HOST EXTRACELLULAR RNASES SHAPING THE REGULATORY RNA NETWORK DURING INFECTION

THE SPREAD OF INFECTIOUS DISEASES RESISTANT TO ANTIBIOTICS REPRESENTS ONE OF THE MAIN CHALLENGES OF BIOMEDICAL RESEARCH. THE DEVELOPMENT OF A NEW GENERATION OF DRUGS THAT EXPLORE ALTERNATIVE STRATEGIES IS URGENTLY NEEDED. THIS PROJECT AIMS TO COMBAT ANTIMICROBIAL RESISTANCE BASED ON A BETTER UNDERSTANDING OF BOTH OUR OWN IMMUNE SYSTEM AND THE MECHANISMS OF RESISTANCE BY PATHOGENS. DURING INFECTIOUS PROCESSES OUR INNATE DEFENSE SYSTEM RELEASES A GREAT DIVERSITY OF ANTIMICROBIAL PROTEINS; THESE INCLUDE SECRETORY RIBONUCLEASES, A FAMILY OF VERTEBRATE-SPECIFIC ENDONUCLEASES. OUR RESEARCH TEAM HAS PIONEERED THE STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF ANTIMICROBIAL RNASES. DURING THE LAST YEARS WE HAVE INCORPORATED NEW METHODOLOGIES IN THE LABORATORY FOR THE STUDY OF RNASES EXPRESSED IN MACROPHAGES, AS WELL AS SELECTIVE SEQUENCING TECHNIQUES OF RNAS THAT ARE PRODUCT OF CLEAVAGE BY ENDONUCLEASES. BASED ON THE COMPARATIVE STUDY OF A NATIVE MONOCYTIC CELL LINE AND ITS COUNTERPART DEFECTIVE IN HUMAN RNASE 2, WE HAVE CHARACTERIZED THE DIGESTION PROFILE WITHIN THE POPULATION OF NON-CODING RNAS (NCRNAS), IDENTIFYING ITS CUTTING PATTERN. THE RESULTS REPRESENT THE FIRST EVIDENCE OF THE SELECTIVE CLEAVAGE OF NCRNAS, AND IN PARTICULAR TRNAS, ASSOCIATED WITH THE EXPRESSION OF RNASE2 IN MACROPHAGES. RNA CLEAVAGE PRODUCTS ON ITS TURN MAY ACT AS SIGNALING MOLECULES, ACTIVATING THE IMMUNE RESPONSE. REGULATORY RNAS REPRESENT A NEW LANGUAGE THAT WE ARE JUST BEGINNING TO GRASP ITS MEANING. THEIR PARTICIPATION WOULD BE ESSENTIAL, BOTH TO ORCHESTRATE THE HOST'S IMMUNE RESPONSE AND TO FACILITATE THE COHESION AND SURVIVAL OF MICROORGANISM COMMUNITIES. PATHOGENS WOULD ACHIEVE A RAPID ADAPTATION TO THE ENVIRONMENT BY TAKING PROFIT OF A NETWORK OF REGULATORY RNAS THAT WOULD FACILITATE THE DEVELOPMENT OF ANTIBIOTIC RESISTANCE MECHANISMS. IN THIS PROJECT WE PROPOSE TO ANALYZE THE POPULATIONS OF NCRNAS, PRODUCT OF THE ACTIVITY OF THE ANTIMICROBIAL RNASES DURING THE PROCESS OF HOST-PATHOGEN INTERACTION. WE WILL FOCUS ON TWO SECRETION RNASES SELECTIVELY EXPRESSED IN MACROPHAGES IN RESPONSE TO INFECTION. TWO MODELS OF RESPIRATORY TRACT INFECTION, PREVIOUSLY CHARACTERIZED IN OUR LABORATORY, WILL BE USED: MYCOBACTERIUM AURUM, AS A SUBSTITUTE FOR TUBERCULOSIS AND THE RESPIRATORY SYNCYTIAL VIRUS, A COMMON SINGLE-STRANDED RNA VIRUS THAT CAN CAUSE BRONCHIOLITIS. THE RESULTS IN EDITED CELL LINES WILL BE VALIDATED IN VITRO AND THE THREE-DIMENSIONAL STRUCTURE OF RNASE COMPLEXES WITH RNA ANALOGUES WILL BE SOLVED. WE PROPOSE TO IDENTIFY: A) THE RNAS ACTIVATING THE IMMUNE RESPONSE AND THOSE ESSENTIAL FOR THE SURVIVAL OF THE PATHOGEN; B) THE PATTERNS OF SELECTIVE RECOGNITION OF HOST/PATHOGEN RNA AND C) THE STRUCTURAL DETERMINANTS OF THE RNASE-TRNAS INTERACTION. FINALLY, WE PLAN TO VALIDATE THE RESULTS IN A TRANSGENIC MOUSE MODEL AND APPLY THEM TO THE DESIGN OF ACTIVATABLE PROBES FOR THE DETECTION OF RNASES IN VIVO AND THE TARGETED RELEASE OF DRUGS. IN SUMMARY, WE AIM TO IDENTIFY THE MECHANISMS OF SPECIFIC RECOGNITION OF RNA BY ENDOGENOUS RNASES OF MACROPHAGES AND THEIR ROLE IN THE ERADICATION OF INFECTION. THE EXPECTED RESULTS WILL CONTRIBUTE TO DEEPENING THE UNDERSTANDING OF THE CELL SIGNALING SYSTEM MEDIATED BY REGULATORY RNAS AND WILL LAY THE FOUNDATIONS FOR THE DESIGN OF ALTERNATIVE ANTI-INFECTIVE AGENTS.