Areas of research
My research program has combined training experiences in both organismal and molecular biology to develop two major focuses: 1) host-bacterial symbiosis; and, 2) the 'design' of tissues that interact with light. The experimental strategy for both areas of research relies on methods that have been developed for the study of the squid-vibrio association over the past 20 years. In addition, I have a continuing interest in the history and development of the field of microbial symbiosis and its impact on biology; a focused effort in this area promises to drive an unprecedented integration across biology as a whole. Such integration will revolutionize the way we think about all aspects of the biosphere. Below, I describe the specific research emphases of my laboratory that relate to these issues.
In the study of symbiosis, my projects have centered on the development of the squid-vibrio association as a model for the establishment and maintenance of the chronic colonization of animal epithelia by gram-negative bacteria. This type of association is the most common in the animal kingdom and the squid-vibrio partnership offers opportunities not available in other symbiotic systems. Most importantly, it is simple, i.e., one host and one microbial species, and both partners can be cultured independently under laboratory conditions. Using this model, my laboratory has explored the most critical questions about the phenomenon of beneficial animal-microbe associations:
- With each generation, how does the animal harvest the often rare symbiont from the environment upon birth or hatching?
- How do the host and symbiont recognize one another?
- How does the bacterial partner influence the developmental program of the host?
- How is stability achieved and maintained in the mature association?
- What are the principal differences between how an animal interacts with pathogenic bacterial species and beneficial ones?
A general principle emerging from our studies has been the finding that in all aspects, from the onset of the association through its maturation, the themes typically associated with bacterial pathogenesis are recurrent. Specifically, hallmarks of pathogenesis dominate each stage of the symbiosis, from the cell to the molecular biology. Because biologists are now recognizing that beneficial symbioses are far more prevalent than pathogenesis, it is likely that most pathogens subvert a pre-exisiting dialogue that host animals have with their beneficial microbial partners.
Over the past several years, we have used microarrays to decipher the molecular dialogue between the host and symbiont. Thus far, we have characterized host responses to the first fully symbiotic state, i.e., at 18 h following colonization, as well as host-symbiont interactions over the day-night cycle in the mature association. These studies provide a foundation for defining the dialogue over the trajectory of development. In our current efforts, we have begun to use next-generation sequencing approaches to understand the first conversations between a host and its bacterial symbionts as the association is initiated. Specifically, in collaboration with Philip Rosenstiel (U Kiel, Germany), we are using RNASeq to determine the total transcriptome of the squid epithelial cells that first contact the colonizing bacteria. Under the natural conditions of ~106 non-specific bacterial cells/ml in the surrounding seawater, significant changes in the gene expression of these host epithelial cells occur within 3 h of hatching in response to as few as 1-5 adhering symbiont cells. Characterization of these remarkably specific responses promises to provide the first insights into the initial molecular interplay between a host's ciliated epithelium and its symbiont cells. Our long-term goal, in collaboration with EG Ruby, is to couple these findings with single-cell transcriptomics of the adherent bacterial cells to define both sides of the host-symbiont dialogue in these earliest partner interactions.
My laboratory remains committed to exploiting the strengths of the squid-vibrio system as a symbiosis model. Not only does the system allow us to dissect the animal-bacterial partnership with unparalleled precision, but this association is one of the few in which the events of symbiosis can be studied under relatively natural conditions. Our goal is to provide concepts and experimental avenues that can be easily extrapolated for the study of the colonization of animal tissues by other more complex symbiotic consortia, or even pathogenic infections.
In my second research focus, which is a minor component of my program, I have studied the 'design' of tissues that interact with light. The symbioses that I have studied have always been ones in which luminous bacteria associate with an animal host. In these systems, evolutionary tinkering has led to the formation of an organ that bears remarkable similarity to the eye, with cornea, lens, choroid, iris, and tapetum analogues. The principal difference is the presence of photoreceptive tissue in the eye (the retina) and photogenic tissue in the light organ (the epithelium harboring luminous bacteria). We have studied this convergence at the biochemical and molecular levels and found that it extends beyond the morphology of the organs. The eye and light organ share many proteins to modulate light, and recently we have reported that the light organ expresses the principal proteins of the visual transduction cascade and itself responds physiologically to light stimulus. These data suggest that the light organ has the molecular, biochemical and physiological potential to behave as a second 'eye'. Currently, we are studying the developmental specification of the eye and light organ to determine whether the embryogenesis of these two organs is under the same inductive forces. Thus far, we have found that at least one isoform of each of the the eye specification genes, pax6, eya, six and dachshund, is expressed in the postembyonic light organ. We are currently comparing the expression of these genes in development of the eye and light organ through embryogenesis and postembryogenesis. In addition, we are determining the effects of the symbiont, both wild-type and luminescence defective mutants, on the expression of these genes and their downstream targets.