High Throughput Marine EST Data Analysis Pipeline
Chen Y.1, Mckillen D.1, Wu S.1, Robalino J.2, Gross P.2, Warr G.2
and Almeida J.1
1 Department of Biometry and Epidemiology, Medical University of South Carolina, Charleston, SC 29425
2 Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston, SC 29425
Abstract
Expressed sequence tags (ESTs) are valuable tools for gene discovery, developing Sequence Tag Sites (STSs) for genome mapping, and hunting for disease/stress-response genes. We implemented an EST pipeline to automate the high throughput EST data analysis process for several marine organisms. A user-friendly web-based interface was implemented to process batch EST sequence uploading, editing, and displaying. An XML-based database was integrated to store the processed sequences and annotations. Vector screening, quality control, database searching, clusters of redundant sequences, and submission to GenBank are performed automatically. The integration of web-based front end and database back end offers ease and flexibility of customization for each individual marine organism. Registered users can further dynamically update the attribute information and edit the EST sequences and annotations. The gene-oriented clusters (UniGenes) of redundant sequences are generated and could further be used for microarray probe design to explore the expression profiles for marine organisms under different stress and environmental conditions. The deposited ESTs and their blastable database are available in the public domain. This integrated pipeline will not only organize the large amount of local sequence data but also link to the wider public-domain bioinformatic resources (GenBank, GO, Swiss-Prot).
Unique chemical synthesis approach to the characterization of a novel class of antimicrobial peptide
Brandon Cuthbertson1, Erika Büllesbach2 and Paul Gross1,2
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston SC.
2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston SC.
Penaeidins are a unique family of antimicrobial peptides composed of multiple, divergent classes. As important components of the shrimp immune system penaeidins target bacteria and fungi that are potentially pathogenic to shrimp. Penaeidins are expressed at high levels in hemocytes and secreted at sites of immune challenge. We have recently discovered a novel penaeidin class 4 that is expressed in multiple species of Penaeid shrimp. Class 4 is highly conserved across species, but has a unique primary sequence and structure when compared to other known penaeidin sequences. We describe here the chemical synthesis and subsequent analyses of a penaeidin class 4 isoform from Litopenaeus setiferus. Solid phase peptide synthesis was used to produce two segments equivalent to the proline-rich and cysteine-rich domains of class 4. The two domains were then combined through native chemical ligation to form the 47-amino acid reduced polypeptide. Free sulfhydryl groups were subsequently oxidized in order to produce a chemically defined and highly pure, bioactive penaeidin 4. This isoform displays some interesting differences in spectrum of activity when compared to previously described penaeidins. Surprisingly, the proline-rich domain of class 4 alone has antimicrobial function and shows an interesting effect on the fungus Fusarium oxysporum that inhibits spore production and alters vegetative growth. Our results demonstrate that the novel chemical synthesis approach we have exercised is a most efficient method for production of a chemically defined and highly pure penaeidin class 4 antimicrobial peptide.
Cloning of an elasmobranch urea transporter-2 (ElasmoUT-2) from the kidney of the bluntnosed stingray, Dasyatis sayi
Emily E. Cwengros1, Michael G. Janech1, David W. Ploth2,3 and Wayne R. Fitzgibbon2
1Marine Biomedicine and Environmental Science and 2 Department of Medicine, Medical University of South Carolina, and the 3Ralph H. Johnson VAMC, Charleston, SC
Marine elasmobranch fishes (sharks, skates and rays) are ureosmotic, i.e., as part of their osmoregulatory strategy they synthesize and retain high concentrations of urea (~350 mM) in their body fluids and tissues. The maintenance of the high urea concentration is due in large part to efficient renal tubular urea reabsorption. Interestingly, the regulation of tubular urea reabsorption contributes to fluid homeostasis of marginally euryhaline and euryhaline marine elasmobranchs that exploit estuarine and/or riverine habitats. The mechanisms by which urea is reabsorbed by the renal tubules have yet to be elucidated since both passive and active processes have been proposed. Passive absorption of urea occurs via facilitated urea transporters (UT). Two isoforms of a facilitated urea transporter are expressed in the elasmobranch kidney. The longer isoform (ElasmoUT-1) is 431 amino acids (aa) in length and has only been identified in the kidney of the euryhaline Atlantic stingray (Dasyatis sabina)1. In contrast, the shorter isoform (ElasmoUT-2) is expressed in the kidneys of a stenohaline skate (378 aa protein)2, a marginally euryhaline shark (380 aa protein)3 as well as the Atlantic stingray (379 aa protein)4. Whether the ElasmoUT-1 isoform is unique to D. sabina or expressed at low levels in the kidneys of other elasmobranchs has yet to be determined. It is possible that the longer isoform may be the predominantly expressed isoform in Dasyatid rays. We tested this latter possibility by utilizing molecular cloning (PCR and 5’/3’ rapid amplification of cDNA ends (5’/3’ RACE)) to identify the predominantly expressed UT in the kidney of a marginally euryhaline, Bluntnose stingray, Dasyatis sayi. Initially, we performed RT-PCR (using primers based on the published D. sabina urea transporter cDNA sequence) to identify and sequence a partial UT PCR product from D. sayi kidney mRNA. We then performed 5’/3’ RACE with gene specific primers designed to the PCR product to obtain overlapping cDNAs containing the open reading frame (ORF) and either the 5’ untranslated or the 3’ untranslated regions. The cDNA sequence was confirmed by sequencing overlapping PCR products. The full length D. sayi UT cDNA is 1728 nt in length. The first putative open reading frame (ORF) is the longest (1137 nt). The 5’ untranslated region (5’UTR) is 117 nt in length, and the 3’ untranslated region (3’UTR) excluding the poly(A)+ tail is 471 nt long. The ORF is predicted to encode a 379 amino acid protein that was designated D. sayi(str)UT-2. This protein included a repeated LP motif common to all known UTs. The D. sayi urea transporter was found to be 98% identical to the ElasmoUT-2 expressed in the kidney of the Atlantic stingray (D. sabina(str)UT-2), 88% identical to the Winter skate renal UT (L. ocellata(sk)UT-2), and 83% identical to the Spiny dogfish shark renal UT (S. acanthias(sh)UT-2). Further, the sequence of the carboxy-terminal region of the D. sayi(str)UT-2 was similar to that of the shark, skate and Atlantic stingray ElasmoUT-2s, and distinctly different from that of the D. sabina(str)UT-1. These findings indicate that, contrary to our proposal, an ElasmoUT-2 is the predominant urea transporter isoform expressed in the kidney of the Dasyatid ray, D. sayi. Further, these findings support the conclusion that ElasmoUT-2 is a common urea transporter isoform expressed in the kidneys of elasmobranchs.
Characterization of a unique penaeidin class 3 like antimicrobial peptide expressed in Litopenaeus setiferus, the Atlantic white shrimp
Kizee Etienne3, Brandon Cuthbertson1, Erika Büllesbach2 and Paul Gross1,2
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston SC.
2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston SC. 3Department of Biology, Mercer University, Macon GA.
Penaeidins are shrimp antimicrobial peptides (AMP) that target Gram(+) bacteria and fungi including marine and terrestrial pathogens of plants and animals. Recent assessment of EST libraries generated from L. setiferus hemocytes revealed novel penaeidin-like sequences that share a highly conserved leader sequence with previously identified penaeidins but show substantial divergence in the functional domain encoding sequence. Analysis of the encoded peptide sequences and comparison with known penaeidins revealed that penaeidin class 3 like sequences from L. setiferus show dramatic differences in length and amino acid composition when compared to the well characterized class 3 from Litopenaeus vannamei. Class 3 like sequences are the most divergent of penaeidins shared by L. setiferus and the closely related L. vannamei. These differences between representatives of the same class across species are likely sources of structural variability in penaeidin peptides and putative functional differences between members of the same class. In an effort to examine the relevance of expressed penaeidin diversity in microbial target specificity we have produced a penaeidin class 3 isoform from L. setiferus (Ls3k) for the purpose of examining the antimicrobial activity spectrum of this new AMP. We developed a chemical route to reproduce native penaeidin peptides. In general, short peptide segments were produced and combined by native chemical ligation to the full-length AMP. Thereafter the disulfide bonds were formed by DMSO-mediated oxidation at neutral pH. Through variations of this scheme we have completed the synthesis of a chemically defined, antimicrobially active penaeidin (Ls3k). The peptide is identical to the native structure and not compromised by adjustments to recombinant protein expression systems. In particular, the chemical synthesis allowed for the C-terminal amidation, a post-translational modification in the biosynthesis of penaeidins, thus far not accomplishable for recombinant proteins. These qualities make our synthetic Ls3k directly comparable to a recently characterized Ls4d, a representative of a new penaeidin class 4. Comparative analysis of the different penaeidin classes against a panel of microbial species will permit defined primary structure to function relationship inferences. Target specific regions of penaeidins are of interest for future studies due to recent findings that the different classes do demonstrate some species specificity. By producing Ls3k we have made possible a more thorough comparative analysis of specific elements of the L. setiferus immune system, a relatively un-explored, but economically and environmentally important, immune model system. Further evaluation of penaeidin structure function relationships may also provide important information for the design of possible antimicrobial therapeutics.
The USS Arizona: Microbial Degradation of Bunker C Crude Oil
A. Graham1; T. McDonald2; K. Blair3; K. Crawford4, M. Russell5, L. Murphy5, P.J. Morris1,6
1Marine Biomedicine and Environmental Sciences, Medical University South Carolina, Charleston, SC; 2Texas A&M University, College Station, TX; 3Towson University, Towson, MD; 4The Citadel, Charleston, SC; 5National Park Service’s Submerged Resources Center, Santa Fe, NM; 6National Ocean Service, Charleston, SC
Before the Pearl Harbor attack, the USS Arizona’s 4,630 ton fuel tanks had been filled with Bunker C Crude, a No. 6 fuel oil. A significant amount of oil remains in the ship today, and is leaking from several different locations. In 2000, an environmental assessment was undertaken with oil and sediment samples taken from the ship and adjacent areas to examine the extent of oil weathering as well as the microbial degradability of the leaking oil. The first objective was to analyze the oil leaking from different points of the ship, as well as to assess the oil composition in surrounding Pearl Harbor sediments. The second objective was to determine the degradability of Bunker C crude oil by in situ microorganisms from Pearl Harbor and to characterize the microbial community structure responsible for degradation. Sediments were subjected to a 16 h soxhlet extraction and analyzed by gas chromatography, and revealed a peak ubiquitous in all sediment extracts. Further analysis of the peak by mass spectrometry identified the peak as butylated hydroxytoluene (m/z 205.3), an antioxidant used in petroleum and food products. Aerobic microbial enrichment cultures were developed using sediments collected from different ship locations and oil from the ship as the sole source of carbon. Enrichments were grown at 30°C and 200 rpm and transferred (4%) monthly for three consecutive months. Following the third monthly transfer, gravimetric analysis of oil extracted from enrichment cultures indicated 30% average degradation of the heavy crude oil. Mass spectrometric analysis further demonstrated extensive degradation of the n-alkanes, branched alkanes, and polyaromatic hydrocarbons compared to control samples. Denaturing gradient gel electrophoresis analysis of enrichment cultures revealed multiple banding patterns with an average of 12 bands per enrichment culture. On-going studies are focused on the phylogenetic characterization of the microbial community. To date, these studies suggest microorganisms associated with the USS Arizona are capable of substantial degradation of numerous components of Bunker C crude oil.
Structural determination of a novel toxin produced by the dinoflagellate, Alexandrium monilata
Michelle H. Hsia1, S. Morton2, P. Moeller2
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC, 2Marine Biotoxins Program, National Ocean Service, Charleston, SC
Abstract.
The chain-forming dinoflagellate Alexandrium monilata was first found on the east coast of Florida in 1951 and later observed in 1955 on the coast of Texas where it has been associated with the formation of red colored water and elevated fish mortality. Past studies have found that A. monilata produces a harmful substance(s) that is contained in the cell mass and increases toxicity when the organism cytolyses. The structure and nature of the toxin has yet to be established. Studies indicate that the toxin has low water solubility, casting doubt on the presence of saxitoxin-like toxins that are water soluble. A. monilata toxin appears to be a novel toxic substance of uncharacterized mode/site of action, which is most efficiently extracted with organic solvents. Using techniques of TLC and GH4C1 rat pituitary cell bioassays, we have identified cytotoxic activity. We are currently in the process of determining the structure of this novel toxin through the use of Preparatory TLC, HPLC, NMR and Mass Spectrometry. We present here the spectral data to date on this novel bioactive material.
A second urea transporter isoform (strUT-2) from the kidney of the euryhaline, Atlantic stingray, Dasyatis sabina.
Janech, MG1, Paul, RV2, Nowak, MW3, Miller, DH3, Ploth, DW2, 4, and Fitzgibbon, WR2.
Medical University of South Carolina 1Marine Biomedicine and Environmental Sciences 2Department of Medicine, Nephrology, 3Department of Pharmacology and Experimental Therapeutics, 4VAMC, Charleston, SC, USA.
We have previously identified and characterized a 431 amino acid, phloretin-sensitive, urea transporter (strUT) from the kidney of the Atlantic stingray (1). This urea transporter has a distinct carboxy-terminus compared to that observed in the kidneys of other lower vertebrates. To date, only a single UT has been identified in the kidney of each lower vertebrate species examined, whereas, several isoforms have been identified from the kidneys of a number of mammalian species. The aim of this study was to determine if multiple UT isoforms are also expressed in the kidney of the Atlantic stingray.
Primers were designed to the 5’ UTR (5’-ACA AAA TCC ATT CAT GGA GCA-3’) and 3’ UTR (5’-TAA CAC TGT GCC ATG CAA GGT TCA G-3’) of strUT. Using RT-PCR amplification of stingray kidney mRNA, we observed cDNAs of 1.8 and 3 kb in size. The 1.8 kb product encoded for strUT. The 3 kb product contained an open-reading frame of 1137 nt that was predicted to encode for a UT of 379 amino acids. This short isoform (designated strUT-2) was identical to the N-terminal 377 amino acids of strUT and included a double LP box signature sequence common to all UTs. Interestingly, even though the strUT-2 transcript was considerably larger than that reported for the shark isoform, the putative strUT-2 is almost the same size, has a similar carboxy terminus, and has 79% sequence identity to the shark UT.
The findings of this study suggest 1) multiple UT isoforms are expressed in the kidney of the Atlantic stingray and 2) a similar UT isoform is present in the kidneys of both Myliobatiform and Squaliform elasmobranch fishes.
Discovery of a Defensin Homolog in the Hemolymph of the Eastern Oyster, Crassostrea virginica
Jessica E. Jones1, Matthew J. Jenny1, Kevin L. Schey3, Paul S. Gross2, Gregory W. Warr2
1Marine Biomedicine and Environmental Science, Medical University of South Carolina, 221 Fort Johnson Rd, Charleston, SC, USA, 2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC, USA, 3Department of Pharmacology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC, USA
The eastern oyster, Crassostrea virginica, relies upon an innate, non-adaptive immune system to survive in an estuarine environment under a variety of stresses, such as high salinity, extreme temperatures, tidal changes, metal and other toxicities, and bacterial, fungal, and viral influxes. The innate immune response, both cellular and humoral, is very important to these organisms as a primary mechanism for stress response. The mRNA coding for a novel peptide with homology to the defensin family of antimicrobial peptides exists in the hemolymph of the oyster. The defensins are generally cationic peptides with three disulfide bridges. This defensin-like gene sequence was discovered through screening of EST oyster libraries. With the complete gene and peptide sequences, further analysis has been done on the expression levels of this possible antimicrobial homolog via semi-quantitative PCR. These data reveal increased expression levels of the message with immune challenge to the oyster. The next challenge is to discover the translated peptide that is probably cleaved from its original full sequence, from the oyster hemolymph. The search for the translated peptide is being accomplished through screening of the hemolymph using HPLC and MALDI mass spectrometry and through the use of a commercial antibody to the previously characterized marine defensins. We hypothesize that this peptide plays a role in oyster innate immune response, and that it possesses its own unique range of antimicrobial activity. Further analysis of this peptide is needed to determine its expression, mode of action, and activity in order to support its identification as a defensin homolog.
The Microbial Ecology of Pseudopterogorgia americana, a Common Gorgonian Coral
Christopher Johnston1, Garriet W. Smith2, Cheryl M. Woodley1,3, and Pamela J. Morris1,3
1 Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC
2 Department of Biology, University of South Carolina, Aiken, SC
3 Center for Coastal Environmental Health and Biomolecular Research and Hollings Marine Laboratory, National Oceanic and Atmospheric Administration, Charleston, SC
Pseudopterogorgia americana is a gorgonian coral commonly found in shallow reef environments throughout the Caribbean. Gorgonian corals are susceptible to fungal infections by Aspergillus sydowii and are often used as an indicator species for unhealthy coral reefs. Symbiotic, nonpathogenic microbial communities living in the surface mucus layers of corals are believed to play a role in preventing disease. Gorgonians also produce an array of bioactive natural products that are currently being investigated for their antimicrobial, anti-inflammatory, and anti-aging properties. Here we take a molecular approach to characterize the microbial community associated with the surface mucus layers of healthy P. americana collected from San Salvador Island in the Bahamas and the Florida Keys. Total nucleic acids were extracted directly from the coral mucus using a combination of approaches to reduce co-precipitation of the mucus polysaccharides along with the DNA. From the extracted DNA, a 323 bp sequence of the 16S rRNA gene was amplified using universal primers for the bacteria domain. Amplified gene segments were separated by denaturing gradient gel electrophoresis (DGGE) in order to obtain a profile of the bacterial community. Selected bands were then excised from the gels, re-amplified, and sequenced in order to determine the type of bacteria represented. These results suggest that a member of the β-Proteobacteria subdivision is an important member of the microbial community in healthy P. americana mucus. On-going efforts are focused on developing a quantitative Polymerase Chain Reaction (qPCR) method to determine the relative abundance of each microorganism of interest, and continuing excising and sequencing DGGE bands in order to phylogenetically characterize the members of the mucus microbial community. This study is part of a larger effort to determine the dynamics of microbial communities associated with corals, and how they affect coral health and disease.
Peko Kauppert1,2, David Wingard1 and Thomas Walle1,3
1Medical University of South Carolina, 2Marine Biomedicine and Environmental Sciences Program, 3Department of Pharmacology
Benzo[a]pyrene (BaP) is a contaminant ubiquitously found in the environment, including air (e.g., engine exhaust and tobacco smoke), water (e.g., creosote contamination and boat exhaust), soils and sediments (e.g., creosote and oil spills), and even in foods for human consumption (e.g., charbroiled meats). As such, a variety of animals are exposed to BaP, including fish, dolphin and humans. In order to transform BaP into its ultimate carcinogenic form, and to allow DNA-binding thus exerting many adverse biological effects, carcinogen-bioactivating enzymes such as cytochrome P450 1A (CYP1A) and epoxide hydrolase are necessary. While this process has been thoroughly investigated in humans, much less is known in fish, and very little has been examined in marine mammals.
Previous findings in our lab
found CYP1A(1) activity in fish liver microsomes to be more than ten times
higher than in human liver microsomes. DNA-binding to calf thymus DNA using
3H-labeled BaP was more than 45-fold higher in fish compared to human
liver microsomes. However, while BaP has been implicated with liver cancer in
Fundulus heteroclitus, in humans the compound is more closely associated
with development of lung cancer. The question, if marine mammals such as the
bottlenose dolphin have an increased risk to develop lung cancer through
elevated CYP1A(1) activity, has not been addressed before. The present study
investigates presence and activity of BaP-bioactivating enzymes in human liver
and lung microsomes and compares the results to fish liver and dolphin lung
microsomal preparations.
Analysis of Expressed Sequence Tags (ESTs) from the Dinoflagellate Karenia Brevis
Kristy Lidie1, Fran Van Dolah2, James Ryan2, Dave McKillen1, Ann Cheny1
1Medical University of South Carolina, Charleston, SC 2NOAA/National Ocean Service/CCEHBR, Charleston, SC
Sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery. To understand genetic information carried on a unicellular microalgae, Karenia brevis, a normalized cDNA library was constructed from cells in logarithmic growth phase and a total of 1392 5¢-end sequence tags were established. These sequences were grouped into 1072 independent EST species. Similarity search against the public non-redundant protein database indicated that 936 of these species showed similarity to registered sequences. The remaining 136 species were defined as novel and unclassified ESTs. These cDNA clones and EST sequence information provide powerful sources for future genome-wide functional analysis studies in K. brevis.
Effect of
Pyocyanin on the Functional Diversity of a Crude Oil Degrading Microbial
Community
R. S. Norman*1,2; P. Moeller2; T.J. McDonald3; P.J. Morris1,2
1Marine Biomedicine and Environmental Sciences, Medical University South Carolina, Charleston, South Carolina;
2National Ocean Service, Charleston, South Carolina; 3B & B Laboratories, College Station, Texas
Pseudomonas aeruginosa strains are frequently isolated from petroleum contaminated sites and are capable of producing virulence factors that enhance competitiveness and survival. One factor, pyocyanin (1-hydroxy-5-methyl-phenazine), has demonstrated antimicrobial activity against a range of microorganisms. Thus, under conditions promoting pyocyanin production, growth of P. aeruginosa and subsequent production of pyocyanin may alter community functional diversity. Pyocyanin was first identified in crude oil degrading cultures by HPLC fractionation and mass spectroscopy as a single ultraviolet absorbing species eluting at 25.62 min and having a protonated molecular ion cluster at m/z 211/212. To examine the potential effects of pyocyanin on the functional diversity of crude oil-degrading communities, a time course experiment was established using crude oil-degrading cultures (DLC) grown in minimal media supplemented with 2 mg×ml-1 crude oil with and without 9.5 μM pyocyanin. Tubes were shaken at 200 rpm at 30°C for up to 50 days. Enrichment cultures were analyzed by denaturing gradient gel electrophoresis (DGGE) for microbial community structure and gas chromatography (GC-MS) for oil degradation. DGGE and phylogenetic analysis of the microbial community throughout the time course revealed 7 distinct bands. Microorganisms represented by bands B1-B6 are distributed among the α, β, and γ-Proteobacteria subclasses of the Proteobacteria while the microorganism represented by band B7 was found to be a member of the high-G+C subgroup of the Gram-positive genera. Microorganisms B1, B5 and B7 demonstrated pyocyanin-induced growth suppression while microorganisms B2, B3, B4, B6 were not affected. GC-MS analysis of the oil indicated that pyocyanin-amended cultures had decreased ability to degrade heterocyclic (dibenzothiophenes), aromatic (C1-C4 naphthalenes) and cycloalkane (C29 and C30-hopane) compounds compared to the unamended cultures. These data suggest that the presence of pyocyanin altered the functional diversity of the cultures resulting in decreased oil degradation.
Histones as antimicrobial proteins in Litopenaeus vannamei
Severine A. Patat1, Robert Chapman2, Paul S. Gross3, Gregory W. Warr3 and Kevin L. Schey1
1 Department of Cell and Molecular Pharmacology & Experimental Therapeutics, MUSC
2 Hollings Marine Laboratory, Marine Resources Research Institute, SCDNR
3 Department of Biochemistry and Molecular Biology, MUSC
Bacterial or viral infections can be catastrophic not only for shrimp aquaculture farms but also for natural stocks, therefore a better understanding of the shrimp immune system is needed for prevention and control of diseases. Although penaeidins, small bacteriostatic and antifungal peptides, have been characterized, they are mostly active against Gram positive bacteria. Peptides active against Vibrio species, a major bacterial pathogen in the shrimp, have not yet been reported.
In preliminary studies of hemocytes from Litopenaeus vannamei, high levels of histones H2A, H2B, H3 and H4 were found. In some vertebrates, including fish and amphibians, histone proteins have been described to have antimicrobial activity. Also, fragments of histone proteins are active against some bacteria. This is the case for three peptides derived from the N-terminus domain of H2A, buforin I, parasin I and hipposin from the asian toad stomach, the catfish and the halibut skin mucus, respectively.
The objective of this study was to determine if shrimp histones from the hemocytes play a role in their antimicrobial defenses. Mass spectrometry and DNA cloning were used to determine if shrimp histones were similar to known antimicrobial histones as well as to characterize any posttranslational modifications. Liquid growth inhibition assays were used to assess their antimicrobial activity against various Gram positive and Gram negative microbes.
Results: Litopenaeus vannamei histones H2A, H2B, H3 and H4 were purified by HPLC. Histone H2A was cloned and its N-terminus is highly similar to the known antimicrobial peptides. A 39 amino acid synthetic peptide identical to the N-terminus of the shrimp H2A was produced. Mass spectrometry confirmed that shrimp histones are very similar to other species. Endogenous histones H2A, H2B and H4 have activity against the Gram positive bacteria Micrococcus luteus and the synthetic peptide has activity against M. luteus, Bacillus subtilis, B. megaterium and Aerococcus viridans at less than 10 M.
Histone posttranslational modifications, localization in the hemocytes, and activity against other pathogens are being investigated.
Tracking of marine pathogens in the environment: A study of anthropogenic and physical factors involved in disease transmission
Sara C. Polson1, Shawn W. Polson1, Cheryl M. Woodley1,2
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC
2NOAA/National Ocean Service, Charleston, SC
Little is known about the manner in which transmissible infectious agents are transported in the marine environment. Potential modes of transport include currents and animal vectors, as well as human activity including diving and boat/ship traffic. Some circumstantial evidence exists for transport of disease by currents. Evaluation of field studies showed that the die-off of the sea urchin Diadema antillarum in the Caribbean during the early 1980’s, appeared to spread in the direction of the prevailing water currents. No defined experiments were performed to identify the pathogen or confirm this mode of transmission. This deficiency in the understanding of pathogen transmission severely limits any attempts to determine possible sources of pathogens and to manage outbreaks of disease. In this study we propose to use a combination of epidemiological and molecular approaches including PCR-based detection and fluorescent in situ hybridization assays to track known pathogens in the marine environment. Special emphasis will be placed on determining the role that recreational diving and boating may have in pathogen transmission and transport.
Recent Mortality of Acroporid Corals in the Florida Keys: A Disease Investigation
Shawn W. Polson1, Mats L. Lundqvist2, Samantha L. Ryan3, Sara C. Polson1, Dana E. Williams4, Margaret W. Miller4, Cheryl M. Woodley1,3
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC
2Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC
3NOAA/National Ocean Service, Charleston, SC
4NOAA/National Marine Fisheries Service, Miami, FL
During the spring of 2003, a rapid mortality of Acroporid corals inhabiting reefs in the Florida Keys was reported. The mortality was characterized by a line of sloughing tissue, which moves across the coral, rapidly denuding the skeleton. Symptoms were first noted on the staghorn coral, Acropora cervicornis, on reefs in the Florida Keys National Marine Sanctuary near Key Largo. A similar pattern of mortality was subsequently noted on A. cervicornis colonies at Biscayne National Park and on A. prolifera at Dry Tortugas National Park. A coordinated effort between agencies allowed for a timely sampling of both affected and unaffected corals. Each sample was collected and processed with its role in future laboratory experiments already determined. This allowed samples to be collected and handled in a manner that served to minimize degradation of the component(s) of interest, in an effort to optimize laboratory analysis. Samples were collected for microbial culturing, microbial DNA analysis, RNA analysis, protein analysis, and histological examination. These samples will not only be used to study the microbial aspect of the mortality (i.e. possible pathogen involvement, community shifts, etc.), but should also provide information on the coral responses involved in the mortality.
Robalino J.1, Prior S.2, Metz D.2, Shepard E.2, Chapman R.2, Browdy C.2, Gross P.1, Warr G.1
1Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston, SC 29425
2Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC 29412
Shrimp are commercially important crustacea, as well as essential components of many marine ecosystems. Viral diseases affect shrimp and other crustacea; pathogenic viruses have been devastating for the shrimp farming industry and are also a severe threat to wild crustacean populations. We are interested in understanding crustacean antiviral immunity in order to 1) facilitate the development of better strategies for disease control in aquaculture, 2) provide useful information for the design of environmental management practices, and 3) provide new insights into the evolution of innate antiviral immunity. Because nothing is known about antiviral immunity in any invertebrate, we are utilizing functional genomics tools and approaches to explore the molecular bases of the antiviral response in the marine shrimp Litopenaeus vannamei. We have generated a collection of 2800 unigenes from this species by characterizing tissue specific libraries prepared both by standard cDNA library construction methods and by Suppression Subtractive Hybridization (SSH). Three tissues of importance to the immune system of the shrimp have been used in our studies: hemocytes, hepatopancreas, and gills. SSH was performed after viral and microbial challenges, and by subjecting virally-infected animals to hyperthermia (32°C); which is known to enhance resistance of shrimp to White Spot Syndrome Virus, the most pathogenic crustacean virus known to date. As a result of this gene collection effort we have identified components of signal transduction pathways known to participate in the immune response of both vertebrates and invertebrates. Our sequence analysis also suggests that many of the genes expressed in the shrimp are quite divergent from genes known in other organisms. The shrimp unigene collection has been arrayed into a cDNA chip, which will be used to study the expression patterns of these 2800 genes during viral infection. Characterizing the changes in gene expression associated with the shrimp-virus interaction should provide insight into the molecular nature of the antiviral response in these animals. Furthermore, these analyses should provide the basis for future studies on novel genes and pathways likely involved in crustacean antiviral immunity.
Characterization of a Water-Soluble Toxin Produced by the Cyanophyte Trichodesmium thiebautii
Tracey B. Schock1,2, Steve Morton2 and Peter D. R. Moeller2
1Department of Marine Biomedicine and Environmental Science, Medical University of South Carolina (MUSC), 2Marine Biotoxins Program, National Ocean Service (NOS)
Trichodesmium thiebautii is a toxin producing nonheterocystous cyanobacterium ubiquitous in tropical, subtropical, and temperate seas. It is known for its ability to fix nitrogen and for its massive blooms, making it a major component of oceanic primary production and nitrogen cycling. The toxin(s) produced by this cyanophyte has been observed as a potential cause of detrimental effects to local flora and fauna, as well as humans. To date, a T. thiebautii toxin has not been isolated or structurally characterized. Here, we report the extraction of a toxin(s) from T. thiebautii cell mass. We have demonstrated cytotoxic activity of T. thiebautii extracts using GH4C1 rat pituitary cells, dectected presence of toxin by thin-layer chromatography (TLC) and have obtained partial structural data using nuclear magnetic resonance (NMR). The characterization of the T. thiebautii toxin(s) may lead to the development of pharmaceuticals possibly for use as anticancer, antimicrobial, or antifungal products.
Tapia, H.1,4, R.S. Norman2,3 , P. Moeller3, P.J. Morris2,3
1University of Texas at El Paso, 2Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, 3National Ocean Service, Charleston, South Carolina, 4Medical University of South Carolina Undergraduate Research Fellowship
Abstract
Rhamnolipids are a class of biosurfactants secreted by Pseudomonas aeruginosa that are proposed to facilitate the degradation of crude oil. Two P. aeruginosa isolates were recovered from polyaromatic hydrocarbon (PAH) contaminated soils from Fairhope, AL. and designatedU1 and U3. U3 was used for preliminary experiments due to its reduced lag time when grown on oil.. U3 was grown under three different conditions. The conditions are, PPGAS, which enhances rhamnolipid production; LB media, which is a nutrient rich media, and Bonny Light crude oil (BLC). During growth on each media, rhamnolipid production was monitored using the orcinol assay and surface tension measurements. Orcinol assays demonstrated the highest concentration of rhamnolipids for U3 cells grown on LB (28.0417 μg rhamnose/ml). Surface tension measurements of culture supernatants showed a drop in surface tension corresponding to an increase in rhamnolipids. When U3 was grown on LB, surface tension dropped from 53.67 dynes/cm to 34.33 dynes/cm. At stationary phase, rhamnolipids were extracted from culture supernatant using ethyl acetate and analyzed by thin layer chromatography (TLC). Two distinct spots were visible on the TLC plates for all three types of media (Rf = .9667 and .9). Rhamnolipids from each extract were further purified using high performance liquid chromatography (HPLC). HPLC analysis of the different extracts demonstrated distinctive peaks at 214 nm. HPLC fractions were collected every minute during analysis and each analyzed using the orcinol assay to verify the presence of rhamnolipids. On PPGAS, fractions collected at 4 and 6 minutes tested positive with 5.426 and 12.361 μg/ml of rhamnose respectively. On LB, fractions collected at 6 and 14 minutes tested positive with 9.6982 and 3.5503 μg/ml of rhamnose respectively. On crude oil, fractions collected at 2 and 39 minutes tested positive with 5.4556 and 13.7574 μg/ml of rhamnose respectively. Further analyses will be done by NMR and Mass spectrometry to determine the chemical structure of the rhamnolipids and determine whether the rhamnolipids produced by P. aureginosa growing on rich media are the same as those growing on crude oil.
The Effect of Nickel and pH on Protein Expression in Burkholderia cepacia PR1301
J. D. Van Nostrand1, M. Swartz2, J. Arthur2, A. G. Sowder3, P. M. Bertsch3, P. J. Morris1,4
1Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, SC 29412, 2Department of Medicine, Medical University of South Carolina, Charleston, SC 29412, 3Savannah River Ecology Laboratory, The University of Georgia, Aiken, SC 29802, National Ocean Service, Charleston, SC 29412
Divalent cationic metals, such as nickel (Ni), are necessary micronutrients for bacteria; however, higher concentrations can interfere with their normal function including translation, transcription and protein synthesis. Ni is a common co-contaminant at waste sites, many of which are also acidic due to the presence of weathered soils and sediments or the co-disposal of acidic material. We have been studying the impact of pH on the toxicity of Ni to Burkholderia cepacia PR1301 at pH 5, 6 and 7. PR1 is able to grow in the presence of 50 mg L-1 Ni at pH 7, while at pH 5 PR1 is able to grow with 1000 mg L-1 Ni. To understand how PR1 is able to grow in higher concentrations of Ni at pH 5, two-dimensional gel electrophoresis was used to examine changes in protein expression in PR1 in the absence and presence of Ni (200 and 500 mg L–1) at pH 5. We hypothesized that specific proteins upregulated during exposure to Ni are involved in Ni-resistance and that proteins upregulated during growth at pH 5 are providing cross-protection, allowing PR1 to grow in higher concentrations of Ni. Total cellular protein was extracted from PR1 using a differential extraction protocol designed to extract as many types of proteins as possible employing both detergent lysis and sonication. The proteins were first separated based on isoelectric point over a pH range of 3 to 10. The focused proteins were then separated on an 8 to 16%T acrylamide gel, and spots were visualized using SYPROTM Ruby fluorescent staining and digitally analyzed. Results show increased expression of specific proteins with a corresponding decrease in expression of other proteins in cells grown in the presence and absence of Ni. These results demonstrate that specific changes in protein expression occurred in the presence of Ni, suggesting that these proteins may be involved in enhancing Ni resistance at low pH. On-going studies will characterize these proteins, and address the role these proteins are playing in providing resistance to Ni.
Peripheral Blood Mononuclear Cells From the Atlantic Bottlenose Dolphin Can be Infected With Lentiviral Vectors
Margie Peden-Adams, Lauren Heesemann, Patricia Fair, Mark Kindy, Sebastiano Gattoni-Celli
Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC
The field of marine mammal biology seems ripe for significant progress and the Atlantic bottlenose dolphin (BND) provides a great opportunity to perform high quality research aimed at establishing better parameters to measure the health of these mammals as well as the quality of the environment in which they live. However, in order to advance the BND research field it seems imperative that permanent cell lines are established from various tissue sources (e.g., epidermis and peripheral white blood cells). These cell lines would allow us to validate existing in vitro assays that are diagnostic of the BND health, and to provide a reliable and controlled source of material (DNA, RNA, Proteins) for genomic and proteomic analyses. We believe that the wealth of reagents and techniques routinely employed in modern biomedicine can be brought to fruition to advance BND cell biology and immunology by the establishment of BND cell lines, that would allow a more systematic exploration and study of this important “sentinel” species. Lentiviruses are human retroviruses that have attracted enormous interest because of their ability to infect non-dividing cells like neurons, hepatocytes and quiescent lymphocytes. After successful infection, lentiviruses integrate their genetic material in the form of proviral DNA into the genomic DNA of the host. The most extensively studied lentivirus is the human immunodeficiency virus type 1 (HIV-1), from which most lentiviral vectors have been derived. Here we report evidence that peripheral blood mononuclear cells from the BND can be infected with lentiviral vectors expressing the green fluorescence protein (GFP) as a transgene. These results pave the way for additional studies aimed at establishing BND cell lines using lentiviral vectors carrying immortalizing (onco)genes.