Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2004 Isolation and characterization of carbonic anhydrase from Ostertagia ostertagi Andrew Allan DeRosa Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation DeRosa, Andrew Allan, "Isolation and characterization of carbonic anhydrase from Ostertagia ostertagi" (2004). LSU Doctoral Dissertations. 1926. https://digitalcommons.lsu.edu/gradschool_dissertations/1926 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. ISOLATION AND CHARACTERIZATION OF CARBONIC ANHYDRASE FROM OSTERTAGIA OSTERTAGI A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural Mechanical College in partial fulfillment of the requirements of the degree of Doctor of Philosophy in The Interdepartmental Program inVeterinary Medical Sciences through the Department of Pathobiological Sciences by Andrew A. DeRosa B.S. Louisiana State University, 1992 M.S. Louisiana State University, 1997 August, 2004 ACKNOWLEDGEMENTS I want to thank Dr. James C. Williams for providing the opportunity to work and train in the field of ruminant parasitology. If it were not for the foundational training in parasitology he instilled in me and the resources provided by his efforts, none of this work would have been possible. I am also very thankful for the encouragement, patience and intellectual advice of Dr. Fredrick Enright during my time in the Department of Veterinary Science as an employee and a student. I would like to thank Dr. Thomas Klei for his mentoring and advisory role throughout this venture. In addition, I am most thankful to Dr. Klei for adopting me into his laboratory and allowing me access to his laboratory facilities and resources. I would like to thank Dr. Richard Cooper for his willingness to serve and advise on my graduate committee. His input was of great value. I am very grateful to Dr. Louis Gasbarre for his donation of the pure Ostertagia isolate as well as his efforts that resulted in the public access to Ostertagia ostertagi expressed sequence tags. In addition this work could not have been completed without the surgical expertise of Dr. Phil Hoyt. I would also like to thank my graduate advisor, Mrs. Lyn LeJeune for her unwavering guidance and advice through my graduate studies. The training and knowledge base and techniques that I gained in molecular biology is attributed entirely to the tremendous teaching and guidance of Dr. Sharon Chirgwin. This work would not have been attempted and subsequently not completed without her patient guidance, training and focus. I would like to thank my parents for instilling in me a strong work ethic and discipline. Finally, all glory and thanks be to Jesus Christ for all is by His grace and grace alone. ii TABLE OF CONTENTS ACKNOWLEDGMENTS……………...………………………………………………………..ii LIST OF TABLES…………………...………………………………………………………….iv LIST OF FIGURES……………………………………...………………………………………v ABSTRACT……………………………………………………….……………………………vii INTRODUCTION…………………………………….…………………………………………1 CHAPTER 1 LITERATURE REVIEW...…………………………………………..………………6 2 IN VIVO EXSHEATHMENT KINETICS OF OSTERTAGIA OSTERTAGI……………………………………………………………………..……...59 3 ISOLATION AND QUANTITATIVE MEASUREMENT OF GENE EXPRESSION OF CARBONIC ANHYDRASE FROM OSTERTAGIA OSTERTAGI DURING IN VIVO EXSHEATHEMENT OF INFECTIVE LARVAE………………………….………………...……….………74 4 OSTERTAGIA OSTERTAGI CARBONIC ANHYDRASE PROMOTER CHARACTERIZATION AND PARTIAL GENOMIC ORGANIZATION……………………………………………………..……………...108 SUMMARY...………………………………………………………………………………….130 CONCLUSIONS………………………………………………………………...…………….133 APPENDIX 1 OOCAIII FULL LENGTH TRANSCRIPT.…….…….…………..………..…..…..138 2 GENOMIC DNA SEQUENCE………..………………………………………….....139 VITA………………………………………………………………………………….………..140 iii LIST OF TABLES 1.1 Summary of carbonic anhydrase isoforms isolated from Caenorhabditis elegans….……...44 3.1. Listing of primers used in isolation of full length cDNA encoding carbonic anhydrase from exsheathed third stage Ostertagia ostertagi………………………………….……………..81 3.2. Sequences of the primers and probes used to quantitate carbonic anhydrase and actin mRNA levels in Ostertagia ostertagi L3 during the process of exsheathment……………..88 3.3. First five results obtained from blasting Ostertagia ostertagi carbonic anhydrase against the translated nucleic acid database……………………………………………………..……...93 3.4. The amino acid composition of the Ostertagia ostertagi (OoCA) hydrophobic core, compared with that of Caenorhabditis elegans (CAH-6) and human carbonic anhydraseIII (HCAIII)……………………………………………………………………………………96 3.5. Amino acid composition of the active site of Ostertagia ostertagi carbonic anhydrase (OoCA), compared to those of Caenorhabditis elegans carbonic anhydrase (CAH-6) and human carbonic anhydrase III (HCAIII)…………………………………..……….…….…97 3.6. The number of exsheathment studies carried out to quantitate carbonic anhydrase expression in Ostertagia ostertagi third stage larvae exposed to the rumen of a cannulated steer while the steer was on a grass or grain ration…………………………………………98 4.1. Restriction enzymes used to produce digested genomic DNA libraries for use in the GenomeWalker protocol to obtain the promoter and non-coding regions of the Ostertagia ostertagi carbonic anhydrase gene………………………………………………………...112 4.2 Primers used in the isolation of the genomic DNA sequence of Ostertagia ostertagi carbonic anhydrase III……….………………………………………………………….....116 4.3 Binding sites for cis-acting elements found within the first 200 bp of the sequence immediately 5’ to the OoCAIII………………………………...……………...…………...122 iv LIST OF FIGURES 1.1. Subcellular location of active carbonic anhydrase isoforms in mammalian cells……….…39 2.1. The cannulated steer used in the exsheathment studies…………………………………….63 2.2. The dialysis bags and the PVC tube used in performing the exsheathment experiments in the cannulated steer……………………………………………………………………………..66 2.3. Percent exsheathment of Ostertagia ostertagi exposed in vivo to (A) grain derived rumen content or (B) grass derived rumen content. Exsheathment was 2 fold more rapid when larvae were exposed to grass derived rumen content. Bars represent standard deviations of the means…..………………………………………………………………...…………..…67 3.1. Alignment of the Ostertagia ostertagi (Oo) expressed sequenced tag (Genbank # BQ097482) and the Caenorhabditis elegans cah-6 (Ce) (Genbank # NM_058788.1.)……………………………………………………………..…………..…..89 3.2. Primer location relative to the nucleic and amino acid sequence of the Ostertagia ostertagi CA expressed sequence tag (BQ097482)…………………………………………………..89 3.3. Schematic diagram of the cDNA clones amplified by PCR using multiple primer pairs......91 3.4. Entire DNA and putative amino acid sequence of the carbonic anhydrase isolated from Ostertagia ostertagi third stage larvae………………………………………...…….….…..92 3.5. Alignment of the Ostertagia ostertagi carbonic anhydrase (OoCA) with the eukaryotic consensus sequence (EUK)…………………………………..…………………………..…93 3.6. Alignment of the putative protein sequence of Ostertagia ostertagi carbonic anhydrase (OoCA) with cah-6 from Caenorhabditis elegans and human CA III (HCAIII)…….….…94 3.7. BLAST alignment of Ostertagia ostertagi actin amino acid sequence (OoACT) with that of Panagrellus redivivus (PgACT) (AAQ89578.1) and Caenorhabditis elegans (CeACT) (NP_505817.1)……………………………………………………………………………...95 3.8. Expression of Ostertagia ostertagi carbonic anhydrase in relation to the exhsheathment kinetics of O. ostertagi L3 on (A) grass diet or (B) grain diet………………………….…..99 4.1. The adaptor sequences ligated onto the ends of digested Ostertagia ostertagi genomic DNA for use in the GenomeWalker protocol for the isolation of the promoter region….……...111 4.2. The adaptor sequences ligated onto the ends of digested Ostertagia ostertagi genomic DNA for use in the GenomeWalker protocol for the isolation of the promoter region. Also shown are the sequences of adaptor primer 1 (AP1) and adaptor primer 2 (AP2)……………......113 v 4.3. Electrophoretic analysis of amplicons isolated from Ostertagia ostertagi genomic DNA using primers specific to OoCAIII………………………………………………………...118 4.4. Schematic of amplicons obtained using OoCAIII-specific primers and Ostertagia ostertagi gDNA. The size of the amplicons suggests intron 3 is approximately 1600 bp in size….118 4.5. Schematic of genomic DNA structure of the Ostertagia Ostertagi CAIII (OoCAII), Caenorhabditis elegans carbonic anhydrase cah-6 and human carbonic anhydrase 3 (HCAIII)…………………………………………………………………………………..119 4.6. DNA sequence of the promoter region of OoCAIII showing some of the cis-regulatory elements reported. Start codon is in italics……………………………………………..…121 vi ABSTRACT The first event in the infection process of Ostertagia ostertagi in cattle is the process of exsheathment. Before trichostrongylid nematodes can transition from a free-living infective stage larva (L3) on pasture to a parasitic existence within the ruminant host, it must first undergo exsheathment. Exsheathment is the process whereby the L2 cuticle retained from the previous molt is cast from the L3. Exsheathment enables the developmental