Harris D. Bernstein, Ph.D.

Senior Investigator

Protein Biogenesis Section, Genetics and Biochemistry Branch

NIDDK

Building 5, Room B120
5 Memorial Drive
Bethesda, MD 20814

301-402-4770

harris_bernstein@nih.gov

Research Topics

Research Goal

Our goal is to understand how proteins are transported across the cell membranes of both pathogenic and nonpathogenic bacteria.

Current Research

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.

Biography

  • Ph.D., Massachusetts Institute of Technology, 1987
  • B.A., Harvard University, 1980

Selected Publications

  1. Pavlova O, Peterson JH, Ieva R, Bernstein HD. Mechanistic link between β barrel assembly and the initiation of autotransporter secretion. Proc Natl Acad Sci U S A. 2013;110(10):E938-47.

  2. Ieva R, Tian P, Peterson JH, Bernstein HD. Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain. Proc Natl Acad Sci U S A. 2011;108(31):E383-91.


This page was last updated on December 3rd, 2018