Competence is the type of horizontal gene transfer in which a bacterium acquires DNA from the environment and includes this new genetic material in its genome. While this is a common phenomenon in many bacterial species, the mechanism used to ensnare and internalize environmental DNA is unknown. Other studies have shown that type IV pili (T4P), hair-like appendages, are somehow involved in competence but their role is unclear. A highly competent species, Acinetobacter baylyi (ADP1), expresses proteins that comprise the T4P and produce pili on its surface that are morphologically similar to T4P in their diameter and propensity to bundle. To examine the role these pili may play in the process of competence, we exposed ADP1 cells to a variety of environmental DNA concentrations and evaluated pilus production. We expected an increase in abundance or distribution of pili in the presence of more DNA. Pilus production was measured quantitatively by imaging the cells and their pili with atomic force microscopy and semi-automatically tracing the pili. Both the total length of all pili and the number of pili per cell were calculated. Our data demonstrate that ADP1 did not increase pilus production when exposed to more DNA. The cells consistently produced an average of 0.15 +/- 0.05 µm of pili per µm^2 of open mica with a few outliers. Additionally, the Primary pili produced per µm of shoreline was similar across the tested DNA concentrations and minor variations did not follow a trend of increasing Primary pili with increasing DNA concentrations. Slight increases in pilus production were seen at DNA concentrations of 200:1 and 1800:1 DNA molecules:cells and slight decreases seen at 100:1 and 400:1 DNA molecules:cells. Overall, our results do not demonstrate the involvement of pili in the acquisition of environmental DNA as measured by an increase in pilus production when cells were exposed to more DNA. Therefore, more research is needed to elucidate the role of pili in competence.
Diabetes Mellitus is one of the most prominent and rapidly increasing diseases in society today. Peripheral and autonomic neuropathy are prominent in any person presenting this disease, and therefore it is imperative to understand how these manifest in the diabetic. This study investigated dermal manifestations of T1D peripheral/autonomic neuropathy in comparison with healthy controls. Sudomotor dysfunction often presents complications in patient’s suffering from Diabetes Mellitus and therefore we emphasized observation of sweat glands for the purposes of investigating this disease. Using iontophoresis we were able to test the efficacy of Pilocarpine and the neurotransmitter Acetylcholine for Sweat Gland (SG) induction. Post-induction, skin biopsies were harvested from volunteers for the purpose of immuno-staining and confocal analysis. There was previously no study that used a horizontal sectioning method for the purposes of investigating diabetic neuropathy. Using Confocal Microscopy and Light microscopy, we took an in depth look at structural components surrounding the sweat gland, such as innervation, vasculature and vesicular traffic. We found that diabetic patients exhibit severe structural degeneration in comparison to healthy controls. Additionally, our diabetic volunteers displayed evidence of lipofuscin, increased presence in fibroblasts and deficient sudomotor functional ability.
The use of tobacco products is directly linked to the increased risk of developing several life threatening complications, like myocardial infarction (MI) and ischemic stroke (IS). MI and IS are both caused by spontaneous clot formation within the blood vessels, leading to the occlusion of arteries supplying nutrient rich blood to either cardiac muscle or cerebral tissue. An important molecule used for platelet-platelet signaling during clot formation is 5-hydroxytryptamine (5-HT). Previous studies have shown that sympathetic nervous system (SNS) stimulation, induced by nicotine, results in elevated concentrations of 5-HT within the blood, but the link between this increase and the increased risk for MI and IS has yet to be made. Therefore, we hypothesize that elevated concentrations of 5-HT stimulated by nicotine, induce a hyperactive platelet phenotype in smokers resulting in the increased risk for MI and IS. To test this hypothesis, we incubated isolated platelets from nonsmoker’s in a high concentration cocktail of 5-HT and other known platelet signaling molecules released after SNS stimulation, to see if these elevated concentrations were inducing the hyperactive platelet activity observed in smokers. Incubation of nonsmoker’s platelets in the cocktail resulted in more platelets aggregating together after stimulation with collagen than platelets from nonsmokers without addition of the cocktail. Next we looked to block individual receptors on the platelets known to be used for platelet-platelet signaling during clot formation. After blocking these receptors, we were able to reduce the effect of the cocktail on nonsmoker’s platelets. Complete negation of the cocktails effect on the magnitude of the aggregation cascade on nonsmoker’s platelets was only achieved when all three receptors were blocked at the same time indicating that the receptors are working independently of each other. Interestingly a time dependent relationship between the cocktail and triple block was observed indicating that platelets exposed to the cocktail first aggregate at a higher magnitude even in the presence of the triple block than when the platelets were exposed to the triple block first. These finding have implication in potential therapies or screenings for high risk MI or IS patients as reduction of reactivity of platelets to clotting factors within the blood vessels could reduce the chance of both MI and IS from occurring.
Acinetobacter baylyi are highly competent Gram-negative bacteria that are easy to culture in vitro, making them model organisms for studying natural transformation. Natural transformation is a means of genetic exchange for bacteria that occurs by the uptake and incorporation of extracellular DNA into a bacterium’s own genome to enhance fitness and survival. It is hypothesized that hair-like appendages known as type IV pili (T4P) or homologous structures make up the competence machine that facilitates natural transformation, and these appendages are also associated with the bacterial form of movement known as twitching motility. While studies have investigated the role of pilin proteins and general competence proteins in the competence machine, little is known about how the cytoskeleton influences its assembly and function. The cytoskeleton includes shape-determining proteins that specify cell shape and maintain intracellular organization. These proteins might influence natural transformation and twitching motility in A. baylyi because the assembly, extension, and retraction of T4P requires localization of the necessary components, which is facilitated in part by the cytoskeleton. Specifically, I tested the importance of two shape genes, mreC and rodA, on transformation and twitching motility by comparing knockout mutants to wild type A. baylyi cells. The results indicate that RodA plays an important role in natural transformation and twitching motility, as deletion of rodA decreased both processes. The mreC knockout did not have a significant impact on transformation or twitching motility. The data suggests that cellular organization facilitated by shape-determining proteins like RodA impacts the proper assembly and function of the competence and locomotive machine.
Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is the etiological agent of listeriosis, a food-borne illness. In healthy individuals, listeriosis results in mild symptoms, but can be life-threatening for immunocompromised individuals such as the elderly or pregnant women in which case listeriosis can even lead to abortion. During infection with intracellular pathogens like L. monocytogenes, host cells upregulate a plethora of genes involved in combatting the infection. In infection with L. monocytogenes, one such gene is interferon stimulated gene 15 (ISG15), a ubiquitin-like protein, which has been shown to have antiviral activity. Previous work from our group demonstrated, through its effect on cytokines, that ISG15 is also antibacterial. However, we wanted to understand more about the protein’s specific targets. Our group previously identified proteins targeted by ISG15 during L. monocytogenes infection using a combination of proteomic strategies. To confirm the modifications by ISG15 during L. monocytogenes infection, we infected mouse embryonic fibroblasts (MEFs) isolated from deconjugase (USP18) inactive mice, ISG15 knock-out mice, and wild type mice. Using infected cells, we found that ISG15 has a protective effect against L. monocytogenes infection in vitro. Additionally, since previous work has shown that approximately 80% of ISGylation targets are integral membrane proteins localized to the endoplasmic reticulum (ER), we focused specifically on morphological changes to the ER caused by ISG15 induction upon L. monocytogenes infection. Our data suggest that the absence of ISG15 may compromise both nuclear integrity and endoplasmic reticulum morphology. Although the role ISG15 plays upon infection with intracellular bacteria remains elusive, a better understanding of the mechanism of action of the small protein and its role during L. monocytogenes infection could lead to breakthroughs in the study of several human diseases due to the multitude of pathways in which ISG15 has been implicated.
Infection with Influenza A virus (IAV) continues to cause morbidity and mortality in children across the globe, in part due to the excessive inflammatory response during pathogen clearance. Using a murine model of IAV infection, this study focuses on the role of the innate immune system in IAV infection through the scope of NLRP3 inflammasome protein activation and assembly. We were able to detect the presence of the NLRP3-inflammasome target proteins ASC, Pro-caspase-1, NLRP3, RIG-1, and IL-1β in both juvenile and adult mice. Notably, we found significantly increased levels of ASC and RIG-1 protein in juveniles compared to adults. This suggests that ASC and RIG-1 are related to the observed excessive inflammatory response upon IAV infection in juveniles. To examine NLRP3-inflammasome assembly, we created multiple mutant constructs of the inflammasome scaffolding protein Vimentin as well as vimentin-/- cells. IL-1β production was greatly inhibited in vimentin-/- cells compared to the wild type upon activation of the NLRP3 inflammasome. The same trend was seen when only the head region of the protein was present. We suggest that the intermediate filament (IF) Vimentin serves as a protein scaffold for inflammasome assembly, and that expression of Vimentin is a necessary checkpoint in the innate immune response. Furthermore, we propose the Vimentin-targeting drug withaferin A as a potential treatment for IAV induced acute lung injury. Further studies are to be conducted to compare age-related differences in expression of other inflammasome-related proteins as well as the effect of other Vimentin constructs on IL-1β production. A better understanding of age-related differences in innate immune signaling as well as the overall structure of the inflammasome will be essential to improve care of and treatment for this high-risk population.
Eosinophilic Esophagitis (EoE) is a chronic, allergic inflammatory disease that presents with elevated numbers of eosinophils in the esophagus, and is caused by contact with food allergens. However, little is known about the mechanism by which food allergens diffuse into the esophagus to contact and activate immune cells, leading to the recruitment of eosinophils. A better understanding of this mechanism of diffusion would allow for the development of more targeted treatments for EoE. The objective of this project was to establish a working assay to determine the diffusion rates of various fluorescent dyes through regenerated cellulose membranes that can later be applied to fluorescently labeled food proteins and human esophageal tissue. In testing our assay, we found the observed diffusion rates of different fluorescent molecules across synthetic membranes fit our mathematical model for rates of diffusion. In preliminary studies, we demonstrated that our assay and mathematical model for diffusion rates could be applied not only to synthetic membranes, but also to human esophageal tissue. The results of this project have provided the lab with a model that can be applied to different membranes and fluorescently labeled molecules to measure diffusion rates with a relatively high level of accuracy. Future steps will be to fluorescently label food proteins commonly implicated in EoE, and measure the diffusion rates through normal human esophageal tissue, as well as the diffusion rates after administration of various potential EoE treatments such as steroids and antihistamines.