Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli.

Research in my lab seeks to elucidate how cells make decisions in response to environmental cues. My particular focus is on how networks of molecules interact within free-living microbial cells. These networks govern the decision to grow when conditions are optimal or deploy damage repair systems when faced with stress. I study microbial stress responses in extremophiles of the domainArchaea, which represent extreme examples of microbes surviving damage by multiple stressors. These organisms remain viable on the extreme end of the gradient of environmental stress (e.g. high temperature, saturated salt, nutrient starvation). However, extremophiles also adapt during wide variations in conditions and nutrients and therefore provide a study system for both constant and dynamic stress resistance mechanisms. Because archaea resemble life’s earliest ancestors, they can teach us about the origins of stress response features shared amongst all life. In my recent and future work, I compare across species how networks function to regulate important aspects of cell physiology such as growth and division during stress. Ultimately, I seek to uncover how environmental conditions shape the regulatory network over evolutionary time. I use a combination of quantitative and experimental biology approaches, including computational modeling, functional genomics and molecular microbiology. I work across the disciplines of systems biology, microbial stress response, and archaeal molecular biology. My lab group and I are also actively involved in developing microbiology and bioinformatics workshops for various communities (K-12, teachers, researchers).

Enterotoxigenic E. coli (ETEC) causes traveler's diarrhea and infant mortality in underdeveloped countries, and Pseudomonas aeruginosa is an opportunistic pathogen for immunocompromised patients. Like all gram negative bacteria studied to date, ETEC and P. aeruginosa produce small outer membrane vesicles that can serve as delivery "bombs" to host tissues. Vesicles contain a subset of outer membrane and soluble periplasmic proteins and lipids. In tissues and sera of infected hosts, vesicles have been observed to bud from the pathogen and come in close contact with epithelial cells. Despite their association with disease, the ability of pathogenic bacteria to distribute an arsenal of virulence factors to the host cells via vesicles remains relatively unexplored.

In our lab, we focus on the genetic, biochemical and functional features of bacterial vesicle production. Using a genetic screen, we have identified genes essential in the vesiculation process, we have identified specific proteins that are enriched in vesicles, and we have identified critical molecules that govern the internalization of vesicles into host cells. Using biochemical analysis of purified vesicles from cell-free culture supernatants, we have found that heat-labile enterotoxin, an important virulence factor of ETEC, is exported from the cells bound to the external surface of vesicles. Presented in this context, it is able to mediate the entry of the entire ETEC vesicle into human colorectal tissue culture cells. We have also discovered that the ability of vesicles to bind to specific cell types depends on their strain of origin: for example, P. aeruginosa vesicles produced by a strain that was cultured from the lungs of a patient with Cystic Fibrosis adhered better to lung than to gut epithelial cells, whereas a strain that was isolated from sera showed no such preference for lung cells. The vesicles stimulate epithelial cells and macrophages to elicit a cytokine response that is distinct from that of LPS (a major component of the vesicles) alone.

These studies will provide new insights into the membrane dynamics of gram-negative bacteria and consequently aid in the identification of new therapeutic targets for important human pathogens.