Virulence plasmid-borne spvB and spvC genes can replace the 90-kilobase plasmid in conferring virulence to Salmonella enterica serovar Typhimurium in subcutaneously inoculated mice

Abstract

In a mouse model of systemic infection, the spv genes carried on the Salmonella enterica serovar Typhimurium virulence plasmid increase the replication rate of salmonellae in host cells of the reticuloendothelial system, most likely within macrophages. A nonpolar deletion in the spvB gene greatly decreased virulence but could not be complemented by spvB alone. However, a low-copy-number plasmid expressing spvBC from a constitutive lacUV5 promoter did complement the spvB deletion. By examining a series of spv mutations and cloned spv sequences, we deduced that spvB and spvC could be sufficient to confer plasmid-mediated virulence to S. enterica serovar Typhimurium. The spvBC-bearing plasmid was capable of replacing all of the spv genes, as well as the entire virulence plasmid, of serovar Typhimurium for causing systemic infection in BALB/c mice after subcutaneous, but not oral, inoculation. A point mutation in the spvBC plasmid preventing translation but not transcription of spvC eliminated the ability of the plasmid to confer virulence. Therefore, it appears that both spvB and spvC encode the principal effector factors for Spv- and plasmid-mediated virulence of serovar Typhimurium.

FIG. 1

Physical and genetic maps of virulence plasmid sequences and recombinant plasmids used in this study. The top line depicts the insert of pGTR061, which carries spvRABCD, whose open reading frames are indicated by arrows with the direction of transcription shown. Restriction sites indicated below pGTR061 are as follows: B, BamHI; C, ClaI; E, EcoRI; M, MscI; P, PstI; and S, StuI. Note that pGTR061 extends to the XhoI site on the right. The rightmost BamHI site is indicated for reference to plasmids depicted below. The extent of the ΔspvB30 deletion and the location of the spvC22::Tn5 insertion are shown. The ClaI insert of pGTR040 corresponds to the Δspv::tet deletion of UF110. The arrows next to the insertion sequences of each clone indicate the direction of transcription of the promoters of the respective vectors. For pGTR333, pGTR337, and pGTR338, the broken line indicates that the EcoRI-PstI fragment carrying the spvA promoter has been juxtaposed to the spvB open reading frame by the PstI deletion. Details of plasmid construction are presented in Table 1.

FIG. 2

Recombinant spvBC complements Δspv::tet after s.c. inoculation of mice. Mice were inoculated s.c. with wild-type strain χ3306 and Δspv::tet strain UF110 containing either spvBC-bearing pGTR356 or the vector pMW119. Four days later, spleens were removed, homogenized, and plated to enumerate CFU. P values are for the mean paired difference (MPD) being greater than 0. Error bars represent standard deviations. n = 5 (for all groups).

FIG. 3

Recombinant spvBC complements virulence plasmid-cured serovar Typhimurium after s.c. inoculation of mice. Mice were inoculated s.c. with wild-type strain χ3306 or virulence plasmid-cured strain χ3337 containing spvBC-bearing pGTR356, spvRABCD-bearing pGTR061, or no plasmid. Four days later, spleens were removed, homogenized, and plated to enumerate CFU. P values are for χ3337(pGTR356) being different from the other strains. Additionally, χ3456 and χ3337(pGTR061) were each significantly greater than χ3337 (P < 0.0001), and χ3337(pGTR061) was significantly greater than χ3456 (P < 0.02). Error bars represent standard deviations. n = 5 (for all groups).

FIG. 4

spvC is required for virulence conferred by spvBC-bearing pGTR356. Mice were inoculated s.c. with 10⁵ CFU of either wild-type χ3306, virulence plasmid-cured χ3337, χ3337(pGTR356), or χ3337(pGTR357). Six days later, spleens were removed, homogenized, and plated to enumerate CFU. The spvC mutation of pGTR357(∗) significantly decreased infection, compared with spvBC-bearing pGTR356 (P < 0.01). N.S., χ3306 was not significantly different from χ3337(pGTR356) (P > 0.3). Error bars represent standard deviations. n = 5 (for all groups).