My research with C. elegans was specifically chosen with a primarily undergraduate institution (PUI) in mind. Most animal models in immunology require large budgets and special protocols from Animal Care and Use committees. However, the usefulness of “alternate” models has become apparent in recent years, including important immunological studies involving fruit flies (Drosophila melanogaster), plants (Arabidopsis thaliana) and nematodes (Caenorhabditis elegans). These models provide inexpensive means to answer important questions regarding innate immune responses and host-pathogen interactions.
As a model system, C. elegans has some unique advantages to other organisms. Through its use in various fields, many tools are widely available for C. elegans research, including a completely sequenced genome, a cell fate map, many genetic tools, and consortia that provide knockout or promoter-green fluorescent protein (GFP) fusion animals to researchers at no cost. Because the C. elegans community freely shares its resources, I am able to conduct research at a PUI that many others would find difficult.
C. elegans research in the laboratory requires minimal specialized equipment. C. elegans is propagated in the laboratory using agar-containing petri plates inoculated with an attenuated strain of Escherichia coli. Animals are incubated at specific temperatures to control growth, and manipulation of animals requires a simple dissection microscope and platinum wire pick. If research must be halted, C. elegans can remain in a dormant larval stage for weeks, or can be frozen at -80ºC for longer periods of time.
My research with C. elegans investigates its immune responses to the pathogen Salmonella typhimurium. To test the specificity of a Salmonella response, other pathogens such as Enterococcus faecalis, Pseudomonas aeruginosa, or Cryptococcus neoformans are utilized once a disease phenotype is seen using Salmonella typhimurium. All of these organisms are attenuated BSL2 laboratory strains, which can be found in many undergraduate Microbiology laboratories. They do not pose a serious health risk to students. Basic rules of hand washing and absence of food or drink in lab, combined with training in aseptic techniques will ensure student safety while handling these bacteria. If BSL2 organisms are an issue, there are nematode-specific pathogens or plant pathogens which can be studied as an alternate means to explore the immune responses of C. elegans.
I am specifically interested in the role of the GATA transcription factor, ELT-2, in the C. elegans immune response. My postdoctoral research has concentrated on the role of c-type lectins in the C. elegans immune response. We have found that a subset of these lectins is controlled by ELT-2. I have shown that ELT-2 is important in the C. elegans immune response to multiple pathogens, and therefore is a likely regulator of many important immune defense genes. My project has reached an exciting phase, where we are beginning to piece together the genetic pathways that control these c-type lectin responses in the context of ELT-2. There are many avenues to explore, all of which can be conducted with undergraduate research:
1. Examine the genetic pathways through which ELT-2 and candidate lectins are expressed. This will be done by measuring RNA levels from elt-2 and candidate lectin genes by RTPCR in nematodes in which known immune pathways are mutated. Alternatively, we can perform chemical mutagenesis of lectin promoter-GFP fusion marker strains or an ELT-2 promoter-GFP fusion strain, and screen for altered GFP expression. These mutations can then be mapped by crossing nematodes and performing SNP analysis by PCR and use of restriction enzymes.
2. Determine other genes controlled by ELT-2. This will be initially done using bioinformatics to screen candidates in the C. elegans genome based upon intestinal expression and presence of GATA binding sites on candidate gene promoters. Candidate genes can then be cloned and knocked down in the nematode by RNA interference, and animals screened for susceptibility to pathogens.
3. Determine Salmonella genes that are important in the infection of C. elegans. This will be accomplished by transposon mutagenesis of Salmonella typhimurium and a subsequent screen for increased survival of C. elegans. Candidate mutants will be sequenced to determine transposon position, and reconstitution experiments will be performed to show a direct correlation of candidate genes with disease phenotype. Subsequent experiments will concentrate on genes that directly act at the host-pathogen interface to circumvent the C. elegans immune response and cause disease.
Because of its use in so many fields of research, I am excited about the collaboration possibilities of my model system at a PUI. Nematodes are routinely used to study neurobiology, developmental biology, animal behavior, and aging, as well as host-pathogen interactions. As such, C. elegans can be used to augment a variety of course laboratory exercises, or confirm colleague’s data in a whole organism model, if desired.
I am very excited about my projects, as I think they will enable me to conduct quality scientific research within a PUI. This is exactly the type of experience undergraduates need to solidify their future plans in the sciences.