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

Abstract

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.

FIG. 1

Kinetic study of flagellar mutants recovered from the spleen following oral inoculation of mice. C57BL/6J mice were inoculated with SL3201 (triangles) or SL3201 flhD::Tn10 (circles) at a dose of approximately 10⁷ CFU per mouse. At various times postinoculation up to 14 days, mice were sacrificed and the number of CFU per spleen was determined. Each symbol represents data from an individual animal. The horizontal line indicates the GM for the group of five mice. Asterisks indicate mice that had died; values of 10⁸ CFU/spleen were assigned to these mice. The 95% CL of the GMs for the two groups overlapped at all time points including day 14 (data not shown). Limit of detection = 10² CFU/spleen.

FIG. 2

Survival in mouse resident peritoneal macrophages. Macrophages were infected in vitro with SL3201 (triangles), SL3201 flhD::Tn10 (circles), or SL3201 fljB::MudJ fliC::Tn10 (squares) at a MOI of approximately five opsonized bacteria per cell. The numbers of viable bacteria and macrophages per well were determined at 0, 4, and 24 h postinfection. The ratio of CFU/macrophage and one SE of the mean are shown. Note that the SE bars are covered by the symbols at 0 and 4 h postinfection.

FIG. 3

Secretory response and PMN influx elicited by serovar Typhimurium strains in bovine intestinal loops. Approximately 1.5 × 10⁹ CFU of the wild-type strain SL3201 (WT), SL3201 flhD::Tn10(D-) or SL3201 fljB::MudJ fliC::Tn10 (B-C-) was injected into each loop and left for 12 h before analysis. LB broth (LB) was also used as a negative control. The secretory response is shown as volume per length of loop. The PMN influx is a ratio of the PMN radioactivity within the infected loops to the PMN radioactivity within the control loops. Each mean is calculated from either nine loops in three calves (secretory response) or six loops in two of the three calves (PMN influx) and is presented with the SE.