Characterisation of the pathogenesis and immunogenicity of two novel attenuated mutants of Salmonella Typhimurium

Salmonella enterica causes many diseases in many different animals. Previously we used our own novel technology to test every gene in a particular type of S. enterica called serovar Typhimurium, for whether or not that gene is required for the bacteria to cause disease in a mouse model of infection. Having identified the extent to which almost every gene in the bacterium contributes to virulence we now need to follow up some of the more interesting genes to find out how they work in the disease process. We also need to understand why mutants of the bacteria lacking the genes in question cannot cause full infection, why they do not cause the inflammation that is normally seen with Salmonella infections, and possibly most importantly, to what extent and how the mutant bacteria act as live vaccines. In this project we propose to characterise atpA and trxA mutants. In preliminary studies we have shown that these mutants grow much more slowly than normal salmonellae in mice (known as being highly attenuated), and that they protect mice against subsequent infection with normal, virulent salmonellae, i.e. they can act as good vaccines. Both of these mutants induce minimal to no inflammation in the spleens of infected animals, thus using these mutations in vaccine strains may be a way of reducing adverse reactions. In this study the basis for the attenuation, lack of inflammation and ability to act as a vaccine will be thoroughly characterised using microbiological, cell biological, and immunological techniques. The atpA gene exists in the DNA of salmonellae next to several other similar atp genes, and mutants in these other genes will be constructed and tested for the ability to cause infection. Similarly, there are other trx genes in salmonellae that contribute to the function of the trxA gene product, and these too will be mutated and the effect of this on the ability of salmonellae to cause infection tested. To try to understand what it is that the mutants lack that makes them unable to cause severe infection, they will be subjected to a range of tests and assays. The atp genes are involved in a form of resistance to acid stress, amongst other things, so resistance to acid will be tested. The trx genes are involved, amongst other things, in systems that salmonellae have for dealing with different levels of oxygen, and so resistance to different types of oxygen will be tested. The ability of the mutants to survive in particular cells called macrophages from normal mice and mice that lack certain mechanisms for fighting bacterial infection will be tested to see whether these mechanisms are being countered by the bacterial gene products. The ability of the mutants to induce signals that are normally required to start inflammation in cells and animals of various genetic types will be tested to investigate the mechanism by which inflammation is not induced. Finally, antibody and immune cell responses will be tested to try to understand what it is that these mutants are doing that induces protective immunity against normal Salmonella infection. This project will provide detailed information about these new candidate vaccine strains, as well as considerable data concerning the mechanisms of attenuation, virulence, inflammation and protective immunity in invasive salmonellosis.

Salmonella enterica causes a wide range of diseases in many different hosts. Previously we used novel technology to screen the genome of S. enterica serovar Typhimurium to identify genes required for full virulence of the bacterium in a mouse model of invasive salmonellosis. We must now follow up this study by characterising thoroughly some of the more interesting mutants. We thus propose to characterise atpA and trxA mutants, which we have shown in preliminary studies to be highly attenuated and protective when used as live vaccines. Both of these mutants induce minimal to no inflammation in the spleens of infected animals, thus using these mutations in vaccine strains may be a way of reducing adverse reactions. In this study the basis for the attenuation, lack of inflammation and immunogenicity will be thoroughly characterised using microbiological, cell biological, and immunological techniques. Mutants in the other genes in the atp operon and in other trx genes will be constructed and tested for their degree of attenuation. Mutants will be subjected to a range of tests and assays, for example resistance to acid and reactive oxygen species, to investigate whether their lack of virulence results from reduced resistance to these challenges. The ability of the mutants to survive in primary macrophages from wild-type and knock-out mouse strains will be tested. The ability of the mutants to induce pro-inflammatory signals in cells and animals of various genetic types will be tested to investigate the mechanism by which inflammation is not induced. Finally a full range of immunological parameters will be tested and correlated with the different degrees of protection provide by these mutants. This project will provide detailed information about these new candidate vaccine strains, as well as considerable data concerning basic parameters and mechanisms of attenuation, virulence, inflammation and protective immunity in invasive salmonellosis.