Role of diffusible signals secreted by macrophages in chemotaxis of Escherichia coli

My PhD thesis explored bacterial chemotaxis to diffusible signals secreted by macrophages, using the model organism Escherichia coli. While an extensive knowledge exists on the physiology of this microorganism, less research has been done to understand its interaction with secreted signals from cells of the immune system. My research aimed to identify metabolites secreted in the culture supernatants of macrophages and systematically, test their ability to elicit chemotactic response in E.coli. To this end, I performed a comparative metabolomics study on the secreted metabolites of M1 macrophages (microbicidal/inflammatory phenotype), M2 macrophages (anti-inflammatory phenotype), and Mφ macrophages (resting phenotype). My data revealed that the secreted metabolites in the culture supernatants were predominantly products of tryptophan degradation. To determine whether E. coli exhibited chemotaxis towards any of these supernatants, I conducted a side-by- side comparison after mixing them with E. coli cells in a microfluidic chip. I found that E. coli showed an attractant response towards supernatants from stimulated macrophages (M1 and M2). However, these responses could not be attributed to the tryptophan degradation products suggesting that putative chemoeffectors secreted in the medium are from different pathways. PCA uncovered that the metabolite content, both in terms of quantity and quality, were similar for Mφ and M2. In contrast, M1 macrophages were distinct from both M2 and Mφ. This distinction was primarily due to the extensive degradation of tryptophan and secretion of kynurenine metabolites. Therefore, while the types of metabolites secreted by all three macrophage phenotypes were similar, they differed significantly in the levels of kynurenine metabolites. Additionally, compounds from serotonin metabolism were highly secreted in macrophage SUMMARY supernatants. We also found that M1 macrophages secrete components of melanin synthesis and a chemoattractant for inflammatory recruitment of neutrophils—5-Hydroxyindoleacetic acid (5-HIAA). Nucleosides are known to be secreted in cultures of mouse bone- marrow derived macrophages (BMDMs). To test chemotactic responses to nucleosides, I employed a FRET-based chemotaxis assay using the E. coli W3110 strain as the model organism. The major finding of my chemotaxis experiments was that E. coli exhibited an attractant response to nucleosides. Experiments with chemoreceptor mutants confirmed that pyrimidine nucleosides are sensed by the Tap chemoreceptor. Dynamic response measurements revealed that this sensing occurs through indirect binding to Tap, suggesting the involvement of an unknown nucleoside binding protein in the periplasm that interacts with Tap to mediate chemotaxis to nucleosides. Among the tested nucleosides, E. coli exhibited a chemotactic response to pyrimidine deoxyribonucleosides—thymidine and deoxycytidine— with high sensitivity. Uridine and the purine exception— deoxyguanosine—showed similar responses. Interestingly, among the nucleobases, only uracil demonstrated an attractant response. The conclusions drawn from my thesis warrants for further research aimed at elucidating the role of bacterial chemotaxis to nucleosides in context of infections.