Research Forum 2014

Appalachian Student Research Forum, 2014

PRODUCTION OF AN ANTIBIOTIC LIKE ACTIVITY BY STREPTOMYCES SP. COUK1 UNDER DIFFERENT GROWTH CONDITIONS

Olaitan Akintunde and Bert Lampson. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

Streptomyces are known to produce a large variety of antibiotics and other bioactive compounds with remarkable industrial importance. Notable antibacterial, antifungal, anticancer and anti-inflammatory drugs as well as pesticides, herbicides, etc. have been detected and recovered from different Streptomyces species. Streptomyces sp. COUK1 was found as a contaminant on a plate in which Rhodococcus erythropolis was used as a test strain in a disk diffusion assay and produced a zone of inhibition against the cultured R. erythropolis. 16s rRNA sequencing confirmed the identity of the contaminant as Streptomyces. This Streptomyces produces a strong inhibitory compound in fermentation broths as well as solid media. A culture extract from inorganic salt starch agar (a minimal medium) was found to be very active; producing a large zone of inhibition against our gram positive and gram negative test strains. The active molecules in this extract have been detected via TLC and bioautography. The difference in the antibacterial activity and chromatographic properties of extracts recovered from different media suggests that this Streptomyces strain might be able to produce more than one type of inhibitory compound.

CHARACTERIZATION OF EXBB AND EXBD FROM RHIZOBIUM LEGUMINOSARUM ATCC 14479

Valeria Barisic and Dr. Ranjan Chakraborty, East Tennessee State University, Johnson City, TN

Rhizobium leguminosarum is a Gram-negative nitrogen-fixing bacterium that can exist as free-living in the soil or form a symbiotic relationship with leguminous plants. Iron plays an essential role in Rhizobia, not just in maintenance of biological processes such as gene regulation and DNA biosynthesis, but also in the enzyme nitrogenase which uses iron as a cofactor to reduce atmospheric nitrogen. Although quite abundant in Earths crust, soluble ferrous iron (Fe2+) is rapidly oxidized to form insoluble ferric hydroxides under aerobic conditions and at physiological pH, which results in a low concentration of biologically usable iron. To combat this iron limitation, bacteria utilize small compounds called siderophores that scavenge and bind available iron in the environment. In order to transport the iron-siderophore complexes inside the cell, transporters located in the energy-devoid outer membrane must be supplied with energy. This is achieved by the TonB-ExbB-ExbD complex found in Rhizobia and other Gram-negative bacteria. The complex harnesses the energy from the inner membrane and transduces it to the transporters in the outer membrane. However, the mechanism of transduction is still unknown. We have identified putative tonb, exbb, and exbd genes in Rhizobium leguminosarum ATCC 14479. In this study, we attempt to characterize the functional roles of exbb and exbd by creating single and double knockout mutants via splicing by overlap extension (SOE), or crossover PCR. The mutants will be compared with wildtype R. leguminosarum ATCC 14479 in their ability to grow in high- and low-iron media, and in the ability to transport radioactively-labeled Fe55-vicibatin complexes inside the cell.

LACTOBACILLI BACTERIAL ISOLATES FROM AMABERE AMARURANU CULTURED MILK HAVE PROBIOTIC POTENTIAL

B. B. Boyiri and E. M. Onyango, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

Probiotics are viable non-pathogenic microbes that positively effect health including inhibition of infection, development of mucosal barrier, activation of immunity and alleviation of allergies. In this study bacteria from 'amabere amaruranu' cultured milk from Kenya were screened for ability to withstand digestive tract conditions and thence their potential as probiotic candidates. Bacteria were isolated and identified by whole colony PCR squence match analysis of the 16s rRNA gene. Isolates were examined for acid stability, acid and/or bile stability, antimicrobial activity against 5 test strains (Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, Klebsiella pneumonia, Pseudomonas aeruginosa), mucin degradation and sensitivity to 8 antibiotics (ampicillin, bacitracin, chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin, tetracycline). Lactobacillus isolates were acid-and bile-stable, did not degrade mucin and inhibited growth of all test strains. L. rhamnosus was sensitive to all antibiotics tested but L. paracasei was resistant to kanamycin, bacitracin and streptomycin. Bacillus isolates were acid-and bile-stable, did not degrade mucin nor inhibit growth of test strains. Bacillus pumilus was resistant all antibiotics tested but Bacillus safensis was sensitive to all. Acetobacter isolates were acid-and bile-unstable, bile stable and did not degrade mucin. They were resistant to kanamycin, streptomycin and tetracyline and inhibited the growth of E. faecalis, and P. aeruginosa. Staphylococcus epidermidis were acid-and bile-stable , resistant to all antibiotics tested, did not degrade mucin and inhibited the growth of E. coli, E. faecalis and P. aeruginosa. Staphylococcus aureus and Staphylococcus sciuri were acid-and bile-unstable. S. aureus did not degrade mucin, was sensitive to all antibiotics tested and inhibited the growth of K. pneumonia. S. sciuri degraded mucin, was resistant to kanamycin and streptomycin and inhibited the growth of K.pneumonia. In conclusion, Lactobacillus rhamnosus isolates that did not degrade mucin and showed stability in acid/bile conditions and demonstrated antimicrobial activity and were sensitive to antibiotics could be potential probiotic candidates.

ETHANOL DISRUPTS METABOLIC SIGNALING IN LIVER CELLS

Sammie M. Dunlay, Matthew Lee, Patty Atwood, Jonathan M Peterson, College of Public Health, East Tennessee State University, Johnson City, Tennessee

Alcohol abuse is the third leading cause of preventable death in the United States. Excessive intake of alcohol can result to alcoholic fatty liver disease, the number one cause of live related mortalities in the US. The outlining purpose for this project is to determine the alcohol-induced changes in the liver cell protein signaling. For this project, we treated H4IIE rat hepatoma cells (with 100 and 200 mM ethanol overnight). H4IIE cells were chosen because they are a commonly used liver cell culture line that maintains characteristics of intact liver cells. After treatment we collected and prepared the cells for protein signaling analysis, using standard western blotting procedure. A western blot detects relative quantity of proteins in a sample. Briefly, protein samples are separated by size through electrophoresis, smaller proteins move faster through the gel so that the larger proteins are toward the top and smaller towards the bottom. The proteins are then transferred to a nitrocellulose membrane and protein concentration is detected by chemiluminescence. We chose to examine the effects of ethanol on the activation of the key regulator of metabolic signaling, Protein Kinase B/Akt (Akt). Based on our results, ethanol has no effect on the total amount of Akt in the H4IIE liver cells. However, ethanol significantly attenuates insulin-induced activation of Akt in a dose-dependent manner, as seen by a reduction in the amount of phosphorylated Akt. Therefore, we conclude that treatments that increase Akt activation may be a viable option for the treatment of alcoholic fatty liver disease.

DETERMINING RETS REGULATORS IN PSEUDOMONAS AERUGINOSA

Kaci Foster, Dr. Christopher Pritchett, East Tennessee State University, Johnson City, TN

Pseudomonas aeruginosa is an important opportunistic pathogen that is capable of infecting any tissue in the body and is especially problematic in immunocompromised patients.This makes it a common pathogen in cystic fibrosis patients. Because of the intrinsic antibiotic-resistant nature of P. aeruginosa, along with a variety of virulence factors produced by the organism, P. aeruginosa infections are difficult to treat. In order to provide better treatment options, a greater understanding of the organism's genetic circuit that controls virulence gene expression is needed. P. aeruginosa possesses several detection systems that control virulence gene expression. RetS is a protein that controls the expression of several virulence factors such as the type III secretion system and the type VI secretion system. This orphan sensor histidine in P. aeruginosa. Specifically, it negatively regulates type VI secretions in the pathogen and positively activates type III secretion. In order to better understand the regulation of the type III and type VI expression, a transcriptional fusion, retS-lacZ , was constructed. The retS promoter region was amplified and cloned into the integrating vector miniCTX and was then introduced into various P, aeruginosa strains to examine retS expression. In addition, to better understand the regulation of retS, a transposon mutagenesis screen was performed on a retS-lacZ containing strain. These studies will help to identify the regulatory cirucuit that controls expression of many P. aeruginosa virulence factors.

ESTABLISHING THE HIERARCHY IN THE REGULATORY NETWORKS WHICH CONTROL CHRONIC INFECTION IN PSEUDOMONAS AERUGINOSA

Shawn Hooker and Dr. Christopher Pritchett. Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that causes both acute and chronic infections. Acute infections occur in both burn and intubated patients. Over time acute infections can make the transition to chronic infections, as seen in cystic fibrosis (CF) patients. The progression from acute to chronic infection is due to intricate regulatory networks that allow P. aeruginosa to coordinate its gene expression in response to different environmental stressors. The switch to a chronic infection is partially controlled by the Gac/Rsm system, which is a conserved two-component regulatory system (TCS) that controls the production of virulence factors in response to unknown environmental stimuli. The lung environment found in CF patients is a hostile environment, and the stringent response is a mechanism that P. aeruginosa uses to respond to starvation, reactive oxygen species, and fatty acid limitation. This response is mediated through the alarmone (p)ppGpp, which is produced primarily by the enzyme RelA. Little is known about the interactions of the stringent response and the Gac/Rsm system. In order to study the effects of the stringent response on the Gac/Rsm system a relA deletion mutant was constructed in the mucoid P. aeruginosa strain PDO300. Transcriptional fusions using the promoter regions of rsmY, rsmZ, rsmA, and gacA were tested in the PDO300-?relA strain and compared to the wild-type. Overall, the -galactosidase activity for the rsmY, rsmZ, and gacA were significantly decreased. However, rsmA expression was increased at three different time points. The preliminary data suggests that the stringent response influences the Gac/Rsm system. Furthermore, when the stringent response is activated the alarmone causes a decrease in RsmA expression, which causes a transition in P. aeruginosa from acute to chronic infection, and generates the different virulence factors that are associated with this phenotypic switch.

CTRP3 PROTECTS LIVER CELLS FROM ALCOHOL-INDUCED DAMAGE, BUT NOT THROUGH ENHANCED AKT SIGNALING

Matthew L. Lee1 and Jonathan M Peterson2.
1 Department of Chemistry, College of Arts and Sciences,
2 Department of Health Sciences, College of Public Health,
East Tennessee State University, Johnson City, Tennessee

Alcoholic fatty liver disease (AFLD) is a significant public health concern. Excessive alcohol (ethanol) consumption causes liver cell damage and death, which results in eventual failure of the liver and death. AFLD is the number one cause of liver-related mortality in the United States. Our lab works with the novel protein C1q TNF Related Protein 3 (CTRP3), which inhibits non-alcoholic fatty liver disease, however the effects on AFLD are unknown. Therefore, the purpose of this experiment is to determine if CTRP3 prevents ethanol-induced liver cell death. The H4IIE rat hepatoma cell line was chosen for experimentation as a cell culture model of liver tissue. To determine a suitable alcohol level H4IIE cells were treated with 50, 100, and 200 mmol of ethanol for 18-24 hours. Trypan Blue was used to identify the dead/damaged cells, as only dead/damaged cells will be stained blue with this protocol. We observed that 100 mmol of ethanol consistently induced ~10% mortality rate in these cells. Next, we tested the ability of CTRP3 to reduce ethanol-induced mortality. We added purified CTRP3 protein to the cell media along with the 100 mmol ethanol treatment. The addition of CTRP3 reduced the amount of alcohol-induced cell death/damage in the H4IIE cell line by approximately 60%. Our next goal was to determine how CTRP3 reduces ethanol-induced death. The Akt signaling pathway is a well-known inhibitor of cell death. Therefore, to determine if CTRP3 attenuated ethanol-induced cell damage/death through activation of the Akt signaling pathway, another set of cells was treated with 100 mmol of ethanol and CTRP3 (with or without insulin). Western blots were used to compare the amount of active Akt (phosphorylated) in the CTRP3-treated and non-treated cells. A Western blot utilizes an electric current to separate denatured protein samples on a SDS-page gel, separating the proteins based on size. The smaller the protein the faster it migrates across the gel. The proteins are then transferred to a membrane for analysis, through exposure to commercial antigens and chemiluminescence imaging. There was no change in the amount of total or active Akt between the samples treated with or without CTRP3. We conclude that CTRP3 protects liver cells from ethanol-induced damage/death, but not through activation of the Akt pathway.

SIGNIFICANCE OF EXPRESSION OF TRANSCRIPTIONAL FUSIONS IN PSEUDOMONAS AERUGINOSA

Kristen Roark, Tyler Speaks, and Christopher Pritchett, Ph.D.
Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, Tennessee.

Pseudomonas aeruginosa is an important nosocomial pathogen and ubiquitous in natural and man-made environments. Thriving in aerobic and hypoxic conditions, its versatility allows it to infect damaged tissue and/or those with suppressed immunity. Especially in Cytsic Fiborsis patients, P. aeruginosa causes significant morbidity and mortality. Chronic infections, like those in CF patients, rely on different virulence factors than those required for acute infections. Studies with this pathogen can bridge the gap it has created between its virulence and treatment of such. A major determinant of clinical isolates from CF patients is the production of virulence determinants such as alginate and type VI secretion. The type VI secretion system is controlled by the Gac/Rsm system which is comprised of an RNA binding protein RsmA, as well as two small RNAs, RsmY and RsmZ. The two small RNAs are controlled by the GacA/S two-component system. The GacA/S system is thought to be the switch from acute to chronic infection. In order to better understand the switch to chronic infection, we have undertaken a transposon mutagenesis study to determine the regulatory controls that govern the GacA/S system. To accomplish this, we have constructed a gacA-lacZ transcriptional fusion and introduced the transcriptional fusion into a mucoid strain. This strain was subjected to transposon mutagenesis and about 2,000 mutants were screened using a patch-plating method. Mutants displaying differential and unique gacA-lacZ expression were further characterized by determining beta-galactosidase activity via several beta-galactosidase assays. Mutants identified in our screen were tested in triplicate to ensure these mutants were in fact mutated in a regulator of gacA expression. Our future endeavors will be to determine the gene(s) interrupted by the transposon, which will provide us with more research to further our understanding of P. aeruginosa pathogenesis and also provide new potential targets for chemotherapeutics.

THE GAC/RSM REGULATORY SYSTEM IS INCREASED IN CHRONIC PSEUDOMONAS AERUGINOSA CLINICAL STRAINS

Tyler Speaks and Dr. Chris Pritchett, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, Tennessee

Pseudomonas aeruginosa is an environmental pathogen capable of producing a number of diverse virulence factors during chronic and acute infections of immunocompromised individuals. Infections in cystic fibrosis patients become more severe after the bacteria have shifted their cellular machinery towards production of chronic virulence factors like Type-6 secretion and overproduction of the exopolysaccharide alginate. This leads to biofilm formations and the mucoid phenotype. Pseudomonas undergoes a mutation in mucA, a gene encoding an anti-sigma factor. This leaves the sigma factor AlgU available to increase expression of genes involved in production of alginate. The response regulator GacA controls expression of small-RNAs RsmY and RsmZ, which bind and sequester the RNA-binding protein RsmA. This inhibition of RsmA decreases expression of Type-III secretion and leads to increase in chronic virulence factors. The genetic regulation of this system and its role in leading to chronic infection is poorly understood and must be fully characterized to understand the signals responsible for the mucoid phenotype. An rsmA transcriptional fusion showed an increase of rsmA expression in the mucoid background. Increased levels of RsmA were confirmed using an epitope tagged RsmA-HA in Western blot analysis. This increase was dependent upon the AlgU sigma factor. The small RNAs RsmY/Z were also shown to be upregulated in clinical strains via transcriptional fusions. This upregulation was not due to an increase in GacA expression. A primer extension of rsmA identified a possible AlgU dependent promoter. The AlgU binding site was mutagenized and RsmA levels were shown to decrease. The response regulator AlgR was also found to control rsmA expression in vivo. This data shows that the rsmA/Y/Z system is actually controlled by members of the alginate regulatory system. Understanding the hierarchy of genetic regulation leading to chronic infection is paramount in determining novel targets for therapeutics.

THE MPSR/T OPERON INFLUENCES BIOFILM DEVELOPMENT AND MOTILITY

S. D. Stacey and C. Pritchett, East Tennessee State University, Johnson City, TN

Pseudomonas aeruginosa is the leading cause of respiratory failure in cystic fibrosis (CF) individuals. In addition, P. aeruginosa is a major nosocomial pathogen. The intrinsic antibiotic resistant nature of P. aeruginosa is due in part to the formation of biofilms, a scenario that leads to chronic infection in the CF lung. CF clinical isolates commonly overproduce alginate, a biofilm component in CF lungs, and are non-motile. Chronic infecting strains are termed mucoid if they overproduce the exopolysaccharide alginate. Alginate facilitates biofilm formation and also interferes with the innate immune systems clearance mechanisms, such as phagocytosis. Previous studies determined that the mpsR/T operon, composed of a transcriptional regulator (mpsR) and putative membrane transporter (mpsT), affects both biofilm production and motility. Phenotypically, mutants produce significantly less alginate. Interestingly, transmission electron microscopy (TEM) indicated decreased exopolysaccharide production and a single polar flagellum. Flagella expression was unexpected, since most clinical P. aeruginosa isolates are non-motile. In order to determine the mechanism whereby the mpsR/T operon affects biofilm formation and motility, we investigated a transcriptional regulator, AmrZ, that is known to control alginate production and repress the production of flagella in mucoid strains. An amrZ transcriptional fusion to a lacZ reporter was used to determine if defective biofilm formation and expression of flagella was due to reduced AmrZ levels. The results suggest that the mpsR/T operon controls amrZ expression. Because biofilm formation and flagella are important in P. aeruginosa virulence, we assessed the virulence properties of these strains using J774 macrophages. Attenuation in either mutant warrants further investigation of these genes as potential pharmacotherapy targets.

EFFECTS OF WHOLE-BODY RADIATION ON MUCIN PRODUCTION IN THE UTERINE TUBE OF MICE

Sarah Wallace and Dr. Allan D. Forsman, Department of Health Sciences, College of Public Health, East Tennessee State University, Johnson City, TN

The mucin layer that lines the uterine (Fallopian) tube functions for the transport of gametes, and is an active secretory organ whose secretions provide a suitable environment for continued maturation of male gametes, interaction between gametes, and early embryonic development . Changes in this mucin layer could have catastrophic consequences on reproduction. Previous studies conducted on mice flown for 13 days on space shuttle mission STS-113 have indicated the space flight has an effect on the mucin that lines the uterine tube in mice. It has been commonly accepted that when changes are seen in spaceflight experiments, the changes are due to the microgravity of spaceflight. Recently researchers have turned their attention to the relatively high levels of radiation exposure related to spaceflight. Therefore, this study is being conducted to determine the effect of whole-body radiation on the mucin layer thickness and relative pH of the mucin in each portion of the mouse uterine tube. Mice were divided into three groups, wild-type control (WTC), apocynin-treated (APO), and nox2 knockout (KO). Each of these groups was then exposed to 0Gy, 0.5Gy, and 2.0Gy of radiation. Tissues were harvested and mounted on microscope slides using standard histological procedures. The slides were then stained using an Alcian Blue/Periodic Acid Schiff staining procedure. Using this technique neutral mucins will stain magenta, acidic mucins will stain pale blue, and mixtures of acidic and neutral mucins will stain purple. For each animal, five randomized measurements of mucin layer thickness were taken from each portion of the uterine tube, isthmus, ampulla, and infundibulum. A one way ANOVA test was used to compare mucin layer thickness between the three groups of mice and their radiation exposure group. Mucin layer production type was qualitatively determined as acidic, neutral, or a mixture of both based on the stain color of the mucin layer. To date, the initial results indicate that the isthmus has a mixture of acidic and neutral mucins, the ampulla predominantly neutral with some mixture of mucins, and the infundibulum acidic with some mixture of mucins. With regard to mucin layer thickness, the APO group had a significantly thinner mucin layer than WTC mice that had been exposed to 0 and .5 Gy radiation in all three portions of the uterine tube. WTC with 0 and .5 Gy radiation exposures were also found to have thicker mucin layers than KO mice in varying regions of the uterine tube. WTC mice with 2 Gy radiation had thicker mucin layers than some APO and KO mice exposed to .5 and 2Gy radiation only in the ampulla and infundibulum. There was no significant difference in mucin layer thickness determined between any APO mice and KO mice.