Transport of folded proteins across membranes is a feat only accomplished by few biomacromolecular machines. One of these sophisticated machineries is the type II secretion system (T2SS) which spans two membranes and translocates many different folded proteins from the bacterial periplasm into the extracellular milieu. Examples of exoproteins secreted by the T2SS are the cholera toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC). These toxins are key virulence factors for global diseases where infections by V. cholerae can result in death within days or even hours, and ETEC causes infant diarrhea and children's death worldwide. The proposed research uses primarily crystallographic and electron microscopy approaches, complemented by other biophysical techniques. Determining the three- dimensional structure of the T2SS constitutes a tremendous challenge since this system contains multiple copies of 11 to 14 different proteins, resulting in a total of 40-70 polypeptide chains. The T2SS is dynamic and most likely exists only transiently in a fully assembled form. We already made significant contributions to the T2SS field including numerous crystal structures of individual components and subcomplexes, and a cryo-EM reconstruction of the ~0.90 MDa outer membrane channel. Recently, we obtained preliminary electron microscopy class averages of a ~1.44 MDa inner membrane protein subcomplex. Considering the synergy of our team of investigators using complementary techniques, we have both a strong background and the know-how required to obtain profound insight into the architecture and mechanism of action of the T2SS. The T2SS is related to other sophisticated machineries such as type IV pilus system, which is responsible for complex processes such as twitching motility, protein secretion, protein import and DNA uptake in many pathogenic bacteria. These relationships linking the T2SS with other transport systems further enhance the importance of our studies on the T2SS. Given the unique characteristics of the T2SS, our studies will substantially increase the fundamental scientific knowledge of bacterial pathogenesis. Furthermore, the outcomes of the proposed project will provide a framework for structure-based development of therapeutics targeting a range of bacterial infections.