We investigated the attenuating effects of a range of respiratory chain mutations in three Salmonella serovars which might be used in the development of live vaccines. We tested mutations in nuoG, cydA, cyoA, atpB, and atpH in three serovars of Salmonella enterica: Typhimurium, Dublin, and Gallinarum. All three serovars were assessed for attenuation in their relevant virulence assays of typhoid-like infections. Serovar Typhimurium was assessed in 1-day-old chickens and the mouse. Serovar Gallinarum 9 was assessed in 3-week-old chickens, and serovar Dublin was assessed in 6-week-old mice. Our data show variation in attenuation for the nuoG, cydA, and cyoA mutations within the different serovar-host combinations. However, mutations in atpB and atpH were highly attenuating for all three serovars in the various virulence assays. Further investigation of the mutations in the atp operon showed that the bacteria were less invasive in vivo, showing reduced in vitro survival within phagocytic cells and reduced acid tolerance. We present data showing that this reduced acid tolerance is due to an inability to adapt to conditions rather than a general sensitivity to reduced pH. The data support the targeting of respiratory components for the production of live vaccines and suggest that mutations in the atp operon provide suitable candidates for broad-spectrum attenuation of a range of Salmonella serovars.
SummarySalmonella enterica serovar Typhimurium ( S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA , invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-g g g g production compared to S. Typhimurium aroC derivatives.
We have studied the growth suppression seen in early-stationary-phase LB broth cultures of Salmonella typhimurium. Multiplication of small numbers of an antibiotic-resistant S. typhimurium mutant was prevented when the mutant was added to 24-h cultures of the antibiotic-sensitive parent strain, whereas an antibioticresistant mutant of an Escherichia coli strain added to the same culture grew well. A 24-h E. coli culture produced a similar specific bacteriostatic inhibition against E. coli. In older cultures, a specific bactericidal effect similar to that observed by M. M. Zambrano and R. Kolter (J. Bacteriol. 175:5642-5647, 1993) was also observed. Whether incubated statically or shaken, sufficient nutrients were present in the filtered supernatants of 24-h cultures for small inocula of the same strain to multiply to ca. 10 9 CFU/ml after reincubation. Introduction of the rpoS mutation had no effect on the specific bacteriostatic inhibition. Similar specific inhibition was also observed in strains of Citrobacter freundii, Klebsiella pneumoniae, Enterobacter agglomerans, and Shigella spp. Experiments in which the 24-h culture was physically separated from the antibiotic-resistant mutant by using a dialysis membrane were carried out. These results indicated that the inhibition might be mediated by a diffusible but labile chemical mediator.The observed reduction in the rate of bacterial multiplication in broth cultures toward the end of logarithmic phase has been well documented in standard bacteriology texts for many years (8,29), and at least some of the factors that cause it are understood.A combination of composition of the growth medium and particular organisms may induce conditions which are inimical to bacterial multiplication and might even be bactericidal. The low pH that results from the fermentation of carbohydrates by different bacterial groups and the production of inhibitory concentrations of hydrogen peroxide by Streptococcus pneumoniae are well-known examples (29).Limitation of nutrient concentrations is another factor that can induce a cessation of growth rate. Carbon starvation is accompanied by the induction of at least 50 bacterial proteins (12,18,19) and a generalized resistance to other harmful agents such as heat shock and hydrogen peroxide (16). This type of nongrowing, stationary-phase behavior is a result of the starvation conditions to which the cells are subjected. This regulon (9) is complex, and the production of different starvation-induced proteins is regulated by cyclic AMP (23), oxidative stresses (16), osmotic shock (15), and the sigma factor s (RpoS, KatF) (7,17,20). It is, however, unclear whether the conditions that induce these changes are equivalent or similar to those present in early stationary phase in broth cultures, in which nutrient limitation might not always be a major problem.The factors responsible for growth suppression in stationaryphase nutrient broth cultures have been of interest for a long time and were studied in the early decades of this century (for review ...
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