Zhang Lu, Wu Tonglei, Wang Fengjie, Liu Wan, Zhao Guixin, Zhang Yanying, Zhang Zhiqiang, Shi Qiumei
Hebei Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Hebei Normal University of Science & Technology, Qinhuangdao, 066004, PR China.
BMC Vet Res. 2024 Mar 11;20(1):100. doi: 10.1186/s12917-024-03951-x.
Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain's major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model.
Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD values of C50336 and ΔcheV were 6.3 × 10 CFU and 1.25 × 10 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection.
All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.
肠炎沙门氏菌（SE）是一种主要的人畜共患病原体，可在多种宿主中引起感染。开发新型SE疫苗对于根除这种病原体很有必要。基因工程减毒活疫苗具有更强的免疫原性且更安全。因此，为了开发一种减毒活沙门氏菌疫苗，我们构建了SE的cheV基因缺失株（命名为ΔcheV），并研究了cheV在SE毒力中的作用。首先，分析了ΔcheV在体外抵抗环境压力的能力、生物膜形成能力、耐药性和运动性。其次，通过细胞模型进行细菌粘附、侵袭和细胞内存活试验。使用小鼠感染模型进行体内毒力评估。为了进一步评估毒力降低所涉及的机制，利用qPCR分析来检测该菌株主要毒力基因的表达。最后，使用小鼠模型评估ΔcheV的免疫保护率。
与C50336相比，ΔcheV在酸性、碱性和热应激条件下的存活能力显著降低，但在氧化应激条件下的存活情况无显著差异。生物膜形成能力、耐药性和运动性也没有显著变化。发现ΔcheV对Caco-2细胞的粘附能力保持不变，但在RAW264.7细胞中的侵袭能力和存活率显著降低。攻毒试验结果表明，C50336和ΔcheV的LD值分别为6.3×10 CFU和1.25×10 CFU。cheV基因缺失后，fimD、flgG、csgA、csgD、hflK、lrp、sipA、sipB、pipB、invH、mgtC、sodC、rfbH、xthA和mrr1基因的表达水平显著降低。减毒活ΔcheV在小鼠中提供了100%的保护，使其免受SE感染。
所有结果证实，cheV基因的缺失降低了SE的毒力，并在小鼠中提供了显著的免疫保护，表明ΔcheV可能是作为减毒活疫苗进行探索的潜在候选者。
