My laboratory uses Salmonella as a highly tractable model organism to delineate the mechanisms through which pathogens manipulate host cells to either grow within them or exploit them as vehicles for dissemination to deeper tissue. There are two questions in my laboratory:
1) How are human proteins subverted by pathogens to facilitate intracellular growth?
To begin to answer this question, we designed a fluorescence based high throughput RNA interference assay and screened the entire human genome at Duke University for host factors that allow intracellular Salmonella growth. In this screen, we individually depleted host cells of every human protein with siRNA, and infected them with Salmonella expressing the green fluorescent protein. We incubated the infected host cells overnight and then stained them with a DNA stain that lit up the host nuclei and analyzed them for green fluorescence. Cells that had roughly the same amount of the nuclear stain, indicating that host cell health was not affected, but displaying reduced green fluorescence were lacking a protein that Salmonella uses to its advantage. In all, we identified 252 human proteins, that we are beginning to characterize, that permit Salmonella growth within infected, human cells. The long-term goal of this project is to advance our understanding of the Salmonella/macrophage paradigm, which may provide us with insight into the underlying shared logic through which numerous pathogens manipulate host cells. Also, these host susceptibility factors can potentially be exploited as a novel class of anti-microbial targets and finally can illuminate many aspects of fundamental host cell biology.
2) Through what mechanisms do intracellular pathogens disseminate within hosts to cause serious, systemic disease?
The long-term goal of this project is to develop and test comprehensive, global models that account for reverse transmigration and its regulation. Reverse transmigration is a naturally occurring process in which dendritic cells traverse endothelium in the basal to apical direction. While Salmonella can spread from the gastrointestinal tract to the bloodstream within these cells, it is conventionally thought to be passive on the part of the microbe. We have uncovered evidence however, indicating that Salmonella can actively parasitize reverse transmigration to expedite bacterial colonization of internal organs, creating a much more serious, systemic infection. This is likely relevant to the spread of numerous pathogenic microbes not only from the GI tract, but also from lung tissue and the oral mucosa to the systemic circulation. Reverse transmigration also functions in the invasion of the bloodstream by some cancerous cells and in excessive inflammation disorders such as autoimmune and graft versus host disease, which piques our long-term interest in designing drugs that either inhibit or augment the process.
We recently developed an in vitro model of reverse transmigration. In this assay, endothelial cells are grown into confluent monolayers on membranes with small pores. They are then placed upside down in a blind well and another membrane is coated with a natural extracellular matrix and placed right side up in the device. Infected dendritic cells are then added to the top compartment and the reverse transmigration of these cells across the endothelial monolayer into the bottom compartment is measured. This models the invasion of the bloodstream by Salmonella-infected dendritic cells associated with the gastrointestinal epithelium. In prior work, we discovered a Salmonella protein, which is secreted into infected host cells to stimulate reverse transmigration. We will use this assay to characterize this virulence factor and to screen for new ones. Also, this assay will be used to identify and characterize host factors that facilitate and inhibit reverse transmigration and to study how Salmonella perturbs them to its advantage.
Office: You can find me in room 355 of the Shumaker Research Building on the main campus (Belknap). tel. 502-852-0966 email: micah dot worley at louisville dot edu
Laboratory: You can find us in room 311 of the Shumaker Research Building on the main campus (Belknap).
The link address is: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045245
The link address is: http://www.pnas.org/content/103/47/17915.full