Protein Transport Across Membrane by Bacterial Pathogens

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Project Summary This research seeks to understand the fundamental process of protein translocation across membrane barriers in bacteria. To establish an infection or exchange antibiotic resistance genes, bacteria must transport macromolecules including protein across multiple membrane barriers: their own and the host cell’s. In response to the universal requirement for macromolecule export, bacteria have evolved elaborate machineries called secretion systems that use energy to move macromolecules from the bacterial cell out into the extracellular milieu or directly into a host cell. In this proposal, I focus on the Type IV secretion system (T4SS). This family of secretion systems is unique in that there are T4SSs that can transport nucleic acid and/or protein cargo. As an important example of a T4SS, I will first investigate the defect in organelle trafficking / intracellular multiplication (Dot/Icm) T4SS in Legionella pneumophila. This system is essential for pathogenesis, which can result in the potentially fatal pneumonia Legionnaires’ Disease. The Dot/Icm T4SS is composed of 30 proteins in different copy numbers. It secretes over 300 protein substrates in order to evade the host cell’s immune system and scavenge nutrients. This represents a much larger repertoire of substrates than observed in other secretion systems that transport proteins out of the bacterial cell. Thus, the Dot/Icm T4SS is an ideal model system for determining how these numerous substrates are engaged and transported. Protein transport by T4SSs has traditionally been studied using cell-based assays. The cellular environment, however, does not allow for precise control and manipulation of reaction conditions. The field needs rigorous biophysical assays with which to dissect the molecular mechanism of protein translocation. I propose to combine determination of high-resolution structures of the Dot/Icm T4SS by cryoEM and thermodynamics and enzyme kinetics studies of the system. These approaches will complement traditional genetic and cell biological strategies and will lead to mechanistic insights into how this secretion system transports protein. For example, transient state kinetics experiments observing the ATP-dependent translocation of a fluorescently labeled substrate protein will answer questions such as “which signal sequences are recognized by which motor protein(s),” “are protein substrates unfolded during transport,” and “which kinetic steps are coupled to ATP binding and hydrolysis?” This approach to investigating complex cellular machinery by integrating biochemical, biophysical, structural, and genetic approaches will shed new light on the fundamental process of translocation across multiple membranes, an important feature of bacterial pathogenesis. While this work aims to understand the fundamental mechanism of protein translocation, our findings could lay the foundation for scientists to develop anti-virulence drugs, the next generation of tools fighting bacterial disease, and to engineer the targeted delivery of gene and protein therapeutics to eukaryotic cells by secretion systems.