Appalachian Student Research Forum, 2015

IDENTIFICATION OF algR REGULATORS

Kimberley Araujo, Sean D. Stacey, and Dr. Christopher Pritchett, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of infecting any tissue in the human body. The AlgZR two-component regulatory system has been associated with several virulence factors in both acute and chronic Pseudomonas aeruginosa infections. AlgZR positively regulates virulence factors such as type IV pili, pyoverdine, hydrogen cyanide synthesis, rhamnolipids, and alginate production. In a septicemia and pneumonia model, algR mutants are attenuated. While AlgZR plays an important role in virulence, only a few regulators of algZR expression are known. To identify possible algR regulators, transposon mutagenesis was performed on an algR-lacZ fusion strain. The algR-lacZ fusion strain produced blue colonies on media containing X-GAL; mutants with altered algR-lacZ expression were identified by change in the intensity of their blue or white color. Two mutants were identified in this screen and were further studied. We performed quantitative -galactosidase assays of the mutants versus the wild -galactosidase activity compared to the parent strain, suggesting that an algR negative regulator was interrupted. To identify the interrupted gene, arbitrary PCR was performed and the resulting amplicon was cloned and sequenced. The interrupted gene will be identified through BLAST analysis, and future studies will involve constructing a non-polar deletion mutant as well as further characterization of this mutant strain. Future goals are to further determine the mechanisms that control algR expression. Because AlgR regulates many P. aeruginosa virulence factors, our study could identify new potential drug targets.

ESTABLISHING THE BINDING OF CTRP3 TO LIVER CELLS

Patty B. Atwood1 and Dr. Jonathan M. Peterson2 . 1Department of Allied Health Sciences, College of Clinical and Rehabilitative Sciences, East Tennessee State University, Johnson City, TN; 2Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

C1q TNF Related Protein 3 (CTRP3) is an adipokine, meaning that it comes from adipose tissue and circulates through the blood. Our lab has previously observed CTRP3 prevents fatty liver disease, however at this time; it is unclear if CTRP3 binds directly to hepatocytes (liver cells) or functions through an indirect mechanism. We want to know if the effect of CTRP3 is direct or indirect. Hypothesis: CTRP3 binds directly to hepatocytes. Methods: We used purified recombinant FLAG tagged CTRP3 protein to test our hypothesis. Briefly, immunocytochemistry and flow cytometry was used to detect the binding of CTRP3 H4IIE hepatoma cells. H4IIE hepatoma cells are a well-established in vitro model of hepatocytes, which is useful for metabolic research as this cell line mirrors the liver-like insulin regulated glucose and lipid metabolism found in the liver. Further, we use immunohistochemistry and immunofluorescence to detect the binding of CTRP3 to the liver in paraffin embedded and frozen tissue sections, ex vivo. Results: The binding of recombinant CTRP3 protein to hepatocytes was detected by both immunocytochemistry and flow cytometry. Further, we showed that the binding of CTRP3 to hepatocytes was specific as we were unable to detect the binding of a purified recombinant CTRP1, a closely related adipokine, to hepatocytes using identical techniques and concentrations. Further, flow cytometry analysis of CTRP3 treated hepatocytes showed a six-fold increase in mean fluorescent intensity compared to control treatments. However, using standard immunohistochemistry and immunofluorescence techniques we were unable to detect the binding of recombinant CTRP3, ex vivo. Conclusion: Our lab was able to confirm using two separate techniques that CTRP3 protein is able to bind to hepatocytes, in vitro. However, whether CTRP3 also binds to intact liver cells has yet to be detected. A key limitation of this study was that liver sections were treated with recombinant CTRP3 protein ex vivo (frozen or paraffin embedded slides). Future studies will examine the CTRP3 binding to the liver in vivo. Briefly, purified recombinant CTRP3 will be injected directly into a mouse and then liver section analyzed.

This Poster Won 2nd Place in the undergraduate Biomedical and Health Sciences Division

CTRP3 PREVENTS ETOH TREATED HEPATOCYTE APOPTOSIS

Samantha Dunlay and Dr. Jonathan Peterson. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN. 

Alcohol abuse is the third leading cause of preventable death in the United States. Our lab works with the novel protein C1q TNF Related Protein (CTRP3), which is secreted from adipose tissue and circulates in the blood stream. Our past lab experiments show that CTRP3 prevents non-alcoholic fatty alcoholic liver disease. Nevertheless, the effects of CTRP3 on alcoholic fatty liver disease (AFLD) are still unknown. Our lab to date, has determined that CTRP3 prevents alcohol-induce hepatocyte (liver cell) death. The purpose for this project is to understand how. Previous research has demonstrated that ethanol consumption increases apoptosis (programmed cell death) in hepatocytes through an inappropriate elevation in apoptotic signaling. Our working hypothesis is that CTRP3 prevents ethanol-induced hepatocyte cells death by suppressing ethanol-induced elevations in pro-apoptotic signaling. To test our hypothesis we examine the Page 40 2015 Appalachian Student Research Forum effects of ethanol plus/minus CTRP3 treatment on the Bcl-2 family of apoptotic regulatory proteins. The Bcl-2 family can either induce (pro-apoptotic) cell death or inhibit cell death (anti-apoptotic). Methods: Hepatocytes were treated with ethanol (100 mM) plus/minus recombinant CTRP3 and Bcl-2 family protein content was determined through western blot analysis. We measured the specific apoptotic regulator proteins: Bcl-2-associated X protein (BAX), which is pro-apoptotic, and B-cell lymphoma 2 (BCL-2) and (BCL-XL), which are anti-apoptotic. Western blots were performed using standard techniques; briefly, protein samples are separated by size through SDS-PAGE (PolyAcrylamide Gel Electrophoresis). The proteins are then transferred to a nitrocellulose membrane and relative protein concentrations are detected by chemiluminescence. Results: ethanol treatment increases the amount of BAX and decreases the amount of BCL-XL but not BCL-2 protein content in the hepatocytes. However, treatment with CTRP3 stopped the ethanol-induced elevations to BAX and reductions in BCL-XL. In conclusion this data supports our hypothesis that CTRP3 prevents ethanol-induced hepatic cell death.

ETHANOL FEEDING REDUCES CIRCULATING CTRP3 LEVELS

Christina K. Fleming1 , Kristin Roark2 , and Dr. Jonathan M. Peterson2 . 1Department of Biological Sciences, College of Arts and Sciences, East Tennessee State University, Johnson City, TN; 2Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

Alcohol-induced fatty liver disease (AFLD), a leading cause of liver related mortalities, is an increasing public health concern resulting from excessive alcohol intake. Unfortunately, there are no currently available pharmaceutical treatments for the treatment/prevention of AFLD. C1q TNF Related Protein 3 (CTRP3), a novel adipokine, has been demonstrated to prevent diet-induced fatty liver disease by acting directly on liver cells to increase lipid oxidation, reduce lipid synthesis, and ultimately protect the cells from alcohol-induced damage/death. However, the effects of CTRP3 on preventing/treating AFLD are unknown. This project is working towards determining if CTRP3 can be used to prevent alcohol-induced liver lipid accumulation. HYPOTHESIS: Alcohol consumption reduces circulating CTRP3 levels. METHODS: Mouse models of acute and chronic ethanol feeding protocols were used. Briefly, acute ethanol feeding was accomplished by a single gavage of ethanol with blood collections completed at 0, 1, 2, and 4 hours post gavage. The chronic ethanol exposure was completed by a 10-day ethanol containing liquid diet (5% ethanol) followed by a gavage of ethanol with the serum collected 9-hours post gavage. For both groups control mice were given a gavage of maltodextrin (to match caloric intake of ethanol). To test circulating CTRP3 levels, an immunoblot was conducted on 1 microliter of serum and the proteins were transferred to a membrane for analysis with antigens and chemiluminescence imaging. RESULTS: Both acute and chronic ethanol exposure displayed reduced circulating CTRP3 levels by ~30% and ~40%, respectively. CONCLUSION: Ethanol suppresses circulating CTRP3 levels, thus supporting our labs long term hypothesis that CTRP3 is a relevant treatment for humans diagnosed with AFLD. Further research is needed to determine if restoration of CTRP3 levels will prevent alcohol-induced liver lipid accumulation.

This Poster Won 1st Place in the undergraduate Biomedical and Health Sciences Division 

EFFECTS OF T6SS ON THE OVERPRODUCTION OF ALGINATE TO PROMOTE MUCOIDY AND THE MICROBICIDAL EFFECT OF T6SS ON OTHER PSEUDOMONAS

Abigail Hughes and Christopher L. Pritchett. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

Pseudomonas aeruginosa, a leading cause of respiratory failure in cystic fibrosis (CF) patients, and is an important nosocomial pathogen. The biofilm produced in the lung of CF patients, is frequently composed of alginate. Alginate is an exopolysaccharide responsible for protection against the innate immune system. Additionally, P. aeruginosa has intrinsic antibiotic resistance. Overproduction of alginate leads to the mucoid strains associated with the chronic infection. Another putative virulence determinate of chronic clinical isolates is type VI secretion (T6SS). T6SS is composed of several proteins including, VgrG and Hcp. It has been proposed that the VgrG proteins form trimetric complexes that could be used as puncturing devices to perforate membranes and allow the passage of proteins or macromolecular complexes. Bacteria supposedly prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the toxins. In the Gac/Rsm signal transduction pathway, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. The aim of this study was to determine if T6SS leads to alginate production, promoting mucoidy, and if T6SS is involved in killing other Pseudomonas. To accomplish this goal, we have developed a bioassay to detect alginate production and/or killing due to P. aeruginosa strains that constitutively express T6SS. The retS strain overexpresses the T6SS and will be used to antagonize another P. aeruginosa strain to determine if T6SS can induce mucoidy or can kill other P. aeruginosa. PAO1 rsmZ-lacZ strain was used as a marker strain that expresses high levels of - galactosidase activity and can be discerned from the retS strain. The retS and the marker strain PAO1 rsmZ-lacZ were mixed together and serial dilutions were plated onto PIA to examine colonies for alginate production. The PAOI rsmZ-lacZ transcriptional fusion will also be screened using X-Gal plates to determine the number of cells remaining after incubation. Because T6SS is a suspected virulence factor of P. aeruginosa, understanding its role in virulence is important.

Adipogenesis may be influenced by Lactobacillus cultured milk isolates

J Kotala, EM Onyango.  Department of Health Science, College of Public Health, East Tennessee State University, Johnson City, Tennessee

Obesity has reached epidemic levels around the world.  There is a need for a better understanding of the different factors in the digestive tract environment that may modulate adipogenesis.  Our goal is to understand the influence of potential probiotics on the differentiation and subsequent lipid storage of adipose cells.  This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from amabere amaruranu, a traditional Kenyan cultured milk, on 3T3-L1 cell line. Cultures of 3T3-L1 cells were treated with bacterial isolate cell extract, filtered supernatant from overnight bacterial culture or with a PBS control.  Cells were allowed to differentiate in the presence of different dose levels of appropriate treatments for 10 days.  Cells were collected, lysed and the resultant lysate analyzed for adipogenic-related transcription factors using western blot. Protein expression levels indicated that the treatment with bacterial isolate cell extract, but not the bacterial culture supernatant, downregulated the expression of various adipogenic-related transcription factors in a dose-dependent manner. The highest dose of the bacterial cell extract downregulated peroxisome proliferator-activated receptor-, sterol regulatory element-binding protein 1, and adipose triglyceride lipase by 1.6, 1.5, and 1.8 fold, respectively. The cell extract did not affect the level of expression of CCAAT/enhancer binding protein-. In conclusion, bacterial cell extract from, Lactobacillus rhamnosus, from amabere amaruranu cultured milk showed anti-adipogenic properties, and could be used for anti-obesity management.  This study shows that the L. rhamnosus isolate warrants further studies in vivo.

THE PA2028 TRANSCRIPTIONAL REGULATOR INFLUENCES VIRULENCE

Ryan Shankle and Christopher Pritchett, Ph.D. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

Pseudomonas aeruginosa is the leading cause of respiratory failure in cystic fibrosis (CF) patients. P. aeruginosa is also one of the most common nosocomial pathogens and can cause a great number of illnesses. Chronic infections, such as those found in the CF lung, rely on a wide array of virulence factors. Two important chronic virulence factors are alginate production and type VI secretion. Both of these mechanisms are controlled by the Gac / Rsm system. This system is comprised of an RNA binding protein, RsmA, as well as two small RNAs, RsmY and RsmZ. Little is known about how RsmA expression is regulated. In order to better understand RsmA expression, an rsmA-lacZ transcriptional fusion was constructed to make a reporter strain. A transposon mutagenesis was then performed on the strain and >2,000 mutants were screened. Those mutants showing a change in rsmA-lacZ expression were more extensively characterized by further -Galactosidase assays. One mutant identified in the transposon mutagenesis screen contained an insertion in an unknown response regulator, PA2028. A non-polar deletion mutant was constructed and the rsmA-lacZ transcriptional fusion assayed to confirm the transposon screen. PA2028 has since been characterized further with the addition of separate transcriptional fusions in both the wild type and 2028 strains. -Galactosidase assays have shown PA2028 to be a transcriptional regulator for all three components of the rsm system. In these assays, rsmA and rsmZ show a decreased level in the 2028 strain while rsmY shows increased levels. Further studies are proposed to determine if regulation is direct by testing the ability of purified PA2028 to bind to the rsmA promoter region. Because the Gac/Rsm system controls important virulence factors and PA2028 regulates rsmA expression, it could be a potential new target for future antibacterials. Further studies will indicate exactly how PA2028 controls the expression of these components and what other functions it has.

ALGR CONTROLS RSMA IN CHRONIC PSEUDOMONAS AERUGINOSA

Tyler Speaks and C.L. Pritchett. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN 

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas, leading to ~80% mortality. P. aeruginosa uses Type VI secretion and alginate production as virulence factors during chronic infection. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. Pseudomonas often uses post-transcriptional regulation to control timing and to tune virulence factor gene expression. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors, such as Type III and Type VI secretion. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of RsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We describe a previously unknown regulatory mechanism in P. aeruginosa, which sheds light upon the complex hierarchy regulating gene expression during infection.

THE IMPORTANCE OF AN ON/OFF SWITCH: ANALYSIS OF GACA PHOSPHOMIMETIC MUTANTS IN PSEUDOMONAS AERUGINOSA

Sean Denver Stacey and Dr. Christopher L. Pritchett. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN.

Pseudomonas aeruginosa is an opportunistic pathogen able to colonize most host tissues. P. aeruginosa accomplishes this feat with its wide array of virulence factors. The virulence factors are part of a complex network of gene regulation the bacterium uses depending on the environment. Various virulence factors are regulated via two-component systems, composed of a histidine kinase and a response regulator. Of all bacteria, P. aeruginosa has the most two-component systems in its genome. Of the 64 systems, we decided to focus on the GacA/S system. GacS is the histidine kinase, which upon sensing an unknown environmental chemical signal, becomes phosphorylated. From here, GacS transfers this phosphate to GacA, the response regulator. It has been show previously that GacA directly regulates the small RNAs RsmY and RsmZ, but whether in a phosphorylated or unphosphorylated state is unknown. We aligned GacA with other known response regulators to find the active site in which it is phosphorylated. This residue is the aspartate located at position 54. We propose by making both a GacA D54N (unphosphorylated) and D54E (phosphorylated) phosphomimetic mutants, that the phosphorylated mutant will be the active form of the response regulator. In order to do this, we used two transcriptional fusions for the small RNAs, RsmY and RsmZ. These fusions were analyzed in two laboratory strains PAO1 and its isogenic mutant mucA22. mucA22 has been shown to have a significant increase in both small RNAs. We also compared our results to a gacA mutant in both strains, which shows no production of RsmY or RsmZ. As expected, the GacA D54N mutant showed no production of RsmY or RsmZ. In contrast the GacA D54E mutant showed the small RNAs being expressed in vivo. Further analysis is warranted in the attenuation, if any, in both backgrounds. If a GacA D54N mutant is attenuated, this unphosphorylated state is a potential drug target, which could lead to compromising the entire response regulator network in this formidable pathogen. 

This Poster Won 1st Place in the Graduate Biomedical and Health Sciences Division 

USING GENOMICS TO INFER THE IDENTITY OF AN INHIBITORY COMPOUND

Amber Ward and Dr. Bert Lampson. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN 

Recent research has shown that the most common methods being used for small molecule discovery in bacteria only accesses, at best, 10% of the potential small molecules available. This is supported by the many small molecule biosynthetic gene clusters of unknown function that have been discovered from sequenced bacterial genomes. Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown from our lab to produce a strong inhibitory compound with activity against most strains of Rhodococcus, including the pathogen R. equi., as well as, against closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. 2,306 mutant strains were screened by an agar extraction method to identify non-producing mutants. These strains were also screened for auxotrophic mutants to ensure that random mutations were being created by the pTNR transposon. Of the 2,306 mutants screened, 8 were identified as not producing the inhibitory compound and 7 were identified as auxotrophic mutants. Southern blot analysis of restriction digested chromosomal DNA indicated the pTNR transposon inserted into different locations among the 8 non-producing strains. Flanking DNA at the pTNR insertion site was cloned and sequenced for each of the non-producing mutants. Two of these clones showed significant homology to polyketide synthase genes from Streptomyces. Based on the analysis of non-producing mutants, the inhibitory compound produced by Rhodococcus sp. MTM3W5.2 is a product of a polyketide synthase gene similar to these found in Streptomyces. Future cloning and sequencing of this polyketide synthase in its entirety may reveal clues to the identity of this inhibitory compound.