Tag: Karen Guillemin

The Role of Microbiota in the Development of Insulin-Producing Cells in Drosophila melanogaster

Presenter: Nicole Mullen – Neuroscience

Faculty Mentor(s): Karen Guillemin, Steph VanBeuge

Session: (Virtual) Poster Presentation

Resident gut bacteria have the capacity to influence aspects of animal metabolism. Previous research in the Guillemin lab showed that in zebrafish, gut bacteria promote the expansion of insulin- producing cells (IPC) in the pancreas through a secreted bacterial protein, Beta-cell expansion factor A (BefA). This research investigates the role of gut bacteria and BefA to promote IPC development in the fruit fly, Drosophila melanogaster. In Drosophila, there are ~7 IPCs located in each lobe of the brain. Our first aim was to test the effect of germ-free (GF) rearing on IPC numbers in Drosophila. Our second aim tested whether feeding flies BefA could restore IPC numbers in GF flies. We compared the number of IPCs present in GF, conventionally-reared (CV), and GF flies fed BefA. Tissue-specific GAL4UAS/GFP in all groups made IPCs visible after dissection. Our results showed that GF flies have fewer IPCs per lobe than CV flies, indicating that microbiota is required for normal IPC numbers. Further, feeding BefA caused a statistically significant increase in IPC numbers in GF larvae compared to CV. However, transgenic expression of BefA, using the UAS/GAL4 system, yielded a trending but not a significant expansion of IPCs in GF flies. This could be due to the low levels of BefA produced through transgenic expression. These results indicate that the microbiota has a powerful effect on metabolic pathways, and cell development, and can influence the normal development of the fly brain.

Characterization of a Pro-Proliferative Microbiota in Transgenic Drosophila

Presenter: Zoë Wong

Faculty Mentor: Karen Guillemin, Tiffani Jones

Presentation Type: Oral

Primary Research Area: Science

Major: Biology, Psychology

Gastric adenocarcinoma, or stomach cancer, is responsible for the second highest number of cancer-related mortalities. Infection with Helicobacter pylori, a human pathogen, is the strongest risk factor and results in clinical responses that vary from no symptoms, to ulcers, to gastric cancer. One major determinant of disease expression is the presence of a bacterial virulence factor, CagA. Transgenic Drosophila that express the CagA gene were generated in the Guillemin laboratory and can be used to genetically disentangle H. pylori infection from cagA expression. A simple microbial community, short generation time, easily manipulated genome, and the ability to be raised germ-free make Drosophila an ideal model organism.

The goal of my research is to determine the effect of dysbiotic, or altered microbiota of CagA transgenic flies. The dysbiotic microbial community of CagA transgenic flies consists of Lactobacillus brevis and Acetobacter pomoroum. To address this, I reared control and CagA lines germ-free. I inoculated flies with either L. brevis, A. pomorum, or the combined CagA community. Adult flies were dissected after 7 days and gut contents were plated to quantify the abundance of each strain within the gut. Overall, CagA transgenic flies harbored a greater amount of total microbes within their gut, and interestingly flies that received the combined CagA community showed an increased total abundance of microbes regardless of fly genotype.

Effect of Pyomelanin Production on Oxidative Stress in an Aeromonas Zebrafish Gut Isolate

Presenter: Helena Klein

Faculty Mentor: Karen Guillemin, Cathy Robinson

Presentation Type: Poster 72

Primary Research Area: Science

Major: Biology

Bacteria have rapid growth rates, which allow us to study their adaptive evolution in the lab to selected environments. By studying the genetic changes that occur during the evolutionary process, we can learn about selective pressures experienced by bacteria when evolved in selected conditions. Through passaging of a strain of a natural zebrafish gut isolate, Aeromonas ZOR0001, we isolated one strain that has increased pigmentation compared to the wildtype. This pigmentation is a result of accumulation of pyomelanin in the extracellular environment. We confirmed through genetic sequencing a mutation in the isolate’s metabolic pathway known to be involved in pyomelanin production. Pyomelanin production has been shown in other organisms to be linked to increased resilience to oxidative stress. Therefore, we hypothesized this mutation confers a fitness advantage to this evolved isolate compared to the ancestral strain. Therefore we generated a genetic knockout of the mutated gene in the ancestral strain and used hydrogen peroxide to simulate oxidative stress in vitro. We found that the knockout strain did better than the wild type strain. This demonstrates that pyomelanin production confers resistance to oxidative stress in Aeromonas ZOR0001 which suggests an adaptive advantage for in vivo growth. Knowledge of genes that increase fitness in bacterial strains in the gut can ultimately allow for better probiotics to be developed with wide repercussions for human health.

Elucidating the Active Domain of a Novel Anti-inflammatory Protein Produced by Intestinal Bacteria

Presenter: Lila Kaye

Faculty Mentor: Karen Guillemin, Annah Rolig

Presentation Type: Poster 71

Primary Research Area: Science

Major: Biology

Bacteria play an important role in the health of their host organism; pathogenic lines signal danger, which induces
a host immune response, while commensal, or resident, bacteria signal to reduce or prevent such an immune response. The ability of a resident organism to communicate with its host to alter immune system function is a burgeoning topic in the molecular biology field, offering a potentially rich source of biotherapeutics to treat illnesses. Researchers in the Guillemin laboratory identified a novel bacterial protein, produced by resident bacteria, which reduces intestinal innate immune responses in zebrafish hosts. Zebrafish are a good model organism for humans because of high replicability, easy gnotobiology, and their translucent larvae allow easily monitoring of host cells. I
aim to carry out a structure-function analysis of this protein. I will determine which domain has the anti-inflammatory activity using systematic deletions of the gene sequence. The protein has one region with homology to a human anti- inflammatory cytokine, a protein that controls neutrophil behavior as part of the immune system. We hypothesize that this domain will be important in the bacterial protein’s function. This would suggest that this bacterial resident mimics a host protein to control the host immune response. This anti-inflammatory protein could be a fresh tool to fight chronic inflammatory diseases in humans, ones often caused by disunion between gut microbes and their host.

The Hepatopancreatic Duct: A Conduit for Bacterial Factors from the Gut to Reach Peripheral Organs?

Presenter: Daniel Derrick

Co-Presenters: Jennifer Hampton, Karen Guillemin

Faculty Mentor: Karen Guillemin, Jennifer Hampton

Presentation Type: Poster 56

Primary Research Area: Science

Major: Biology

Funding Source: NIH: National Institute of General Medical Sciences grant, award number P50GM098911, $162,152

Host-microbe interactions are important for normal development of the host, and often, secreted bacterial products play essential roles in these interactions. In zebrafish, the protein BefA is secreted by a subset of resident bacterial species in the gut. Interestingly, BefA is sufficient to induce expansion of pancreatic beta cells during early larval development. Previously, BefA has also been shown to have effects on cultured murine beta cells, suggesting that it may have a direct mechanism of action. This raises an intriguing question: how can a protein secreted by bacteria in the lumen of the gut exert effects on an entirely separate organ? For BefA to act directly on beta cells in vivo, it must somehow travel from the gut to the pancreas. Here, we examined whether a functional hepatopancreatic duct, which connects the pancreas to the gastrointestinal tract, is necessary for BefA-mediated expansion of beta cells. To do so, immunofluorescence labeling and confocal microscopy were used to count pancreatic beta cells in zebrafish that lack an intact hepatopancreatic duct due to a loss-of-function mutation in the sox9b gene. Despite the lack of this duct, sox9b mutants still exhibited a robust response to BefA treatment, which suggests that BefA promotes beta cell expansion independent of the hepatopancreatic duct.

Chitin Binding Protein GbpA promotes Proliferation In The Drosophila Midgut

Presenter(s): Zoë Wong − Biology, Psychology

Faculty Mentor(s): Karen Guillemin

Oral Session 4S

Research Area: Natural Science (Biology)

The microbes that live both in and on us, collectively known as our microbiota, are estimated to include 3.8 ·1013 cells (Sender, et al., 2016). While this considerable community plays an active role in host health, it also contributes to disease phenotypes including states of inflammation and excess cell proliferation. Previous work has shown that secretion of a bacterial chitin binding protein (CBP), GbpA, by Aeromonas veronii is sufficient to induce cell proliferation in zebrafish (Banse et al., unpublished). Chitin serves as an important Carbon and Nitrogen source for hosts and microbes that can breakdown colloidal chitin (Tran et al., 2011). Interestingly, CBPs are found in microbes that are not capable of utilizing chitin as a nutrient source, which suggests that bacteria have an ulterior motive for CBP translation (Tran et al., 2011). To investigate the relevance of GbpA expression, we propose to use Drosophila melanogaster as a model organism because of their short generation time, ability to be reared germ-free, and established assay for measuring cell proliferation in the midgut epithelium (Jones et al., 2017). We hypothesize that the CBP domain of GbpA (domain 1, GbpAD1) is necessary and sufficient for increased cell proliferation in Drosophila. Chitinases and other CBPs are linked to pro-proliferative states of inflammation and we would expect this result to be replicated in Drosophila (Tran et al., 2011). The highly-conserved sequence identity of CBPs makes it an interesting avenue for exploring the intricacies of bacterial-host interactions.

Experimental Evolution of a Bacterial Symbiont to its Host’s Environment

Presenter(s): Helena Klein − Biology

Faculty Mentor(s): Karen Guillemin, Cathy Robinson

Poster 85

Research Area: Natural/Physical Science

Funding: META Grant

The bacteria that live in our guts, and those of other vertebrates, affect our health in a myriad of ways, from aiding in digestion to training our immune system. However, how bacteria first colonize the gut is little-understood. In particular, environment seems to play an important role in host colonization, especially in aquatic organisms. I proposed investigating environmental adaptation to find novel mechanisms for host colonization. I hypothesized that adaptation of a bacterial symbiont to its host’s environment increases host colonization. I tested this hypothesis via experimental evolution by serially passaging a strain of Aeromonas veronii, a zebrafish gut isolate, in fish-conditioned water to quickly and non-specifically find new genes that could affect host colonization. Surprisingly, I found that while the evolved strains grew to higher population densities in the water than the ancestor, these strains had variable gut colonization fitness. In fact, one strain had significantly reduced gut colonization fitness. Genome sequencing revealed that this strain had mutations affecting motility and Type I secretion system membrane protein genes. I recreated the latter mutation in the wildtype bacterial strain and found that it increased Aeromonas fitness in fish water, however gut colonization was comparable to the wildtype. This suggests that other mutations in the evolved isolate, presumably those in the motility genes, are responsible for the reduced host colonization. Future work will further investigate motility mutations among others. This work contributes to our understanding of host colonization dynamics and can lead to the development of probiotics to improve human health.

Novel Bacterial Protein AimA Promotes Mutualism by Increasing Commensal Fitness and Reducing Inflammation in the Host

Presenter(s): Lila Kaye − Biology, Emphasis In Cellular And Mollecular

Faculty Mentor(s): Karen Guillemin, Kristi Hamilton

Poster 50

Research Area: Microbiology

Funding: SPUR 2016, VPRI 2017, Meta, NIH

The microbiota of the gastrointestinal tract is critical for the development and regulation of the host immune system. Some bacterial genera are associated with health and homeostasis, while others have been linked to inflammation and disease. There have been many studies in recent literature investigating the potential role of commensal microbes in autoimmune and gastrointestinal diseases, both preventative and pathogenic. Much less is known, however, about how interactions with the immune system benefit resident microbes. Here I used the zebrafish, Danio rerio, as a powerful gnotobiotic model for investigating host-microbe symbiosis. I investigated the novel immunoregulatory protein aimA, produced by the zebrafish commensal Aeromonas, and show that it facilitates mutualism with the host by reducing gastrointestinal inflammation and increasing bacterial intestinal colonization in both monoassociations and co-inoculation with pro-inflammatory species Vibrio. Using GFP-tagged neutrophils as a reporter for inflammation, I showed that a deletion mutant lacking the gene for AimA (∆aimA) is unable to regulate host immune response and cannot colonize the gut as robustly. Inoculation into immunocompromised MyD88-/-hosts having decreased intestinal inflammation rescues the colonization defect suffered in the absence of aimA, demonstrating reciprocity between control of the host biology and control of the resident bacterial biology. Identification of bacterial products involved in establishing a healthy symbiosis with the host is crucial for understanding how commensal communities are assembled and maintained.

Investigating amino acid-modulated motility of the zebrafish bacterial isolate, Aeromonas veronii

Presenter(s): Emily Ma

Faculty Mentor(s): Cathy Robinson & Karen Guillemin

Poster 53

Session: Sciences

Animals are colonized by communities of microorganisms that influence the health and development of their host. However, the mechanisms of host colonization are still underexplored. To investigate this, previous work in the lab used experimental evolution to adapt a bacterial symbiont, Aeromonas, to the zebrafish gut. These experiments led to the identification of a novel gene, spdE, which significantly impacts host colonization. We found that evolved isolates with mutations in spdE had faster rates of motility and increased host immigration. Sequence analysis revealed that the protein, SpdE, has a domain for sensing extracellular signals and a diguanylate cyclase domain which produces an intercellular signaling molecule that regulates motility. Further biochemical investigation identified that the signal SpdE senses is hydrophobic amino acids, specifically proline, valine, and isoleucine. To further investigate the relationship between SpdE-dependent Aeromonas motility and environmental amino acids, we developed a new technique (“exploration assay”) which is designed to measure differences in motility between strains or conditions. Using the exploration assay, we compared motility of wild type and spdE knockout strains in different amino acid environments. From our results, we found that the wild type strain is more motile in the presence of these amino acids. However, even in the absence of amino acid signal, the spdE knockout is more motile than the wild type. From these data, we have created a model for how SpdE regulates motility in response to amino acids which offers novel insights into Aeromonas biology and the mechanisms of host colonization.

Beta cell regeneration upon the addition of Beta-Cell Expansion Factor A (BefA)

Presenter(s): Robin Black—Biology

Co-Presenter(s):Ian Torrence, Emily Niebergall, Dan Tudorica

Faculty Mentor(s): Karen Guillemin, Michelle Massaquoi

Session 4: Earning your Stripes

All organisms co-exist with a plethora of bacteria, fungi, and viruses living on and within them, collectively known as a microbiota . Previous work has shown that in the absence of the microbiota (after a germ-free derivation), the beta-cells within larval zebrafish fail to develop (Hill et al ., 2016) . Beta-cells are insulin-producing cells found in the pancreas and are vital for glucose uptake in the body . This experiment has practical applications, as Type I diabetes in humans is an autoimmune disease where the body attacks its own beta-cells . Recently, the Guillemin lab has discovered a novel bacterial-secreted protein, Beta-Cell Expansion Factor A (BefA), that is sufficient to rescue beta-cell proliferation within germ-free zebrafish . Although the study found that BefA is critical for beta cell development, its potential role in beta-cell regeneration is unknown . The goal of this study is to test the role of BefA in pancreatic beta-cell regeneration after induced beta-cell death . Using a transgenic line of zebrafish, we validated that we can significantly and specifically ablate beta cells in larval zebrafish . We next plan to ablate beta-cells of conventionally reared (with microbiota) and germ-free zebrafish treated with and without BefA and quantify the number of beta-cells regenerated . We predict that upon the addition of BefA, there will be a rescue in the number of beta-cells . From this experiment we will learn about the potential therapeutic uses of BefA to recover beta-cells and broadly the important roles that gut microbiota play in host homeostasis .