The Genetic Composition and Diversity of Francisella tularensis Pär Larsson Akademisk avhandling som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet för avläggande av medicine doktorsexamen i klinisk mikrobiologi med inriktning mot bakteriologi vid Medicinska fakulteten, framlägges till offentligt försvar vid Institutionen för Klinisk Mikrobiologi, sal E04 byggnad 6, torsdagen den 31 maj 2007, klockan 09.00. Avhandlingen kommer att försvaras på engelska. Fakultetsopponent: Dr. Andrew K Benson Department of Food Science & Technology University of Nebraska–Lincoln Lincoln, Nebraska USA Department of Clinical Microbiology, Clinical Bacteriology Umeå University Umeå 2007 Organization Document type UMEÅ UNIVERSITY DOCTORAL DISSERTATION Department of Clinical Microbiology Date of publication SE-901 87 Umeå, Sweden May 2007 Author Pär Larsson Title The Genetic Composition and Diversity of Francisella tularensis Abstract Francisella tularensis is the causative agent of the debilitating, sometimes fatal zoonotic disease tularemia. Despite all F. tularensis bacteria having very similar genotypes and phenotypes, the disease varies significantly in severity depending on the subspecies of the infectious strain. To date, little information has been available on the genetic makeup of this pathogen, its evolution, and the genetic differences which characterize subspecific lineages. These are the main areas addressed in this thesis. Using the F. tularensis subsp. tularensis SCHU S4 strain as a genetic reference, microarray-based comparative genomic hybridisations were used to investigate the differences in genomic composition of F. tularensis isolates. Overall, the strains analysed were very similar, matching the high degree of conservation previously observed at the sequence level. One striking finding was that subsp. mediasiatica was most similar to subsp. tularensis, despite their natural confinement to Central Asia and North America, respectively. Eight regions of difference (RDs) were found to characterize all European and American isolates of subsp. holarctica. One RD was highly polymorphic and, for the first time, allowed the development of an F. tularensis-specific PCR assay that discriminates between the four subspecies. All RDs were found to have been the result of repeat-mediated excision. Therefore, a screening of loci flanked by direct repeats was undertaken, resulting in the identification of eight additional RDs. Data for the RDs along with a multiple locus sequence analysis suggested an evolutionary scenario for F. tularensis. This indicated a deep divergence of subsp. novicida and a deep divergence of the Japanese strains within subsp. holarctica. Since all virulence-attenuated strains investigated had deletions at two RDs, repeat-mediated excision may represent an important natural attenuating mechanism in F. tularensis. A novel typing scheme for F. tularensis was evaluated, aiming to provide both a high resolution and support robust inference at deeper phylogenetic levels. The concept was based on a combination of MLVA (providing high resolution) and a class of insertion-deletion polymorphisms thought to evolve slowly (providing in-depth structural data). The data indicated that the insertion-deletion markers tested are relatively resistant to homoplasy, and that the statistical robustness could be significantly increased compared to the use of MLVA alone. Finally, the genomic sequence of the highly virulent F. tularensis strain SCHU S4 was determined and analysed. Evidenced by numerous pseudogenes and disrupted metabolic pathways, the bacterium appears to be undergoing a genome reduction process whereby a large proportion of the genetic capacity gradually is lost. It is likely that F. tularensis has irreversibly has evolved into an obligate host- dependent bacterium, incapable of a free-living existence. Unexpectedly, the bacterium was found to be devoid of common virulence mechanisms such as classic toxins, or type III and IV secretion systems. Instead, the virulence of this bacterium is probably largely the result of specific and unusual mechanisms. For example, genes found in the genome sequence suggest that it might possess a poly-γ- glutamate capsule similar to the gram-positive anthrax bacterium Bacillus anthracis. Two perfect copies of a virulence-associated locus, the Francisella pathogenicity island, were also found. Key words tularemia, Francisella tularensis, genotyping, evolution, microarray, genome sequencing, virulence, genome reduction Language: English ISBN: 978-91-7264-288-1-X ISSN: 0346-6612 Number of pages: 51 + 4 papers UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 1094 ISSN 0346-6612 ISBN 978-91-7264-288-1 From the Department of Clinical Microbiology, Clinical Bacteriology, Umeå University, Umeå Sweden The Genetic Composition and Diversity of Francisella tularensis Pär Larsson Umeå 2007 Copyright © 2007 Pär Larsson ISBN 978-91-7264-288-1 Printed in Sweden by Solfjädern Offset AB 2007 I love deadlines. I like the whooshing sound they make as they fly by. Douglas Adams TABLE OF CONTENTS ORIGINAL PAPERS IN THESIS ii SAMMANFATTNING iii ABBREVIATIONS v INTRODUCTION 1 BACKGROUND 2 Historical account 2 Tularemia 5 Taxonomy and geographical distribution 6 Evolution and population structure 10 Genomic typing 11 Genome analysis 13 AIMS OF THE THESIS 14 METHODOLOGICAL CONSIDERATIONS 15 Genome sequencing 15 Comparative genomic hybridisation 16 RESULTS AND DISCUSSION 18 Comparative genomic hybridisations reveals strong compositional similarity within F. tularensis (paper I) 18 Phylogeny and direct repeat-mediated deletions among F. tularensis isolates (paper II and unpublished work) 23 Indel markers for low-resolution genetic typing (paper III) 28 The F. tularensis SCHU S4 genome sequence (paper IV) 32 Reductive evolution 33 Insertion sequence elements 34 IS elements and evolution 35 Virulence mechanisms 37 Whole-genome phylogeny 39 CONCLUSIONS 41 ACKNOWLEDGEMENTS 43 REFERENCES 44 i ORIGINAL PAPERS IN THESIS This thesis is based on the following papers, which will be referred to by their Roman numerals (I-IV). I. Broekhuijsen M*, Larsson P*, Johansson A, Byström M, Eriksson U, Larsson E, Prior RG, Sjöstedt A, Titball RW, Forsman M. Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis. Journal of Clinical Microbiology, 2003; 41: 2924-31. II. Svensson K, Larsson P, Johansson D, Byström M, Forsman M, Johansson A. Evolution of subspecies of Francisella tularensis. Journal of Bacteriology, 2005; 187:3903-8. III. Larsson P, Svensson K, Karlsson L, Guala D, Granberg M, Forsman M, Johansson A Analysis of insertion-deletion markers with canonical properties for safe and rapid DNA-based typing of Francisella tularensis. Submitted. IV. Larsson P, Oyston PC, Chain P, Chu MC, Duffield M, Fuxelius HH, Garcia E, Halltorp G, Johansson D, Isherwood KE, Karp PD, Larsson E, Liu Y, Michell S, Prior J, Prior R, Malfatti S, Sjöstedt A, Svensson K, Thompson N, Vergez L, Wagg JK, Wren BW, Lindler LE, Andersson SG, Forsman M, Titball RW. The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nature Genetics, 2005; 37: 153-9. Reprints were made with the permission of the publishers. *Shared first author ii SAMMANFATTNING Francisella tularensis orsakar den zoonotiska sjukdomen harpest, också kallad tularemi. Alla harpestbakterier visar stora genotypiska och fenotypiska likheter men trots detta skiljer sig bakteriens underarter avsevärt i virulens. Om man smittats av en stam som tillhör underarten tularensis (även kallad typ A), kan utgången i värsta fall bli dödlig. Avhandlingen syftade till att öka vår förståelse för hur harpestbakterier är besläktade, vad som skiljer olika evolutionära grenar av bakterien, och till en bestämning av det genetiska innehållet i en av de mest virulenta stammarna av harpestbakterien. Ett urval av harpestisolat, där alla underarter fanns representerade, undersöktes med mikromatris-baserad hybridisering (eng. ”array-based comparative genomic hybridisations”). Resultaten visade en hög homogenitet vad gäller genetiska sammansättningen hos isolaten. Underarterna tularensis och mediasiatica visade störst likhet, trots att dessa organismers geografiska utbredningsområden är åtskilda och begränsade till Nordamerika och Centralasien. Underarten holarctica (även kallad typ B), som finns spridd över stora delar av norra halvklotet och även i Sverige, var genetiskt mer avvikande. De genetiska regioner som innehöll skillnader, eng. regions of difference (RD), befanns ha uppkommit genom en speciell mutationsmekanism, sk. homolog rekombination. Detta faktum utnyttjades för att identifiera ytterligare mutationsbenägna genetiska regioner. I alla virulens-försvagade stammar upptäcktes att gener tappats i två RD. En hypotes är därför att homolog rekombination kan vara en viktig mekanism för naturlig försvagning av harpeststammar. En modell för hur harpestbakterien kan ha evolverat utarbetades också. Flera analyser indikerade att underarten novicida var den tidigast avvikande evolutionära grenen och på motsvarande sätt indikerades även inom underarten holarctica att japanska stammar avvikit tidigt. Resultaten gav stöd åt tidigare indikationer på ett nära släktskap mellan underarterna mediasiatica och tularensis. Dessutom utvecklades inom avhandlingsarbetet en ny typningsmetod för bestämning av