- Ph.D., Massachusetts Institute of Technology, 1987
- B.A., Harvard University, 1980
Our goal is to understand how proteins are transported across the cell membranes of both pathogenic and nonpathogenic bacteria.
My laboratory has a long-standing interest in understanding how proteins are inserted into or transported across the cell membranes of both pathogenic and non-pathogenic bacteria.
In one project we are investigating the assembly of proteins that reside in the outer membrane of Gram-negative bacteria. While most integral membrane proteins contain one or more α-helical membrane spanning segments, bacterial outer membrane proteins are anchored to the membrane by a single amphipathic β sheet that folds into a distinctive closed cylindrical structure called a “β barrel”. Previous studies have shown that a conserved heteroligomer (the barrel assembly machinery or “Bam” complex) catalyzes the integration of β barrels into the outer membrane, but the mechanism by which it promotes insertion is unknown. In many of our experiments we have focused on the assembly of a specific class of outer membrane proteins produced by pathogenic bacteria called “autotransporters”. These proteins contain a large N-terminal extracellular domain ("passenger domain”) in addition to a C-terminal β barrel. The passenger domains of individual members of the autotransporter superfamily play a variety of roles in pathogenesis; in some cases they function as adhesins, but in other cases they are cleaved from the cell surface and function as soluble virulence factors. We have obtained evidence that the β barrel of a model autotransporter produced by E. coli O157:H7 (EspP) begins to fold in the periplasm (the space between the two cell membranes) and then binds to several subunits of the Bam complex in a stereospecific fashion. We have also found that the Bam complex catalyzes the assembly of the EspP β barrel in two separable binding and integration steps. Finally, although it was proposed many years ago that the passenger domain is secreted through a channel formed by the covalently linked β barrel (whence the name “autotransporter”), our work has strongly challenged this hypothesis by indicating that the Bam complex plays a key role in the secretion reaction.
In a second project we are studying protein secretion by the Bacteroides, a genus of experimentally tractable Gram-negative bacteria that are highly abundant in the human gut microbiome. Although recent studies have suggested that these organisms influence human health significantly, their biology remains poorly understood. We recently found that one member of the Bacteroides genus, B. fragilis, utilizes a very different range of secretion strategies than well characterized Gram-negative bacteria such as E. coli. B. fragilis lacks several of the secretion pathways found in E. coli, and unlike E. coli transports a large number of lipoproteins to the cell surface. We also found that B. fragilis secretes a variety of unique proteins of unknown function. Presumably many of these proteins play important roles in colonization, persistence or communication with other microorganisms. By studying protein secretion in the Bacteroides we hope to gain insight into the remarkable ability of these organisms to thrive in a complex and potentially hostile environment.
Applying our Research
Our studies may lead to strategies to isolate inhibitors of outer membrane protein assembly, which is an essential biological process. These compounds would represent a novel class of antibiotics that could be used to combat infections by a variety of Gram-negative pathogens including multidrug-resistant “superbugs”. Furthermore, our work on autotransporters may accelerate the development of vaccines against specific bacterial pathogens. Autotransporters are excellent vaccine candidates because they contain a large extracellular domain, and one autotransporter (pertactin) is already in use in pertussis vaccines. Our studies on autotransporters may also lead to improvements in “autodisplay” technology, a method in which autotransporters are used for the cell surface presentation of heterologous peptides or proteins. Autodisplay has proven to be a valuable alternative to phage-display technology and may ultimately be useful as a method to present antigens to induce immune protection. Finally, our work on Bacteroides may facilitate the construction of genetically engineered strains that secrete beneficial proteins into the colon.
- BamA forms a translocation channel for polypeptide export across the bacterial outer membrane.
- Doyle MT, Bernstein HD.
- Mol Cell (2021 May 6) 81:2000-2012.e3. Abstract/Full Text
- Bacterial outer membrane proteins assemble via asymmetric interactions with the BamA β-barrel.
- Doyle MT, Bernstein HD.
- Nat Commun (2019 Jul 26) 10:3358. Abstract/Full Text
Research in Plain Language
Living cells can be thought of as extremely sophisticated machines that only function if all of the parts are in the right place. Imagine a car in which the steering wheel is in the trunk and the wheels are on the roof. Such a vehicle wouldn’t be very useful!
Most biochemical reactions are performed by proteins, which are manufactured inside living cells. In addition to folding into a defined three-dimensional shape, many proteins must be transported across one or more membrane barriers or embedded in a membrane in order to reach the location where they perform their jobs. We are currently studying protein localization in Gram-negative bacteria, a large group of unicellular organisms that are bounded by a double membrane. We are especially interested in understanding the process by which proteins that must be transported across the first or “inner” membrane and then inserted into the second or “outer” membrane are localized. In many of our experiments we have focused on the localization of a specific family of outer membrane proteins called autotransporters. These proteins are especially interesting because they contain two segments, one that is embedded in the outer membrane and one that is transported across the outer membrane into the extracellular space. The segment that is exposed on the cell surface enhances the ability of many “pathogenic” bacteria to cause disease. We have found that autotransporter localization is a complex, multistep process involving the participation of several molecular machines that are dedicated to preparing client proteins to insert into (or cross) the outer membrane and to helping them fold correctly. We hope that our work will lead to the development of new strategies to block the localization of autotransporters and other outer membrane proteins and thereby prevent or treat bacterial diseases.
In a separate project we are trying to understand how bacteria that are benign, naturally occurring inhabitants of the human colon transport proteins into the environment. We hope that the knowledge we gain will ultimately enable us to produce modified bacteria that export specific proteins into the colon to fight illnesses such as inflammatory bowel disease.