On the role of ribosomal proteins in stress resistance and fitness of Listeria monocytogenes : a laboratory evolution approach

The production of healthy, nutritious, tasty, and safe foods requires efficient strategies to control foodborne pathogens along the food chain. One of these pathogens is the notorious foodborne Listeria monocytogenes. L. monocytogenes is a robust, ubiquitously present human pathogen, and the cause of life-threatening listeriosis in the very young, elderly, pregnant, and immunocompromised persons, the so-called YOPI population. The incidence of L. monocytogenes infections is low, but the severity of listeriosis and the high mortality rate rank it among the top three causes of death by foodborne disease.There is an inherent heterogeneity in microbial populations, and this heterogeneity gives L. monocytogenes the capacity to cope with stresses during transmission from the environment to the human host. Stochastic differences in stress response between individual cells of a population, lead to the differential survival of cells after lethal stresses such as heat or low pH, and ultimately result in tailing of the inactivation curve. The heterogeneity in a population can be either transient, where certain cells temporarily have different properties, or stable, where individual cells have undergone genetic changes that make them better able to resist (lethal) stress. Cells with genetic changes are called stable variants, and can be isolated from the tail of the inactivation curve.    Almost all research that has been done with L. monocytogenes has focussed on the diversity that is already present in a population. Therefore, in chapter 2, we investigated the rate at which new diversity is generated by mutations. Using a high-throughput protocol, we have experimentally determined the mutation rate of 20 L. monocytogenes strains, and found a mutator strain with an insertion in the DNA mismatch repair gene mutS, that resulted in a 100-1000-fold increase in mutation rate. To our knowledge, this is the first mutator strain of L. monocytogenes isolated from food.In chapter 3 we focussed on two previously isolated multiple-stress resistant variants, both with a mutation in the ribosomal rspU gene, one with a point mutation in the rpsU gene, and one with a deletion of the whole rpsU gene. We described the overlap in the stress response of these two variants, and found that even though the mutation in rpsU was very different, the phenotypic responses were remarkably similar. Both variants were multiple-stress resistant due to massive upregulation of genes under the control of the stress-response regulator SigB. Moreover, both variants showed increased attachment to Caco-2 cells, so potentially more infective, and a significantly lower maximum specific growth rate, i.e., lower fitness.Strains are known to persist in the food processing environment for many years, where they are exposed to continuous selection pressures. In chapters 4 and 5 we used experimental evolution to explore what can happen when these stress-resistant variants with lower fitness are exposed to continuous selection for increased fitness. We focused on the same two variants as in Chapter 3, with a point mutation in and with a complete deletion of rpsU. We were able to select for additional mutations that reversed the phenotype from low-fitness and stress-resistant, to high-fitness with low stress-resistance, thereby revealing a tradeoff between these two states. Complementary whole genome sequencing and SNP analysis showed that in the point mutation variant, the additional compensating mutation occurred in rpsU, while in the deletion mutant, the additional mutation occurred in the ribosomal rpsB gene. Thereby we have revealed a link between the ribosomes and the activation of stress resistance by SigB.In conclusion, the work presented in this thesis highlights various microbial adaptive and evolutionary mechanisms that contribute to the heterogeneous behavior of L. monocytogenes. This thesis revealed a trade-off between stress resistance and fitness in stress-isolated variants and it heightened our understanding of how this notorious pathogen is able to grow and survive in changing environments. A mechanistic understanding of the observed trade-off between stress resistance and fitness will impact fundamental research, and ultimately, incorporation of these trade-offs into risk assessments will add to producing minimally processed foods that are microbiologically safe