Comprehensive Identification of Salmonella enterica Serovar Typhimurium Genes Required for Infection of BALB/c Mice

Abstract

Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing

“…We explored whether Salmonella metabolic requirements for the systemic infection of mice were comparable with the metabolic requirements for fitness within tomatoes. We retrieved data from previous work using transposon insertion libraries in Salmonella enterica serovar Typhimurium (37,38) that had been applied in a mouse model for systemic infection and compared them with our results from the Salmonella-tomato interaction. A total of 327 KEGG Orthology (KO) identifiers were mapped for the genes required for persistence in tomatoes, and 505 KO identifiers required for systemic infection of mice were mapped.…”

“…in the interaction with tomatoes, scavenging of amino acids was required in the systemic mouse infection. Transposon disruptions in just a few pathways (e.g., the urea cycle) impaired Salmonella fitness during systemic infection (37,38). Besides being involved in the synthesis of arginine, the urea cycle is also involved in the incorporation of excess nitrogen.…”

“…Besides being involved in the synthesis of arginine, the urea cycle is also involved in the incorporation of excess nitrogen. (37,38,51). Key metabolic steps are shown as shaded boxes.…”

Vi antigen of Salmonella enetrica serovar Typhi — biosynthesis, regulation and its use as vaccine candidate

“…atpA mutants of S. Typhimurium are strongly attenuated and confer protection against subsequent infections [4, [114][115][116]. The development of regulated, delayed attenuated mutants has further helped in combating this problem.…”

Sugar sulfates are not hydrolyzed by the acid-inducible sulfatase AslA from Salmonella enterica Enteritidis NalR and Kentucky 3795 at pH 5.5

“…Screening of transposon mutant libraries indicated that insertions in STM0084 did not have a strong impact on competitiveness of the mutant strains in in-vivo assays with mice injected with mutants and wild type (Chaudhuri et al 2009), but when chickens, cattle and pigs were infected orally with the transposon mutants, mutants with insertions in STM0084 were less competitive (Chaudhuri et al 2013 In an attempt to further our understanding of the role of aslA and its potentially differential regulation in S. e. Enteritidis Nal R and Kentucky 3795, the gene was deleted in the current study and the utilization of sulfate ester compounds by the mutant and wild-type strains was assayed. The competitiveness of the mutants versus the wild-type strains in cell-invasion assays was also determined.…”