All animals, vertebrates and invertebrates alike, are exposed to a plethora of stresses from their environment. These stresses can include heat, cold, osmotic, heavy metal, physical, toxic and oxidative stress. Some of these stresses (namely toxic and oxidative) can be imparted by disease causing microorganisms while some of them are largely environmental (namely cold). To survive these stresses, it is essential that an animal be able to recognize potential causes of stress in order to avoid or recover from them. Considering the wide range of theses stresses, there is a wide range of corresponding responses.
Stresses imparted by pathogens mainly elicit the immune system for response. All vertebrate animals, humans included, have two immune systems, the adaptive (or acquired) immune system and the innate immune system while invertebrate animals only possess the innate immune system. The innate immune system is the first line of defense against pathogens and activates the adaptive immune system in vertebrates. The infection recognition of the innate immune system can occur through direct binding of the pathogen to pathogen receptors. In sterile body cavities, such as the bloodstream, this is an effective method of recognition, as any microbe present necessitates an immediate response. In non-sterile body cavities which are regularly in contact with and even require the presence of bacteria (such as the epithelial layer of the intestine), this method of recognition is not sufficient because it does not allow for discrimination between harmful and helpful bacteria. This discrimination thus necessitates recognition of pathogen-derived stressors as opposed to the recognition of the pathogen itself.
Stresses imparted by the environment elicit other responses such as metabolic and reproductive. Metabolic response to stress leads to the restoration and allocation of adequate energy and nutrition for the tissues, organs and systems whose functions are essential for survival. Reproductive response to stress may include increased investment in progeny in hope that the progeny would survive the stress if the parent population does not. There is also evidence to support matricide, in which the parent worm dies, is intentional and altruistic in order to support progeny.
In the Powell lab, we use C. elegans, a small invertebrate worm, as a model host organism to study the facets of stress response. C. elegans make a good model host for a variety of reasons. C. elegans are bacterivores and are thus exposed to a diversity of bacteria – some of which are used as nutrition while others are pathogenic. Another important reason is that since they are invertebrates, and therefore lack an adaptive immune response, this allows us to study only the innate immune response. C. elegans also have a digestive tract comparable to that of humans which is able to be infected by human pathogens. This could lead to better understanding of how bacterial infections are combatted in human intestine. C. elegans are also very good lab specimen because they are small and easy to maintain which allows us to work with them in large numbers and they have a short lifespan which allows experiments to be completed in a relatively short time period. C. elegans are also self-fertilizing hermaphrodites which is convenient for studying reproductive response to stress.
In C. elegans there are a variety of known genes involved in stress response pathways, including one which Dr. Powell discovered as a post-doc: fshr-1. The gene fshr-1 codes for a G-protein coupled receptor that is required for the innate immune response for most pathogens. A G-protein coupled receptor is a type of protein that binds to small molecules such as neurotransmitters, neuropeptides, and odorants. This type of protein activates other proteins which can induce neuronal or transcriptional activity. Our work in her lab focuses on characterizing fshr-1. Our main goal is to determine all of the facets of stress response in which fshr-1is involved. Below we briefly describe our own projects, all which contribute to this main goal:
Leah Gulyas ’19
Baby, it’s cold outside…
Specifically 2°C. I am currently continuing research that was begun by Joe Robinson in the Powell lab last summer, in which he cold shocked C.elegans by allowing them to sit at 2 °C instead of their usual 20°C for varying time lengths. Previous studies have assessed immediate survival in the worms following different lengths of cold shock and showed that for shorter durations (about 4 hours) survival was generally high, while longer durations (about 12 hours or so) survival rates dropped off. However, the actual recovery period of the worms has not been accounted for greatly.
Cold shocked worms begin to undergo a series of distinct phenotypic changes following shock; most gradually losing pigmentation and becoming translucent. In general, the worms are not happy campers, as they must wake from a cold coma and cope with damage repair to recover. Our goal is to figure out how they’re completing this recovery process.
Waiting for the worms…
Most of our present experiments involve cold shocking and staining the worms to visualize how lipid concentrations change following the shock. Currently we’re looking at some mutants in an effort to more thoroughly isolate some of the aspects of recovery. However, a bad bout of plate contamination, impossibly slow growing mutants, and persistent incubator malfunctions have been foiling attempts to actually cold shock the mutants so the worms are safe (for now…).
Jenny Giannini ’18
During an infection the host and the infecting microbe have an array of mechanisms they use to in an attempt to overcome one another. While there are many different mechanisms, one mechanism we are particularly interested in, which both host and pathogen can utilize, is the production of reactive oxygen species (ROS). When these ROS come in contact with a cell it causes the cell to be oxidatively stressed. My project utilizes a variety of traditional and molecular genetic tools to study the oxidative stress response and its relationship to the innate immune system’s response to infection by pathogenic microorganisms.
Our model for infection normally consists of allowing one group of C. elegans to eat E. coli (healthy bacteria) and another group to eat P. aeruginosa (pathogenic bacteria). We then use different techniques in an attempt to measure or observe the oxidative stress response occurring during the infection.
Quantitative PCR (qPCR) is the method of choice by many for directly measuring the amount of nucleic acid (DNA or RNA) present in a solution. With this technique, the amplification of DNA by a polymerase chain reaction (PCR) is monitored in real time where the qPCR cyclers is continuously scanning the qPCR plate. It is, in contrast to the conventional PCR, quantitative, meaning that it allows for the exact concentration of the amplified nucleic acid in the sample to be calculated whereas in conventional PCR only the result of amplification after the PCR is completed (end-point detection) can be viewed. Here we use RNA as a template to directly measure gene expression, in which case the RNA needs to be reverse transcribed into DNA before it is amplified with real-time PCR.
Our goal with these experiments is to specifically measure the RNA levels of genes having to do with the oxidative stress response. These are genes that encode for proteins which are capable of detoxifying the ROS present during an infection. Further, we are most interested in detoxification genes which are dependent on our gene, fshr-1. Thus, we measure the RNA levels in wild-type worms (with fshr-1 functional) and fshr-1(-) worms (with fshr-1 nonfunctional) and hope to see that detoxification genes dependent on fshr-1 are expressed at lower frequency than those same detoxification genes in wild type worms.
The Amplex® Red Hydrogen Peroxide Assay is used to detect hydrogen peroxide (a source of ROS) activity in biological samples. The reagents in the assay kit react with hydrogen peroxide to produce a red-fluorescent product called resorufin. Thus, the redder the sample — the more hydrogen peroxide was present. Using the same method of infection described above, we use this as a way to directly measure the ROS present during an infection. If the sample is not red, the worms have either detoxified the ROS present or did not encounter pathogen. We hope to see that fshr-1(-) worms are not capable of detoxifying the ROS present during infection; therefore, the samples containing those worms should be red.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system is originally a bacterial immune system against viruses that has been modified for genome editing. Prior to CRISPR/Cas9, genome editing approaches, such as zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs), relied upon the use of customizable DNA-binding proteins. These approaches required scientists to design and generate a new pair of proteins for every gene they tried to edit. The CRISPR system, which is comparatively much more simple and adaptable, has quickly become one of the most versatile approaches for genome editing as it can be adapted for many plants and animals (including C. elegans!!) and even has therapeutic potential in humans.
While the CRISPR/Cas9 system does not relate directly to our overall goal of characterizing fshr-1 and its role in stress response, learning to use the CRISPR/Cas9 system for genome editing will prove to be very beneficial to us in that we will be able to engineer a variety of useful mutations which could aid us in identifying gene functions (whether that be of fshr-1 itself or identifying genes involved with fshr-1). We could also create insertions around fshr-1 such as fusion of biochemical tags and fluorescent proteins. Essentially, being able to make very specific mutations with the CRISPR system would allow us to study fshr-1 in many ways we were not previously able to. Thus, learning how to use CRISPR effectively and efficiently has also become an important goal for us.
Becky Callaghan ’19
I have spent the majority of the summer working with the gene wdr-23. This gene is a negative inhibitor of skn-1 (which is responsible for the production of detox genes in order to combat the negative effects of ROS production).
There are three types of experiments that I’ve done thus far: killing assays, avoidance assays, and induction assays. In all of these experiments, I’ve used RNAi to knock out the gene wdr-23 in both N2 (wild type) and ok778 (fshr-1 mutant) worms. In killing assays, Pseudomonas aeruginosa (PA14), is spread onto plates and the worms are “picked” into the spot of bacteria. These worms are monitored about 3 times a day, until all are dead. With this type of assay, I am able to compare the survival rate of the different worms, and draw conclusions on how certain genes affect their longevity.
In avoidance assays, the plates are set up a little differently; half of them have a small dot of PA14 in the center, while the other half have a dot of OP50 (the worm’s food). The worms are picked into the center of each dot on both of the plates. 9 hours later, I look at the plates and count the number of worms that have migrated out of the spot of bacteria. This assay shows how well the worms are sensing and recognizing the harmful pathogen and how certain genes are responsible for this behavior.
In induction assays, special strains of worms have to be used. These worms have a GFP (fluorescence) reporter attached to gcs-1 (detox gene). These worms are infected with PA14 and OP50, and their pictures are taken under a microscope. If detox genes are being expressed in the worms, they look like this:
This experiment will show how genes are regulated in response to an infection.
“We are all worms. But I believe that I am a glow-worm.”