Anne Delcour, Ph.D. - US grants

Porins are large channels of the outer membrane of Gram-negative bacteria, which represent the major entry pathway for hydrophilic solutes into these organisms. Porins have classically been considered as permanently open pores, and little is known about their regulation and mode of action. The objectives of this proposal are to study, at the molecular level, the function and modulation of these proteins in Escherichia coli. Bacteria are one of leading agents in human infectious diseases. Because of their abundance and location at the external surface of the bacterial cells, porins may play a role in the host- pathogen interactions involved in bacterial infection and have been shown to be targets for immune responses. Changes in porin expression patterns have also been observed in virulent strains of E. coli causing urinary tract infections. The significance of this project resides in the study of the control of the bacterial outer membrane permeability which plays an vital role in microorganism's survival. The disruption of the normal functional state of the outer membrane by drug-mediated modulation of porin activity can be conceived as a potential strategy for controlling bacterial infection. The patch clamp technique, widely used in the description of eukaryotic channels, will be applied for the real-time measurement, in the sub- millisecond range, of electrical fluctuations across the membrane during the course of activity of a single or small number of channels. Channel activities will be studied in giant spheroplast or giant cells of E.coli, and in outer membrane fractions reconstituted into giant liposomes. Experiments will be conducted to investigate the molecular mechanisms underlying voltage sensitivity and cooperativity of the major porins expressed by the ompF and ompC genes. OmpC activity can be regulated by membrane-derived oligosaccharides (MDOs), a family of piroplasmic sugar polymers synthesized at high osmolarity. The nature of the molecular events taking place at the level of the channel protein during regulation will be studied. We will also determine the class of MDOs which is responsible for the modulation and define the location of the binding site. A search for other regulatory substances will be conducted, in particular form molecules which promote channel opening and inhibition. Some of these should ultimately be relevant to therapeutic approaches. To fully understand the molecular mechanisms underlying the observed ion channels properties, the relationship between structure and function will be explored by the used of mutant channels. A variety of spontaneous mutants will be used first to map the general regions relevant to specific channels functions. Ultimately, site-directed mutagenesis will be implemented to refine these locations. This is an approach which is widely used in the structure/function relationship studies of eukaryotic channels. The advantages of the bacterial system are that mutant channels are to be studied in their natural environment without the need for injection into foreign expression systems, and porins are the first channels for which an X-ray crystallographic structure has been published. This information on the three-dimensional structure of porins will be extremely valuable for the design of genetically engineered channels and the meaningful interpretation of the data.

This proposal is submitted for funding the purchase of a tabletop ultracentrifuge. This type of centrifuge is particularly useful for the isolation of cellular fractions and purification of nucleic acid samples, where small volumes, high speed and short spin times are required. The instrument will be used in three major projects: (1) Membrane preparation: isolation and reconstitution of bacterial membrane fractions will be performed in the context of the electrophysiological study of Escherichia coli porins and other bacterial ion channels. (2) Isolation of RNA by cesium gradient centrifugation will be performed from embryonic explants in the context of the study of gene regulation during neural induction of Xenopus embryos. (3) RNA will be isolated by cesium gradient centrifugation from zebrafish pineal glands in studies of gene regulation by light and the circadian clock The productivity and success of our research efforts will be greatly enhanced by the acquisition of the proposed instrument, which will allow us to perform procedures that are not feasible with the present state of our instrumentation.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.