Project Grant R35GM155280

UNDERSTANDING TYPE IV PILI ON THE SINGE-MOLECULE LEVEL - TYPE IV PILI (TFP) ARE CAPTIVATING BACTERIAL NANOMACHINES THAT ARE BROADLY CONSERVED AND EXHIBIT A DIVERSE ARRAY OF PHYSIOLOGICAL FUNCTIONS. IN THE OPPORTUNISTIC PATHOGEN PSEUDOMONAS AERUGINOSA (PA) FOR EXAMPLE, TFP ARE IMPLIED IN NUMEROUS VIRULENCE ASSOCIATED FACTORS SUCH AS BIOFILM FORMATION, MECHANOSENSING, AND MOTILITY. THE PHYSIOLOGICAL FUNCTION OF TFP RELIES ON REPETITIVE CYCLES OF EXTENDING AND RETRACTING OF A SHORT POLYMERIC FIBER INTO THE EXTRACELLULAR SPACE, FOR EXAMPLE TO ADHERE TO A SUBSTRATE AND PULL THE CELL FORWARD. THESE DYNAMIC CYCLES ARE FACILITATED BY A COMPLEX MOLECULAR MACHINERY CONSISTING OF DOZENS OF DIFFERENT PROTEINS THAT WORK TOGETHER IN A CONCERTED MANNER. ALTHOUGH THE CONSTITUENTS OF TFP ARE KNOWN, THE INTRICATE MOLECULAR PROCESSES GOVERNING THEIR INTERACTION AND HOW THESE INTERACTIONS ENABLE THE PHYSIOLOGICAL FUNCTION OF TFP REMAIN LARGELY ELUSIVE. CURRENT LIMITATIONS OF OBSERVING THE MOLECULAR INTERACTIONS THAT DRIVE THE FUNCTIONS OF TFP ARE INTRINSIC TO THE EXPERIMENTAL TECHNIQUES THAT HAVE BEEN USED TO FAR: THESE TECHNIQUES CANNOT RESOLVE INDIVIDUAL PROTEINS OR TFP COMPLEXES. SINGLE MOLECULE TECHNIQUES HAVE THE POTENTIAL TO FILL THIS GAP AND TO GAIN INVALUABLE INSIGHT INTO THE INTERACTIONS AMONG THE PROTEINS OF THE TFP SYSTEM AND HOW THEY FUNCTION TOGETHER AS MULTI-MOLECULAR COMPLEXES. THE POWER OF SINGLE MOLECULE STUDIES IS TO RESOLVE AND OBSERVE INDIVIDUAL TFP COMPLEXES, THEIR CONSTITUENTS, AND TO PROBE THE RAPID INTERACTIONS BETWEEN TFP AND THEIR EFFECTOR MOLECULES, WHICH HAPPENS ON THE TEN TO HUNDREDS OF MILLISECONDS TIMESCALE. THE LONG-TERM VISION OF MY RESEARCH PROGRAM IS TO LEVERAGE THE POTENTIAL THAT SINGLE MOLECULE LIGHT-MICROSCOPY BASED TECHNIQUES OFFER TO UNDERSTAND TFP AND THEIR PHYSIOLOGICAL FUNCTION AT THE MOLECULAR LEVEL: WE WILL REVEAL HOW TFP MACHINES ARE ASSEMBLED MOLECULE-BY-MOLECULE, HOW INDIVIDUAL MOLECULAR MOTORS AND REGULATORY PROTEINS INTERACT WITH THE STATIC COMPONENTS OF TFP, AND HOW THESE INTERACTION KINETICS ARE CHANGED AND TRIGGER DOWNSTREAM SIGNALING PATHWAYS DURING THE PHYSIOLOGICAL FUNCTION OF TFP. SPECIFICALLY, FIRSTLY, WE WILL USE SUPER-RESOLUTION MICROSCOPY TO RESOLVE INDIVIDUAL TFP COMPLEXES AND THEIR COMPONENTS AND MAP THEIR PRECISE LOCATIONS AND ASSEMBLY STATES MOLECULE-BY-MOLECULE. COMBINED WITH TIMELAPSE FLUORESCENCE MICROSCOPY TO TRACK THE FORMATION OF NEW TFP COMPONENTS THROUGH THE CELL CYCLE, THIS WILL REVEAL THE FIRST STEP TO THE PHYSIOLOGICAL FUNCTION OF TFP: WHEN, WHERE, AND HOW THE DIFFERENT COMPONENTS OF NEW TFP ARE ASSEMBLED AND CONTROLLED. SECONDLY, WE WILL USE SINGLE-MOLECULE FORSTER RESONANCE ENERGY TRANSFER (SMFRET) TO INVESTIGATE THE DYNAMIC INTERACTIONS BETWEEN SPECIFIC PAIRS OF PROTEINS OF THE TFP SYSTEM. THIS WILL REVEAL HOW MOLECULAR EFFECTORS ENABLE AND TUNE TFP DYNAMICS TO REGULATE THE PHYSIOLOGICAL FUNCTIONS OF TFP. LONG TERM, WE WILL COUPLE THESE EXPERIMENTS WITH SINGLE-MOLECULE FORCE MICROSCOPY TECHNIQUES TO REVEAL HOW THE PHYSIOLOGICAL FUNCTIONS OF TFP FEED BACK TO THE MOLECULAR DYNAMICS OF ITS CONSTITUENTS. THE IMPACT OF THIS PROGRAM EXTENDS BEYOND PSEUDOMONAS' TFP AND ENCOMPASSES THE BROADER TFP SUPERFAMILY, INCLUDING SECRETION, ADHESION, AND FLAGELLAR SYSTEMS IN GRAM-POSITIVE AND -NEGATIVE PROKARYOTES, AND ARCHAEA.