建構一株鼠傷寒沙門氏菌fimY與fimZ雙基因剔除菌株並評估其生理功能

Abstract

沙門氏菌是一種食因性與人畜共通傳染疾病病原，屬於腸桿菌科的格蘭氏陰性兼性厭氧桿菌，極易透過環境或畜產品進入食物鏈造成全球性的感染。在感染宿主期間，沙門氏菌會利用其獨特的毒力因子貼附腸上皮細胞或巨噬細胞，除了可以在細胞內繁殖外，也經由巨噬細胞的遷徙導致菌血症、腦膜炎與脊髓炎並造成死亡，沙門氏菌的感染乃是一個重要的公共衛生議題。因此，畜牧產業在預防沙門氏菌上，除了注意環境衛生清理外，疫苗施打也是一個預防疾病的選項。協助沙門氏菌感染宿主的毒力因子中，線毛是類似於毛髮結構的蛋白質，表現於沙門氏菌外膜上，主要功能是為了黏附在細胞上以利後續的入侵作用。在鼠傷寒沙門氏菌中已被發現有13種線毛，其中第一型線毛是最常見的線毛，第一型線毛的產生主要由fimY和fimZ兩正向調控基因進行調控表現。FimY是一種LuxR蛋白質，會和fimZ啟動子上的lux box序列結合。FimZ是一個response regulator，會結合在fimA啟動子上。由於沙門氏菌在入侵細胞的第一步是透過線毛黏附，若同時剔除fimY和fimZ，有可能會讓沙門氏菌的毒力降低，可以評估其作為減毒疫苗候選菌株的可行性。本研究利用allelic exchange的方法建構一株S. Typhimurium LB5010 ΔfimY與ΔfimZ雙基因剔除菌株，並與LB5010 wild type比較其生理功能，測試其對氧化壓力的耐受度、兩者生長曲線差異、細胞膜的安定性、生物膜的形成、黏附與入侵細胞以及細胞內生存之能力。結果顯示，LB5010-ΔfimY-ΔfimZ面對活性氮物質、細胞膜的安定、生物膜形成、入侵及吸附細胞能力都較LB5010 wild type來得差。但在面對活性氧物質及細胞內生存能力，則與LB5010 wild type相似。另外，過去實驗室曾建構fimY和fimZ單獨剔除菌株，藉由轉錄組定序分析，比較LB5010-ΔfimY、LB5010-ΔfimZ與LB5010-ΔfimY-ΔfimZ彼此之間的差異表現基因，推測fimY及fimZ除了調控第一型線毛的表現外，也可能參與調控其他線毛基因，如pef及sti，以及影響其他生理功能基因的表現，但目前研究的結果顯示，沙門氏菌LB5010-ΔfimY-ΔfimZ菌株還須進行其他的試驗，包含動物實驗，才有辦法評估其作為減毒疫苗菌株的可行性。

Salmonella is a food-borne and zoonotic pathogen. It is a gram-negative, facultatively anaerobic bacillus that belongs to the family of Enterobacteriaceae. Salmonella can easily enter the food chain via the environment and animal products and cause infections worldwide. During infection, Salmonella attaches to and replicates within intestinal epithelial cells or macrophages using its unique virulence factors. Besides replicating within cells, it can also cause bacteremia, meningitis, and myelitis through macrophage migration, leading to death. Salmonella infection is a significant public health problem. Therefore, in addition to maintaining environmental hygiene, vaccines are also available to prevent Salmonella infection in livestock and poultry. Among the virulence factors that assist Salmonella in infecting hosts, the fimbriae are hair-like protein structures present on the outer membrane of Salmonella. Their primary function is to adhere to cells and facilitate subsequent invasion. Thirteen types of fimbriae have been identified in Salmonella enterica serovar Typhimurium, with type 1 fimbriae being the most common. Type 1 fimbrial production is mainly regulated by two positive regulatory genes, fimY and fimZ. FimY is a LuxR type protein that binds to the lux box sequences in the fimZ promoter region. FimZ is a response regulator that binds to the fimA promoter. Since the initial step of Salmonella invasion involves fimbrial adhesion, simultaneous deletion of fimY and fimZ could potentially reduce the virulence of Salmonella, making it a candidate strain for attenuated vaccines. In this study, we used allelic exchange to construct a strain, S. Typhimurium LB5010-ΔfimY-ΔfimZ, and compared its physiological functions to the LB5010 wild type strain. We tested their tolerance to oxidative stress, growth curve differences, cell membrane stability, biofilm formation, adherence and invasion of cells, and intracellular viability. The results showed that LB5010-ΔfimY-ΔfimZ exhibited inferior performance in terms of reactive nitrogen species resistance, cell membrane stability, biofilm formation, invasion and adhesion to cells compared to LB5010 wild type. However, it showed similarities to LB5010 wild type in terms of resistance to reactive oxygen species and intracellular viability. Additionally, previous studies in our laboratory have constructed strains with individual deletions of fimY and fimZ. By comparing transcriptome sequencing results, we observed differential gene expression between LB5010-ΔfimY, LB5010-ΔfimZ and LB5010-ΔfimY-ΔfimZ, suggesting that fimY and fimZ may not only regulate the expression of type 1 fimbriae but also participate in the regulation of other fimbrial genes, such as pef and sti, as well as affect the expression of other genes involved in physiological functions. Although fimY and fimZ may act as multifactorial regulatory proteins, current research suggests that S. Typhimurium LB5010-ΔfimY-ΔfimZ requires further testing, including animal experiments, to evaluate its feasibility as an attenuated vaccine strain.