持續表現與不表現第一型線毛的鼠傷寒沙門氏菌株不同生理功能之比較

Abstract

沙門氏菌是一種人畜共通傳染病原，它可以感染人類以及包括溫血和冷血在內的多種動物。沙門氏菌造成的感染在全世界的畜牧業中造成相當大的損失。目前，從環境中消除沙門氏菌是不切實際和無法實現的。良好的飼養管理和疫苗接種計劃是減輕動物感染沙門氏菌症臨床症狀的較佳策略。目前已經開發了數種沙門氏菌疫苗類型。最常見的是通過剔除毒力基因來降低沙門氏菌的致病力，製成減毒的活菌疫苗，其中有些疫苗已經有商品化的產品。最近報導了另一種減毒的方法，稱之為減弱基因表達 (attenuating gene expression, AGE) 的方法，該方法是利用過度表現細菌表面的附屬物來減弱細菌的毒力。第一型線毛是沙門氏菌的主要表面附屬物之一。我們的實驗室已經構建了數個基因缺失的鼠傷寒沙門氏菌 (Salmonella Typhimurium) 菌株，從而產生了會持續表現第一型線毛和不表現線毛的菌株。本研究的目的是比較這些菌株的毒力，以評估其作為減毒疫苗菌株的可行性。來自鼠傷寒沙門氏菌LB5010的fimZ和fimY缺失菌株是不產生線毛的，而fimW、stm0551和stbC缺失菌株是會產生線毛的。研究方法藉由量測菌株的生長速率、對膽鹽和H2O2的敏感性、使用紅黴素或多粘菌素B來檢測菌株細胞膜的穩定性、對低滲透壓的抗性以及細胞吸附和入侵能力來評估上述菌株的毒力。本研究結果顯示，與其他菌株相比，fimY缺失菌株表現出較慢的生長速率。fimY和stbC缺失菌株在含膽鹽的培養基中顯示出較低的存活率。fimY和stm0551缺失菌株在含有H2O2的環境下顯示出較低的存活率。fimZ和fimW缺失菌株對於多粘菌素B均顯示出較低的最低抑菌濃度，表明它們的細胞膜不如LB5010菌株來得穩定。與其他菌株相比，fimY和stm0551缺失菌株在低滲透壓環境中抵抗力較弱，而fimZ和stm0551缺失菌株在PBS環境中對滲透壓的抵抗力較弱。在細胞吸附試驗中，fimY缺失菌株顯示出較弱的吸附能力，而在Caco-2細胞入侵試驗中，fimY缺失菌株也顯示出較弱的入侵能力。由此可知，fimY缺失菌株在各種生理試驗中均顯示弱毒性。將來我們可以進一步評估fimY缺失菌株作為減毒疫苗的可行性。

Salmonella is a zoonotic pathogen and it can infect humans as well as a variety of animals, including warm- and cold-blooded. Salmonella infections in farm animals cause considerable losses in the livestock industry worldwide. Eradication of Salmonella from environment is currently impractical and unattainable. Good husbandry management coupled with vaccination programs is a better strategy to reduce the clinical symptoms of salmonellosis in animals. Several Salmonella vaccines types have been developed. To attenuate Salmonella by deleting virulence genes to develop attenuated vaccines is commonly practiced and some of them are commercially available. Another approach, attenuating gene expression (AGE), attenuates bacterial pathogens through overexpression of bacterial surface appendages had been recently reported. The type 1 fimbriae are one of the major surface appendages of Salmonella. Previously our laboratory has constructed several gene-deleted Salmonella Typhimurium strains, resulting in constitutively type 1 fimbriated and non-fimbriated strains. The objective of this study is aimed to compare the virulence of these strains to evaluate their feasibility to serve as attenuated vaccine strains. The fimZ and the fimY deleted strains derived from S. Typhimurium LB5010 are non-fimbriated, while the fimW, stm0551, and stbC deleted strains are fimbriated. Growth rate, susceptibility to bile salt and H2O2, membrane stability tests using erythromycin or polymyxin B, resistance to low osmotic pressure and cell adhesion and invasion assays were employed to assess the virulence of the aforementioned strains. Our results indicated that the fimY deleted strain exhibited a decreased growth rate compared with other strains. The fimY and the stbC deleted strains demonstrated a lower survival rate in the medium containing bile salt. The fimY and the stm0551 deleted strains showed lower survival rates in an environment containing H2O2. Both the fimZ and the fimW deleted strains exhibited a lower minimum inhibitory concentration (MIC) for polymyxin B, suggesting their membranes being less stable than that of the parental LB5010 strain. The fimY and the stm0551 deleted strains are less resistant to osmotic pressure in the H2O environment compared to other strains, while the fimZ and the stm0551 deleted strain are less resistant to osmotic pressure in the PBS environment. The fimY deleted strain revealed a poor adhesion and invasion ability to the Caco-2 cells. Thus, the fimY deleted strain demonstrated itself a less virulent strain in several physiological function tests. In the future, we can further evaluate the feasibility of using the fimY deleted strain as an attenuated vaccine strain.