Genetic adaptation of salmonella enterica to phagocytic cells

Sammanfattning: Salmonella enterica is a collection of closely related enteric bacteria that as a species is capable of infecting a wide variety of hosts. S. enterica serovar Typhimurium, (S. Typhimurium), causes a systemic disease in mice which in many aspects resembles human typhoid fever caused by S. enterica sv. Typhi. Both these infections are characterized by extensive bacterial growth in the macrophages of the reticuloendothelial system. The pathogenicity of S. Typhimurium has been correlated with its ability to survive and grow in macrophages, so we set out to define the parameters that govern intracellular replication of S. Typhimurium and how alterations in such parameters affect virulence. To accomplish this, we used two very different approaches. The first approach involved the development of a method that allowed RNA extraction from bacteria that had been growing inside eukaryotic cells, and subsequent analysis of gene expression applying microarray technology. This provided a detailed description of the dramatic changes in transcriptional activity that accompanies bacterial transition from an extracellular to an intracellular location. The second approach involved selecting for S. Typhimurium mutants that had increased growth yields in macrophage- like cells. These selection experiments showed that growth advantage mutants indeed could be isolated from phagocytic cells. Phenotypically, the mutations in these bacteria were associated with a modulation in the expression of existing virulence functions, such as the lipopolysaccharide and secreted virulence proteins. Furthermore, we found that most of the mutants that had gained growth advantage, had done so by limiting the host cells production of antimicrobial nitric oxide. Microarray analysis was then used to probe the gene expression profile of one host-adapted strain. This analysis revealed that virulence gene expression was affected. Selected virulence genes were "relaxed" for their expression in this host-adapted strain when grown in vitro, whereas others showed an amplified expression during intracellular growth. Host-adaptation could thus be viewed as an exaggeration of the normal adaptation to an intracellular environment. These changes in the ability to grow inside host cells were also associated with changes in the virulence potential and/or pathogenesis. Whereas some host- adapted mutants were attenuated for virulence, others had decreased lethality, and one mutant was able to cause chronic infection, thus reflecting additional aspects of salmonellosis. These data suggest that S. Typhimurium can indeed alter its virulence potential, and that the spectra of changes that mediate such shifts are broad. However, even if the ability to grow in macrophages was required for the normal pathogenesis, increased growth in macrophages did not necessarily mediate increased virulence, suggesting that adaptation is coupled to trade-offs. In conclusion, the virulence level of S. Typhimurium is well adjusted, and intentional modifications can identify important aspects of this key host-pathogen interaction.