MOLECULAR TECHNIQUES FOR THE STUDY OF HELICOBACTER SP. ENG INTERACTION WITH HOST MICROFLORA IN THE MOUSE MODEL by MASSACI OF Ramahi Sarma-Rupavtarm B. Tech. (Hons) (Biotechnology and biochemical engineering) L11 Indian Institute of Technology, Kharagpur, 1998 Submitted to the Department of Civil and Environmental Engineering in partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2000 @ 2000 Massachusetts Institute of Technology. All rights reserved. A uthor ...............- Civil and Environmental Engineering L-~ De rtment of ( May 19, 2000 Certified by ......... Martin F. Polz Assistant Professor Thesis Supervisor A ccepted by ..................... Daniele Veneziano Chairman, Departmental Committee on Graduate Studies Molecular techniques for the study of Helicobacter sp. interaction with host microflora in the mouse model by RAMAHI SARMA-RUPAVTARM Submitted to the Department of Civil and Environmental Engineering on May 19, 2000 in partial fulfillment of the requirements for the Degree of Master of Science in Civil and Environmental Engineering ABSTRACT Helicobacter spp. are known animal pathogens. Infection by Helicobacter pylori in humans leads to gastritis and ulcers and has been implicated as a causative agent for gastric cancer. Animal models are useful for studying pathogenesis and host-pathogen interactions for pathogens like H. pylori. One such model is the mouse model colonized with a defined group of eight organisms representative of the normal mouse intestinal flora, the altered Schaedler flora (ASF). Infection by a pathogen may lead to changes in the populations of the host microflora, which are difficult to study by conventional culture and biochemical methods. The aim of my work is to develop PCR-based techniques to study the interaction of H. pylori with the ASF microflora in mice. Primers targeting the 16S rRNA gene for the detection of H. pylori and Helicobacter genus bacteria by PCR were developed. The primer pair, 787f -1127r, was evaluated and was found to be specific for H. pylori and was able to detect 40 copies of the gene in a background of mice fecal pellet DNA. Genus specific primer pairs 212f-289r and 212f-1026r were able to detect all the helicobacters tested, but also amplified DNA from other organisms and are being redesigned. Two methods, terminal restriction fragment length polymorphism (t-RFLP) and constant denaturant capillary electrophoresis (CDCE), were assessed for studying the diversity of the ASF species. T-RFLP is based on differences in lengths of terminal fragments obtained by restriction digestion of the amplified 16S rRNA gene from a mixture of different organisms. CDCE separates amplified fragments of the 16S rRNA gene on the basis of melting temperature differences due to sequence variation between different microbes. An artificial mixture of 4 ASF bacteria could be analyzed successfully by T-RFLP. CDCE analysis led to anomalous results due to the use of degenerate primers. The method was, however, very sensitive and is being retried with a different set of primers. These techniques provide a sensitive and accurate means for studying the diversity of ASF bacteria in response to insults like pathogens. Future work is aimed at using these techniques for pathogen ecology research in ASF mice. Thesis supervisor: Martin F. Polz Title: Assistant professor of Civil and Environmental Engineering 2 Acknowledgements Thanks first and foremost to Professor Martin Polz for his guidance and support, to Professor David Schauer for his guidance and help in getting all the DNA samples, to Professor Bill Thilly for letting me use the CDCE instrumentation, to my labmates Vanja, EeLin, Aoy, Linping, Janelle, Stefan and Cathy for their encouragement and help, to Ryan for all the GeneScan help, to Dr. Bruce Paster of the Forsyth Institute for kindly helping me out with DNA samples, and last but not least, to my family and all my friends. 3 Table of contents Abstract ................................................................... ............. 2 Acknowledgements .............................................................................. 3 Table of contents ............................................... .................... 4 List of figures and tables ................................................................ ..... 6 Chapter 1 : Helicobacterpylori and the ASF mouse model: an introduction.............7 History and taxonomy..................................................................... 8 Morphology and pathology............................................................10 P athogenesis................................................................................ 11 V irulence factors .......................................................................... 12 Epidemiology and transmission....................................................... 13 G enom e................................................................................. .. 14 Diagnostic tests for H. pylori ............................................................ 15 Detection methods for H. pylori ...................................................... 17 Animal models in the study of H. pylori ............................................. 18 Germ-free mice and the altered Schaedler flora .................................... 19 Chapter 2: Design and testing of primers for H. pylori and the Helicobactergenus.......22 Introduction ................................................................................ 22 Materials and methods ................................................................. 24 Alignment of sequences....................................................... 24 Prim er design................................................................... 25 PCR analysis of primer pairs ................................................ 26 R e su lts ...................................................................................... 2 8 H. pylori specific primers .................................................... 28 Helicobactergenus specific primers ....................................... 35 D iscussion .............................................................................. 37 Chapter 3: Methods for diversity analysis of ASF species in the mouse model ........... 40 Introduction .............................................................................. 40 Materials and methods ................................................................. 42 t-R FL P .......................................................................... 42 4 CDCE analysis ................................................... 46 Results ...................................................---- 47 t-RFLP analysis.................................................---- 47 CDCE analysis ............................................ 53 Discussion ................................................. 55 Appendix A: Alignments ........................... ..... ---...........--60 Appendix B: Sensitivity calculations for H. pylori specific primer pair 787f-1 127r..... 67 Appendix C: References ............................................ 68 5 List of figures and tables Table 1.1: Helicobacter species known currently 9 Figure 1.1: TEM image of Helicobacterpylori 10 Table 1.2: Bacteria in the altered Schaedler flora 20 Table 2.1: Evaluation of H. pylori primers (empirical) 31 Figure 2.1: Specificity of primer pair 787f-1 127r 33 Figure 2.2: Sensitivity of primer pair 787f-1 127r 34 Table 2.2: Evaluation of Helicobactergenus primers (empirical) 36 Table 3.1: Primers used in analyses 44 Table 3.2: Preliminary results of t-RFLP experiments 49 Figure 3.1: Electrophoretogram of a t-RFLP run for a single species 50 Figure 3.2: Electrophoretograms for t-RFLP analysis of co-amplified mixtures 51 Figure 3.3: Electrophoretogram for t-RFLP analysis of mouse fecal pellet DNA 52 Figure 3.4: Electropherogram of the separation of a 100bp amplicon by CDCE 54 6 Chapter 1 Helicobacter pylori and the ASF mouse model : an introduction History and taxonomy Even in the late nineteenth century, European pathologists had observed curved bacteria in the gastric tissues. However, they were unable to culture them and their work went unnoticed for a long time. In 1979, Robin Warren, an Australian pathologist noticed curved bacteria in gastric biopsy specimens submitted for histological examination. Together with Barry Marshall, he noticed that the bacteria were curved Gram negative rods with morphological similarity to the Campylobacter species (Marshall et al, 1984). Consequently, he used methods for isolation of bacteria of the Campylobacter species. The cultures did not grow over the three day period characteristic of campylobacters but one culture accidentally left over a weekend showed growth (Marshall et al, 1984). The organism was characterized and called Campylobacter pylori. However, cellular fatty acid profiles showed fatty acid types not present in campylobacters. One of the fatty acids found was unique and the overall novelty of the fatty acid profile led to a new name and genus designation, Helicobacter pylori (Goodwin et al, 1985). Bacteriological and molecular studies in conjunction with 16S rRNA gene sequence analysis revealed that there were many differences between Helicobacterpylori and the Campylobacter, Flexispira and Wolinella species which are closely related phylogenetically. Based upon these studies, it was postulated that H.