Our long-term goal is to understand the mechanisms that strengthen the outer membrane (OM) permeability barrier of gram-negative bacteria during stress. The OM represents a severe impediment to antibiotic development, as it is largely impermeable to antibiotics and can become even less permeable during time of stress.
Our general approach is to use genetics and biochemistry to interrogate (i) changes to the barrier during stress, (ii) the mechanisms that strengthen the barrier, and (iii) the role of these changes in antibiotic resistance and pathogenesis.
ECA AND THE OUTER MEMBRANE PERMEABILITY BARRIER
Our current major area of investigation is elucidating the biosynthesis and function of enterobacterial common antigen (ECA). ECA is an invariant carbohydrate produced by members of Enterobacterales (including Escherichia, Shigella, Salmonella, and Klebsiella). Although this molecule was discovered many years ago, its function has remained mysterious.
There are three forms of this molecule, two of which are surface exposed while one is periplasmic. We have recently found at least two forms of ECA to be important for maintaining the barrier function of the envelope in Escherichia coli, especially during cellular stress (please see our recent paper).
The major projects in the lab currently focus on the biosynthesis, function, and role in pathogenesis of ECA.
All three forms of ECA are made from repeating units of three amino sugars. The repeating units are built on an isoprenoid carrier molecule, which tethers them to the inner membrane and allows them to be flipped from the cytoplasmic to periplasmic face of the membrane. This strategy for moving glycans across membranes is highly conserved and used across all domains of life. After polymerization, ECA can be attached to lipopolysaccharide (LPS) or to a phospholipid and transferred to the cell surface or it can be cyclized and remain in the periplasm.
The genes necessary to make ECA units, flip them across the IM, and control their polymerization are found in an operon. However, many of the steps that occur after polymerization of ECA are poorly understood.
How are the different forms of ECA made? What are the genes responsible? How do the surface exposed forms reach the cell surface? Is ECA synthesis regulated? These are some of the questions the lab is interested in.
Our recent work has demonstrated that periplasmic cyclic ECA plays an important role in maintaining the OM permeability barrier. The activity of cyclic ECA on OM permeability is controlled by YhdP, a protein of unknown function. When YhdP is absent, cyclic ECA takes on aberrant activity that damages the barrier.
We are currently using both genetic screens and biochemical approaches to investigate the specific effects of cyclic ECA and YhdP on the OM and the roles that they play in OM permeability. We are also investigating the functions of the other forms of ECA, whose roles remain mysterious.
ECA'S ROLE IN PATHOGENESIS
ECA is produced by throughout Enterobacterales, a family that includes many human pathogens. One form of ECA (ECALPS) is immunogenic and leads to the production of antibodies that can recognize all members of Enterobacterales. Nevertheless, the pathogenic members of this family continue to produce ECA.
Several studies from other labs have convincingly demonstrated that ECA plays an important, yet undefined, role in the pathogenesis of Salmonella enterica Serovar Typhimurium. Further investigating the role of ECA in S. Typhimurium is an exciting new area of investigation in our lab.