Pathogenic bacteria must travel through the highly acidic environment of the stomach before they can reach and infect the intestines. The stomach is therefore an important barricade which helps to kill many bacteria before they can cause illness. In some of the most infectious bacteria, however, the ATP- independent chaperone HdeA plays a major role in aiding bacterial survival at low pH. HdeA's mechanism of action is rather unique, in that it is an unfolded monomeric protein in its activated state. Its job is to protect other proteins from misfolding and aggregating as the cell transitions through the harsh environment of the stomach and into the neutral environment of the intestines. Once the bacteria enter the intestinal tract, HdeA releases these proteins and refolds into its inactive dimer conformation. Biophysical studies have provided clues that HdeA unfolds below pH 3.0 and interacts with its binding partners using hydrophobic residues found at the dimer interface of the folded protein. However, there is a dearth of data that monitors, in detail, the mechanism of monomerization, unfolding and activation at multiple pH values below 3.0. In addition, the properties of instrinsically disordered proteins are generally not well-understood.
Specific aims. We propose to pursue a thorough, atomic-level investigation of the mechanism of activation of HdeA at low pH, using Nuclear Magnetic Resonance (NMR) spectroscopy as our primary analytical tool. Since it is likely that cellular crowding is an important contributor to or understanding of HdeA activity (especially in its unfolded state) we propose to study the mechanism of HdeA activation both in vitro and in-cell. HdeA will also be an excellent model system to improve our understanding of functionality in an intrinsically disordered protein.
Our specific aims are to 1) determine the specific structural and dynamic changes that trigger activation of chaperone activities in HdeA in vitro between pH 3.0 and 2.0 and 2) investigate the differences in structural and dynamic changes that occur in HdeA in-cell or in lysate between pH 6.0 and 2.0 compared to HdeA in vitro. NMR experiments will include titrations to monitor chemical shift changes as a function of pH, hydrogen exchange and 3D experiments to structurally characterize HdeA, and spin relaxation experiments to analyze backbone and side chain protein motions in HdeA at multiple timescales and multiple pH values. Health-related significance. Dysentery, caused by intestinal infection by pathogenic bacteria, kills over one million people per year worldwide. If we can understand how HdeA senses and is triggered by pH changes, we can better understand how this type of acid-stress chaperone helps bacteria survive under extreme conditions. Armed with this understanding we will be able to improve targeting for vaccines or other therapeutics that can disable the activities of HdeA and thereby weaken the infectivity of these pathogenic bacteria.

Public Health Relevance

The stomach is an important barricade that helps to kill many bacteria before they can cause illness, in part by using its acidity to inactivate bacterial proteins. Some bacteria contain a smal chaperone protein called HdeA that helps protect other proteins from becoming permanently inactivated and therefore help bacteria survive and cause infection. We are interested in studying, in-depth, how this protein's function is stimulated by acidic environments; insight we gain may aid future development of vaccines or therapeutics that combat dysentery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
5SC3GM116745-02
Application #
9249639
Study Section
Special Emphasis Panel (ZGM1-TWD-3 (SC))
Program Officer
Zlotnik, Hinda
Project Start
2016-04-01
Project End
2020-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
2
Fiscal Year
2017
Total Cost
$97,875
Indirect Cost
$30,375
Name
California State University Northridge
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
055752331
City
Northridge
State
CA
Country
United States
Zip Code
91330