Isolation of orally attenuated Salmonella typhimurium following TnphoA mutagenesis

Abstract

One hundred fifty TnS IS5OL::phoA (TnphoA) mutants of a mouse-virulent, nalidixic acid-resistant (Nat"), prototrophic Salmonella typhimurium strain, C5 Nalr, were isolated. None of the mutants were auxotrophs. Groups of 8 to 10 BALB/c mice were infected orally with each of 95 mutants with a dose equivalent to 20-fold the 50% lethal dose of the wild-type C5 Nalr strain, and deaths were counted over the next 28 days. Fifteen of the mutants failed to kill any mice, whereas all mice died following challenge with the other mutants. Nine of the 15 attenuated mutants exhibited a defect in lipopolysaccharide biosynthesis. The remaining six mutants were smooth. The TnphoA transposon of each of the smooth attenuated mutants was moved, using P22-mediated transduction, into a fresh C5 background, and all retransductants were still attenuated. Analysis of the membrane proteins of the attenuated mutants failed to reveal any alterations in detectable major outer membrane proteins, although colonies of two of the mutants exhibited a mucoid phenotype following growth on L-agar plates. Individual attenuated mutants differed in their abilities to translocate to livers and spleens of mice following oral infection. All of the smooth TnphoA mutants exhibited increased 50% lethal doses with respect to the wild type following intravenous infection of BALB/c mice. Southern analysis of DNA prepared from each of the mutants suggested that TnphoA had inserted into a number of different sites in the S. typhimurium genome. None of the TnphoA mutants had inserts in the virulence-associated plasmid.

The role of a stress‐response protein in Salmonella typhimurium virulence

“…The JnphoA derivatives of C5. TnphoA 060, 014 and 046, were described previously (Miller et al. 1989).…”

“…Heat-shock proteins are more correctly termed 'stress proteins' in that they are produced in response to a variety of stressful stimuli other than just heat (Lindquist, 1986). Low recovery of htrA mutants from infected tissues (Miller et at., 1989) could reflect an inability to withstand some form of stress experienced within the host. One possibility is that the mutants are unable to survive and/or replicate within the macrophage phagolysosome, a hostile environment and one in which lack of a stress protein may be pleiotropic.…”

“…We have utilized the transposon luphoA to generate S. typhimurium mutants of reduced virulence when giving orally to mice (Miller et at., 1989). 'JuphoA is a Tn5 derivative containing an alkaline phosphatase gene minus the signal sequence (Manoil and Beckwith, 1985).…”

Strategies for isolation of in vivo expressed genes from bacteria

“…The use of this approach to the study of osmotically regulated genes in E. coli identi¢ed two genes already known to be inducible by osmolarity and eight other genes, designated osm. Isolation of orally attenuated S. typhimurium and Vibrio cholerae following TnphoA mutagenesis identi¢ed membrane associated virulence factors following oral infection [16]. Several advantages to the use of TnphoA allowed screening for an e¡ect of a given regulatory parameter at any step of gene expression, including transcription, mRNA processing or stability, and translational or posttranslational modi¢cations [17].…”

The development of oral vaccines based on live attenuated Salmonella strains

“…One such system uses TnphoA, a hybrid Tn5 transposon. We have used this approach to isolate several orally attenuated S. typhimurium mutants [28]. One such strain was characterised and the TnphoA insertion found to be in the Salmonella homologue of htrA, an E. coli stress protein.…”