Transposon-insertion sequencing to identify novel antimicrobial drug targets

The species Streptococcus suis, a Gram-positive lactic acid bacterium, is a leading zoonotic cause of endemic bacterial disease in pigs, with welfare, economic, and public health impacts around the world. The absence of effective vaccines, or effective surveillance tools to understand and interrupt transmission, has led to inadequate prevention and global reliance on widescale use of antibiotics resulting in only partial control. The increasing identification of multidrug-resistant S. suis strains is having an important impact on the pig industry, leading to economic losses. S. suis mainly affects weaning piglets, but is also an emerging zoonotic pathogen with most human clinical cases due to swine contact reported in Southeast Asia and North America. The extensive use of antibiotics in animal husbandry, infectious disease treatment, metaphylaxis and growth promoters which are still used in some countries, is the leading cause of the selection of new drug-resistant strains. Nowadays, S. suis is resistant to some of the main groups of antibiotics, including macrolides, lincosamides, and tetracyclines, among others. This increased resistance highlights an urgent need to develop new antimicrobials, or disease prevention strategies targeting genes required for survival and growth in the environment of the host. The main objectives of this project are (1) to identify the molecular mechanisms that contribute to the intrinsic or adaptive resistance of S. suis to certain antibiotics, other than the acquisition of antibiotic resistance genes by lateral gene transfer; such genes might be attractive drug targets to re-sensitize AMR S. suis and other pathogens. (2). The identification of metabolic or other genes required for S. suis survival and growth in the body fluids of the host. The hypothesis is that the identified proteins and pathways would be good targets for new drugs to treat or prevent infections. Furthermore, the targets identified may be conserved in other bacteria including streptococcal pathogens of humans, broadening the potential societal impact. High-throughput genome-wide screening methods for identification of potential targets are required because metabolic networks are highly complex, containing many genes and protein interactions. We chose a Transposon- insertion sequencing (Tn-seq) approach that requires creation of a saturated transposon library of clones covering the entire genome of S. suis, and relies on identification of mutants that cannot grow in selective media containing antibiotics, and mutants that can no longer survive in pig body fluids ex vivo. These mutants will be subjected to further analysis: the specific gene(s) targeted by transposon tagging will be deleted from the S. suis genome using CrispR/cas9 technology to verify their possible roles in sensitizing S. suis to antibiotics, or infection and survival in the host. Genetic, biochemical and in vitro or ex vivo assays will also be used to determine the functions of selected genes.