际联合实验室，中澳动物健康大数据分析联合实验室，浙江 杭州
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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20250186
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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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