Periodic Reporting for period 2 - TYPHI (From Typhoid Fever to Antimicrobial Mechanisms) Reporting period: 2019-03-01 to 2020-10-31 Summary of the context and overall objectives of the project Despite the dramatic progress of 20th century medicine in fighting bacterial infections (including discovery of antibiotics and development of vaccines), infectious diseases caused by bacterial pathogens (e.g. tuberculosis, typhoid fever, pertussis, leprosy, bacterial meningitis and pneumonia) are still a serious public health problem ( http://www.who.int/epidemic_diseases/meningitis/en/; http://www.who.int/mediacentre/factsheets/fs331/en/; WHO report, 2014). This is due to several factors that include the emergence of antibiotic resistance in bacteria, the difficulty of vaccinating extensive parts of populations in poor areas of the world, and our limited understanding of critical pathogenic mechanisms exploited by many of these bacterial pathogens during infection. Remarkably, how bacterial pathogens are cleared from the infected organism is still an uncertain and controversial issue. Indeed, despite extensive analyses of the recognition and signalling events that lead to pathogen identification and phagocytosis (Reddick and Alto, 2014), the mechanisms that result in killing or inhibition of the bacterial growth once the pathogen is taken up by a phagocytic immune cell are still not clearly understood. One of the major objectives of this proposal is to elucidate mechanisms of pathogen killing by immune cells and in particular will focus on the human pathogen Salmonella Typhi (S. Typhi).Professor Spanò described a novel trafficking pathway that underlies S. Typhi host-restriction. A unique pathogenic feature of S. Typhi is its ability to infect only humans. The mechanisms of S. Typhi human restriction were completely unknown until recently, when she reported that a host trafficking pathway operating in macrophages contributes to prevent S. Typhi infection in mice (Spanò and Galan, 2012). Using a powerful combination of cell biology, genetic and mass spectrometry analyses, she showed that S. Typhi infection is restricted by a host trafficking pathway dependent on the small GTPase Rab32 and Biogenesis of Lysosome-related Organelles Complex (BLOC)-3 (Spanò and Galán, 2012; Fig. 1). Building on these findings this project will address the following questions:1) What killing molecules does the Rab32-dependent trafficking pathway deliver to the S. Typhi vacuole? 2) How is the Rab32-dependent trafficking pathway regulated? 3) Is the Rab32-dependent trafficking pathway active in human macrophages? If so, how does S. Typhi evade killing to replicate in human macrophages? Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During this reported period we have 1) Completed the proteomic analysis of the bacteria-containing vacuoles from both the Salmonella and S. aureus and we have found some protein that are present on the vacuole only when the Rab32 pathway is active.2) Performed a comparative RNAseq of bacteria internalised from macrophages with an active or inactive Rab32 pathway3) Shown that the Rab32 pathway is active in human macrophages and now we are concentrating in understanding how S Typhi counteract that pathway and survive in human macrophage.4)Understood better how the regulation of the Rab32 pathway. In particular we have found a protein that act as inhibitor of the pathway. We have demonstrated that removal of such inhibitor boosts the Rab32 pathway and increase the ability of the immune cells to kill Salmonella.5)Found new Rab32 interactions and we are in the process to validated these interactions and their impact to Salmonella intracellular survival. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The recent finding of a novel traffic pathway that mediates killing of intracellular bacteria in mouse macrophage represents a unique opportunity to identify novel mechanisms of pathogen killing in macrophages and to understand the pathogenic mechanisms that are at the base of S. Typhi human-adaptation.By addressing the following fundamental questions:1) What killing molecules does the Rab32-dependent trafficking pathway deliver to the S. Typhi vacuole? 2) How is the Rab32-dependent trafficking pathway regulated? 3) Is the Rab32-dependent trafficking pathway active in human macrophages? If so, how does S. Typhi evade killing to replicate in human macrophages? This project will:I) Identify novel antimicrobial molecules that kill bacterial pathogens; II) Identify critical regulators of this novel antimicrobial pathway;III) Elucidate how S. Typhi establishes deadly infections in humans and identify new therapeutic targets.