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沙门菌病是由肠杆菌科沙门菌属（Salmonella）引发的全球性人兽共患传染病，其引发的腹泻综合征被世界卫生组织列为四大腹泻致病因素之一。在现行临床诊疗体系中，抗菌药物疗法仍是控制沙门菌感染的核心干预策略。但抗菌药物滥用不仅加速了多重耐药菌株的进化，导致传统治疗方案疗效显著降低，更引发药物残留物在生态环境中的蓄积，形成生物安全风险。在此背景下，基于微生态调节的替代治疗策略逐渐受到广泛关注，其中益生菌疗法通过药效学验证及临床试验证实具有显著的病原菌拮抗作用。本研究基于黏蛋白糖基化修饰理论，利用基因工程技术将唾液酸转移酶ST6GALNAC1编码基因导入干酪乳杆菌（Lactobacillus casei）ATCC393载体，成功构建工程化菌株LC-pPG-ST6。Western blot分析显示，重组菌株在60 kDa处呈现特异性条带，证实目的蛋白的稳定表达。通过建立人结肠腺癌细胞系LS174T体外肠黏膜屏障模型，评估工程菌株的黏附与侵袭能力，并通过荧光定量PCR检测促炎因子及肠道紧密连接蛋白的表达水平。结果表明：工程菌LC-pPG-ST6对LS174T细胞具有较强的黏附能力与侵袭能力，且能有效抑制鼠伤寒沙门菌（Salmonella typhimurium）对上皮细胞的侵袭。LC-pPG-ST6处理组促炎细胞因子IL-6、IL-17、IL-1β及TNF-α的mRNA表达水平呈下调趋势，同时显著上调紧密连接蛋白Occludin、CLDN1和ZO-1基因表达水平。本研究首次阐明ST6GALNAC1介导的黏蛋白唾液酸化修饰在沙门菌-宿主互作中的调控网络，为开发靶向肠黏膜免疫的新型益生菌制剂提供了理论与技术支撑。
Abstract：Salmonellosis is a globally prevalent zoonotic infectious disease caused by Salmonella spp. of Enterobacteriaceae， whose associated diarrheal syndrome has been classified by the World Health Organization as one of the four major etiological factors of global diarrheal diseases. Within current clinical management frameworks， antimicrobial therapy remains the cornerstone intervention strategy for controlling Salmonella infections. However， antimicrobial misuse not only accelerates the evolution of multidrug-resistant (MDR) strains， resulting in markedly diminished efficacy of conventional therapeutic regimens， but also induces bioaccumulation of drug residues in ecosystems， posing biosecurity risks. Under these circumstances， alternative therapeutic strategies based on microbiome modulation are gaining increasing attention， with probiotic interventions demonstrating significant pathogen-antagonistic effects through pharmacodynamic validation and clinical trials. This study employed mucin glycosylation modification theory to genetically engineer Lactobacillus casei ATCC393 through ST6GALNAC1 sialyltransferase gene integration， successfully constructing the engineered strain LC-pPG-ST6. Western blot analysis revealed a specific band at 60 kDa in recombinant strains， confirming stable expression of the target protein. Using LS174T human colorectal adenocarcinoma cells to establish an in vitro intestinal mucosal barrier model， we assessed engineered strain's adhesion and invasion capabilities， with pro-inflammatory cytokines and intestinal tight junction proteins quantified via qRT-PCR. Results demonstrated that LC-pPG-ST6 exhibited robust adhesion and invasion capacities toward LS174T cells， while effectively inhibiting Salmonella Typhimurium invasion of epithelial cells. LC-pPG-ST6 treatment downregulated mRNA expression of proinflammatory cytokines (IL-6， IL-17， IL-1β， TNF-α) while significantly upregulating tight junction proteins (Occludin， CLDN1， ZO-1) at transcriptional level. This study pioneers in elucidating ST6GALNAC1-mediated mucin sialylation regulatory network during Salmonella-host interactions， providing theoretical and technical foundations for developing novel probiotics targeting intestinal mucosal immunity.
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基本信息:
中图分类号:S852.6
引用信息:
[1]张婷婷,王娟,邵鑫,等.表达唾液酸转移酶ST6GALNAC1基因工程益生菌的构建及特性研究[J].经济动物学报().
基金信息:
国家自然科学基金面上项目（32473067）;国家自然科学基金青年科学基金项目（32102681）; 吉林省科技发展计划项目（20220101311JC）
2025-12-12
2025-12-12
2025-12-12