Roberto De Guzman
Department of Molecular Biosciences
University of Kansas
Interactions of Salmonella Needle and Tip Proteins (2008-10)
The aim of this research project is to determine how certain bacterial proteins self-assemble into a structure used by the bacteria to infect humans and cause disease. This knowledge will be useful in the development of novel antibacterial drugs to combat the threat posed by bacteria that have become resistant to current antibiotics.
Many pathogenic bacteria that affect millions of people worldwide, including some that are classified as potential agents of bioterrorism, use a needle-like structure on their cell surface to inject toxic proteins into human host cells. No vaccines are currently approved for general use against any of these pathogens, and the appearance of antibiotic resistant strains makes these pathogens major threats to public health and safety. Because the needle apparatus is critical for virulence and is exposed on the bacterial surface, disrupting this protein assembly is an attractive approach for the development of novel antibiotics. This approach requires a detailed understanding of the protein-protein interactions involved in the assembly of the needle apparatus. The needle is assembled from about 120 identical copies of a protein that are arranged in a helical manner, and the tip of needle is capped by a few copies of a special tip protein.
The objective of the proposed studies is to elucidate in detail the protein-protein interactions involved in the assembly of the needle apparatus. Over the past 2 years, we have completed the NMR structures of two needle monomers: BsaL from Burkholderia pseudomallei, a pathogen associated with biowarfare, and PrgI from Salmonella typhimurium, a pathogen associated with food poisoning. Using NMR mapping techniques, we have identified the key residues involved in the binding interaction between the needle and tip proteins of Shigella flexneri. Our long term goal is to determine precisely how the needle apparatus is assembled from its protein components. We propose here to elucidate the protein-protein interactions of PrgI and SipD, the needle and tip proteins of Salmonella typhimurium, using a combination of NMR, mutagenesis, and bacterial invasion assays in cell culture. Our specific aims are:
- (Aim #1) determine exactly how the needle proteins interact with each other,
- (Aim #2) determine how the needle protein interacts with the tip protein, and
- (Aim #3) use mutagenesis and invasion assays to correlate the structural results to needle assembly and virulence. In the longer term the knowledge gained will be applied to devising small molecule strategies to disrupt needle assembly and thereby render virulent bacteria non-virulent.
Structure and Dynamics of Bacterial Needle Proteins (2005-2007)
Mentor: Mark Richter
The overall goal of this project is to characterize the structures and dynamics of proteins that are involved in bacterial pathogenesis. Many pathogenic bacteria possess a protein transport machinery, the type III secretion system, that is used by bacteria to deliver protein toxins into the host cells. Bacterial pathogens that utilize the type III secretion system for infectivity cause important human diseases, such as bacterial dysentery (Shigella), lung infections (Pseudomonas), plague (Yersinia), food poisoning (Salmonella and Escherichia), and acute infections (Burkholderia). Furthermore, Burkholderia pseudomallei and the B. mallei are Select Agents of the CDC and USDA because of their lethality and potential application in bioterrorism. A central feature of the type III secretion system is a needle-like protein assembly, the needle apparatus, which together with other bacterial proteins, form a physical contact between the bacterium and the host cell. The needle apparatus is about 50 nanometer in length and contains a central channel of 2-3 nm in diameter, and serves as a conduit to transit bacterial proteins into the host cells. The needle apparatus is formed by the polymerization of a single type of protein into a macromolecular assembly.
We have used nuclear magnetic resonance (NMR) spectroscopy to determine the atomic level structure of a monomeric needle protein, BsaL, from Burkholderia pseudomallei and showed that it is composed of a central core domain with a two-helix bundle. Also, our structure revealed that almost ½ of the protein is in partial-helical conformation, suggesting that protein dynamics is an important property of the needle proteins. Our work represents the first report of an atomic level structure for any type III needle protein, and provides the foundation for further NMR studies aimed at characterizing the structures and dynamics of needle proteins, their protein-protein interactions, and their roles in pathogenesis. We hope to provide the details of how these proteins work at the atomic level, and that knowledge can be used to design novel antibacterial therapy targeted at disrupting the type III secretion system.
Dr. De Guzman graduated as an assistant professor from the COBRE program in 2008 when he was awarded an NIH R-01 grant " NMR studies of bacterial needle and tip proteins." He was promoted to professor in 2016.