Contribution of Proton-Translocating Proteins to the Virulence of Salmonella enterica Serovars Typhimurium, Gallinarum, and Dublin in Chickens and Mice

Abstract

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.

Salmonella enterica Serovar Typhimurium Colonizing the Lumen of the Chicken Intestine Grows Slowly and Upregulates a Unique Set of Virulence and Metabolism Genes

“…These models are not strictly comparable, since the streptomycin-treated mouse will retain some gut flora, whereas there was virtually none in this series of experiments. In addition, we have found a degree of host specificity related to the likely route of respiration during intracellular Salmonella infection in chickens and mice (83) and the redox conditions implied therein. Tetrathionate is reduced to thiosulfide and further to H 2 S with the products of ttr, phs, and asr genes.…”

“…Oligonucleotide primers are shown in Table 1. These pDM4 derivatives were maintained in E. coli strain SM10pir (83) and were introduced into the recipient Salmonella strains by conjugation. Transconjugants were isolated on selective medium supplemented with either streptomycin or kanamycin (25 g/ml), and their sensitivities to chloramphenicol were then tested to identify those that resulted from a recombinational doublecrossover event that had not incorporated any pDM4 DNA.…”

“…Selected mutants of S. Typhimurium F98 were tested for their virulence for newly hatched Rhode Island Red chickens. The mutations were transferred by P22 transduction (5) to strain 4/74, which is virulent for mice (SL1344) (83), for assessment of virulence in BALB/c mice. Virulence was assessed by oral inoculation of groups of 20 newly hatched chickens with 0.1 ml or of 10 BALB/c mice with 50 l of a broth culture diluted to contain 10 6 CFU in this volume.…”

“…Insertion mutants using kanamycin or streptomycin/ spectinomycin resistance cassettes were prepared as single mutants using standard procedures detailed elsewhere (82,83,100). Briefly, oligonucleotide primers were used to amplify upstream and downstream fragments, which were then joined together by an additional overlap extension PCR using the same two fragments as a template.…”

DksA potentiates direct activation of amino acid promoters by ppGpp

“…Effects of dksA have been reported on gene expression, chaperonin function, cell division, amino acid requirements, quorum sensing, phage sensitivity, and virulence (11,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Of all these phenotypes, only the effect on rRNA expression has been demonstrated to be direct (11).…”