Salmonella enterica serovar typhimurium strains with regulated delayed attenuation in vivo

Abstract

Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P(BAD) cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.

FIG. 1.

Deletion-insertion mutations resulting in arabinose-regulated virulence traits and deletion mutations altering arabinose metabolism and uptake. Strains are as identified on the panels.

FIG. 2.

Phenotypes of strains with deletion-insertion mutations to enable arabinose-dependent expression of virulence traits. (a) χ9021 with the ΔP_crp527::TT araC P_BAD crp mutation streaked on MacConkey maltose agar without and with 0.2% arabinose. (b) χ8848 with the ΔP_fur33::TT araC P_BAD fur and χ9107 with the ΔP_fur33::TT araC P_BAD fur and ΔP_crp527::TT araC P_BAD crp mutations spotted on CAS agar plates without and with 0.2% arabinose to visualize siderophore production. (c) χ8918 with the ΔP_phoPQ107::TT araC P_BAD phoPQ and χ9108 with the ΔP_phoPQ107::TT araC P_BAD phoPQ and ΔP_crp527::TT araC P_BAD crp mutations streaked on X-P plates without and with 0.2% arabinose to reveal acid phosphatase activity. (d) χ8956 with the ΔP_rpoS183::TT araC P_BAD rpoS and χ9064 with the ΔP_rpoS183::TT araC P_BAD rpoS and ΔP_crp527::TT araC P_BAD crp mutations streaked on glycogen indicator agar without and with 0.2% arabinose and sprayed with iodine indicator solution.

FIG. 3.

Stability of Crp, Fur, RpoS, and PhoP proteins during incubation of cultures induced for expression of these proteins prior to addition of 50 μg/ml chloramphenicol of culture. Rabbit antibodies raised against His-tagged Crp, Fur, RpoS, and PhoP were used for Western blot analyses. χ9021 (ΔP_crp527), χ8848 (ΔP_fur33), χ8956 (ΔP_rpoS183), and χ8918 (ΔP_phoPQ107) were grown in LB broth with 0.2% arabinose for these studies.

FIG. 4.

Decrease in amounts of Crp, Fur, RpoS, and PhoP proteins as a consequence of growth of χ9021 (ΔP_crp527), χ8848 (ΔP_fur33), χ8956 (ΔP_rpoS183), and χ8918 (ΔP_phoPQ107) in the absence of arabinose. The same bacterial strains as used for the results shown in Fig. 3 were grown in nutrient broth with 0.2% arabinose, and at the commencement of sampling to measure the amounts of proteins, the cultures were diluted 1:4 into prewarmed nutrient broth lacking arabinose. Rabbit antibodies raised against His-tagged Crp, Fur, RpoS, and PhoP were used for Western blot analyses. Some synthesis of three of the four proteins continued until after the third 1:4 dilution when the arabinose concentration was 0.003%.