Debra L. Weinstein

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 entericaserovar 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.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

ABSTRACT Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Macrophage defect and inflammatory cell recruitment dysfunction in Salmonella susceptible C3H/HeJ mice

C3H/HeJ mice are homozygous for the Lpsd allele and, as a consequence, are hyporesponsive to all of the biological effects of bacterial lipopolysaccharide (LPS) that have been studied. These mice die in the early phase of infection when inoculated with virulent Salmonella. This susceptibility is also regulated by the Lpsd allele. The mechanism of Lpsd-conferred Salmonella susceptibility was evaluated in these studies. The response of C3H/HeJ mice to S. typhimurium strains of differing virulence was compared in a series of in vivo experiments to the response of: endotoxin-responsive (Lpsn) mice that carry another Salmonella susceptibility gene (Itys) and endotoxin-responsive mice that carry a Salmonella resistance gene (Ityr). The C3H/HeJ mice (genotype Lpsd/Ityr) were more resistant than Lpsn/Itys mice to strains of S. typhimurium of reduced virulence but less resistant than Lpsn/Ityr mice. In addition, C3H/HeJ macrophages cultured in vitro were less able to contain net salmonellae multiplication than were macrophages from Lpsn/Ityr mice. Moreover, histopathological findings revealed that S. typhimurium-infected Lpsn/Ityr animals recruited an abnormally low number of cells into their livers compared to either Lpsn/Ityr mice or Lpsn/Itys mice. These data suggest that the susceptibility of C3H/HeJ mice may be the result of at least two Lpsd-encoded defects: a decreased capacity of macrophages to restrict Salmonella growth and a reduced recruitment of inflammatory cells into liver.