Mutations in the Salmonella Typhi zzz genes impacts on OMVs internalization and uptake kinetics in epithelial cells in culture

Tesis (Magíster en Biotecnología y Ciencias de la Vida)Outer membrane vesicles (OMV) are nano-sized structures formed from the envelope of Gram-negative bacteria (GNB). OMVs are involved in numerous processes are related to bacterial physiology and pathogenesis. Furthermore, OMVs can also be used as biotechnological tools for delivering biomolecules or as a platform to develop vaccines. In general, biotechnological applications are related to how the OMVs interact with the eukaryotic cells. The Host – OMVs interaction depends on the internalization of OMVs by cells. It has been proposed that the OMV size and protein cargo determine the internalization of OMVs by non-phagocytic cells (i.e., epithelial cells). Recently, nine genes have been found to be involved in the OMV biogenesis of Salmonella enterica serovar Typhi. These genes are generically called zzz genes and affect both the OMV size and protein cargo. In the present work, we aimed to identify the differences that mutations in the zzz genes may produce in the Host cells – OMVs interaction. We first evaluated the capacity of the OMVs from the ∆zzz mutants to be internalized by the epithelial cell line HT-29. We observed that all the OMVs could be internalized, and its internalization increased with time. Then we assessed the difference in Host cells – OMVs interaction that the OMVs from the ∆zzz mutants may have between them and the WT strain. We observed a difference in the mean fluorescence that the OMVs exhibited inside the epithelial cells, including the marking pattern inside the cells, the uptake kinetics, and entry in two temperatures (37 °C and 4 °C) for OMV internalization. We also showed that mutations in the zzz genes directly impact the host cells – OMVs interaction. Thus, these results allowed us to better understand how S Typhi OMVs interact with host cells. We expect this knowledge to set the foundations of future developments of a new biotechnological tool based on OMVs technology as well as a better understanding of the impact that OMV have during S. Typhi infection