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LLNL s Ann Holtz-Morris on Studying Y. pestis with Cellular Arrays

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At A Glance

Name: Ann Holtz-Morris

Position: Biomedical scientist, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory

Background: Scientist, Joint Genome Institute, LLNL; Researcher, Clontech Laboratories; Certificate in bioinformatics, California State University, Hayward; MS, plant pathology, University of California, Davis

At last week’s American Society for Cell Biology meeting in Washington, DC, Lawrence Livermore National Laboratory researcher Ann Holtz-Morris presented a new portrait of one of the most infamous pathogens in history, Yersinia pestis — the causative agent of the Black Death. Using a unique microbial cellular array technology, Morris profiled the bacterium under hundreds of different environmental conditions, revealing new information about Y. pestis’ virulence. Holtz took a few moments last week to discuss with Inside Bioassays her research and the technology that enabled it.

How did you develop your interest in studying pathogenesis, and Yersinia pestis in general?

As an undergraduate, I worked in a plant pathology laboratory as a student intern. I did primarily diagnostic work and found that I really wanted to understand how pathogens were able to infect a host, yet could also live in vectors and non-susceptible carrier species largely without harming the latter two. Yersinia pestis has rodent hosts with species-specific varying degrees of susceptibility, is extremely virulent in humans, and causes blockage of feeding in the flea vector. It also has two closely related species, Y. enterocolitica and Y. pseudotuberculosis, that also infect humans, but cause enteric disease and are rarely lethal. These characteristics make Y. pestis an interesting model for understanding pathogenesis.

Yersinia is obviously an infamous pathogen. What is its new relevance today?

Y. pestis has been responsible for three historic pandemics, including the Black Death of Medieval Europe which killed 30 to 40 percent of the medieval population. And it still persists today, though most outbreaks are in underdeveloped nations. There are also natural reservoirs in rodent populations carrying the bacteria throughout much of the world, including the western United States. Public health organizations must continually monitor these. In the 1990s, epidemics broke out again in Madagascar and India. In Madagascar, Y. pestis had acquired natural antibiotic resistance. There are also concerns that Y. pestis could be used as a bioterrorism agent.

You talked about a systems biology approach. What is important about taking a systemsbiology and live-cell approach to studying pathogenesis?

Systems biology is an intellectual framework for understanding biology in general, and pathogenesis more specifically. According to systems biology, each biological process can be described as the result of a collection of molecular interactions, and each individual molecular interaction can only be understood in the context of all of its other potential interactions and the consequences of those interactions. In an individual organism, the genome interacts with the environment through a cascade of protein, RNA expression, metabolic, regulatory, and many other molecular interactions that vary over time. An analogy would be to the interactions of all airplanes flying: weather, wind speed, air traffic control, passengers, pilots, and hub-and-spoke versus direct flight paths — except that’s an easier model. For pathogenesis, there are two organisms that each respond to each other in multiple cascades of events. The vector, or host, represents the microbe’s environment, but as the pathogen attacks the host, it alters its environment.

The big picture is if we can collect all of the information on all of the interactions, we can create computer models, test them, and predict how to interfere with pathogenic progresses, potentially finding new therapeutic targets for plague, and we can define novel diagnostics for early pathogen detection.

What type of studies are you conducting using Yersinia, and what type of technology are you using to do so? What did you have to do prior to this to conduct these types of studies?

I am working on understanding the metabolism of Y. pestis as it behaves under conditions that mimic the flea vector and the human plasma and human intracellular environments. I am comparing and contrasting how individual chemicals affect the growth of the bacteria under those conditions. As a student, I used to do similar experiments one chemical at a time, taking measurements periodically on individual bacterial cultures. Now, I am using a high throughput roboticized system that allows us to measure the growth of the bacteria every fifteen minutes for three days. The roboticized system allows us to use 96-well microtiter plates preloaded with different chemicals in each individual well. Some of the chemicals are metabolites, others are osmotic stressors, and others are antibiotics. I can load up to 50 plates at a time in the robot, so the throughput is 4,800 times faster and is without the data gaps caused by sleep.

What have been your major findings thus far?

We found the bacterium is much more resistant to osmotic stress and antibiotics under the flea conditions than we would have predicted. We’ve also found biochemical activities that were not predicted by the genome annotation, meaning that more experiments need to be done to understand which genes are involved. We’ve found that comparing the flea versus host growth conditions correlate with changes in the patterns of metabolic activities and are similar to published RNA expression pattern changes.

How might an approach like this be translated into finding better treatments for bacterial or viral infections?

I’m part of Dr. Sandra McCutchen-Maloney’s research group at LLNL. By integrating my data with data from other types of experiments done in our group — including proteomic analysis of host response to Y. pestis, comparative protemics of multiple strains of Y. pestis and near neighbors, and real-time characterization of virulence factor expression — we aim to create models predicting how virulence is linked to environment and metabolism. This systems biology view of Y. pestis will provide unprecedented understanding of a virulence mechanism, and will guide our efforts to detect highly virulent or engineered pathogens as well as to detect and predict the effects of emerging threats, such as SARS.

What’s next for your laboratory?

We will be writing this up for publication as well as reiteratively testing chemical combinations and repeating the work with other strains of Y. pestis and the near neighbors to see how general the observations are.