Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

<p>The β-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a β-barrel fold which can vary from 8 to 36 β-strands, with each serving an important role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis has remained elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. Here we present our recent work on the structures of BAM in its near native environment, characterized by cryo-EM, and study its interaction with OMP substrates. Specifically, we focused on the role of BAM-mediated EspP biogenesis, and structurally characterized crosslinked intermediates to atomic resolution, allowing for a more complete understanding of BAM-mediated OMP biogenesis. We also characterized BAM-mediated OmpT and OmpA biogenesis, which further supports a BamA-budding model for OMP biogenesis. Given its essential role in Gram-negative bacteria, BAM is an attractive target for antibiotics, and we contributed to characterizing a novel antibiotic designed against BAM called darobactin, which binds to the lateral gate of BAM, thereby disrupting OMP biogenesis and leading to programmed bacterial lysis.</p>