Absence of All Components of the Flagellar Export and Synthesis Machinery Differentially Alters Virulence of Salmonella enterica Serovar Typhimurium in Models of Typhoid Fever, Survival in Macrophages, Tissue Culture Invasiveness, and Calf Enterocolitis

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

本研究构建了肠炎沙门氏菌鼠伤寒血清型（Salmonella enterica serovar Typhimurium）的鞭毛主调控基因flhD（master flagellar regulator gene）突变株，并通过一系列实验手段对比了该突变株与野生型菌株的毒力，实验涵盖小鼠伤寒感染模型、小鼠巨噬细胞存活实验、肠上皮细胞黏附与侵袭实验，以及小牛肠结肠炎模型。研究发现，flhD突变株在小鼠体内的毒力强于亲本菌株，且在小鼠巨噬细胞感染后的4至24小时内，其净生长速率略快。与之相反，flhD突变株对人源与鼠源肠上皮细胞的侵袭能力减弱；同时在小牛结扎肠段实验（ligated-loop assay）中，其诱导肠液分泌以及触发多形核白细胞（polymorphonuclear leukocyte）应答的能力也有所下降。结合另一组实验结果——即针对不产生鞭毛蛋白（flagellin）但可合成鞭毛分泌装置（flagellar secretory apparatus）的鼠伤寒血清型fljB fliC突变株开展的毒力评估实验结果——本研究表明，无论是鞭毛的存在（如此前所报道），还是鞭毛输出装置（flagellar export machinery）的合成，均非该菌株在小鼠体内致病性所必需的条件。与之相反，细菌在体外组织培养中展现完全侵袭能力，以及在小牛肠道中引发多形核白细胞浸润，均依赖于鞭毛的存在；而在该肠结肠炎模型中，诱导最大程度肠液分泌则同样需要鞭毛分泌组分的参与。该结论的一项推论为：此前虽有相关推测，但尚未通过对同一批突变株的对比研究加以验证——小鼠全身感染实验与巨噬细胞实验所评估的毒力维度，与体外组织培养侵袭实验所评估的维度存在差异，且后者更能预测小牛肠结肠炎模型中的实验结果。