RNA-mediated virulence gene regulation in the human pathogen Listeria monocytogenes Edmund Loh Institutionen för Molekylärbiologi Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå Universitet Umeå 2010 Edmund Loh Institutionen för Molekylärbiologi Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå Universitet S-901 87, Umeå. Sweden Copyright © Edmund Loh ISSN: 0346-6612 New series nr: 1337 ISBN: 978-91-7264-994-1 Printed by: Print & Media Umeå, Sweden 2010 2 To my mother…... (Kepada Ibuku) Holy mother of Jesus! & Now you must work like a dog! (Jörgen Johansson) 3 4 Table of contents ABSTRACT .................................................................................................................................................. 7 SAMMANFATTNING PÅ SVENSKA....................................................................................................... 8 PAPERS IN THIS THESIS ......................................................................................................................... 9 INTRODUCTION...................................................................................................................................... 10 1. LISTERIA MONOCYTOGENES ................................................................................................................. 10 2. PATHOPHYSIOLOGY OF LISTERIA INFECTION ........................................................................................ 10 2.1 Crossing the intestinal barrier...................................................................................................... 11 2.2 Multiplications within the liver and spleen................................................................................... 12 2.3 Bacteraemia .................................................................................................................................. 13 2.4 Crossing the placental barrier...................................................................................................... 13 2.4.1 Neonatal infection...................................................................................................................... 13 2.5 Crossing the blood-brain barrier.................................................................................................. 14 2.6 Other complications of an L. monocytogenes infection ................................................................ 14 2.7 Summary ....................................................................................................................................... 15 3. INTRACELLULAR LIFE CYCLE OF LISTERIA MONOCYTOGENES .............................................................. 15 3.1 Internalisation of L. monocytogenes............................................................................................. 16 3.2 Intracellular growth and spread ................................................................................................... 16 3.3 Virulence factors involved in the intracellular growth cycle of L. monocytogenes ...................... 17 3.3.1 Internalins.................................................................................................................................. 17 3.3.1.1 InlA.......................................................................................................................................... 17 3.3.1.2 InlB.......................................................................................................................................... 18 3.3.1.3 Other internalins..................................................................................................................... 19 3.3.2 Other virulence factors .............................................................................................................. 20 3.3.2.1 Vip........................................................................................................................................... 20 3.3.2.2 p60 .......................................................................................................................................... 20 3.3.2.3 Ami and Auto........................................................................................................................... 20 3.3.2.4 Sortases................................................................................................................................... 21 3.3.2.5 Bsh and BilE ........................................................................................................................... 21 3.3.2.6 Listeriolysin O......................................................................................................................... 22 3.3.2.7 Phospholipases ....................................................................................................................... 23 3.3.2.8 Hpt .......................................................................................................................................... 24 3.3.2.9 ActA......................................................................................................................................... 24 4. REGULATION OF LISTERIA MONOCYTOGENES VIRULENCE GENES ......................................................... 25 4.1 Sigma Factor B ( σB)...................................................................................................................... 26 4.2 PrfA............................................................................................................................................... 27 4.2.1 Transcriptional regulation of prfA expression........................................................................... 28 4.2.2 Translational regulation of prfA expression .............................................................................. 29 4.2.3 Post-translational regulation of PrfA ........................................................................................ 30 5. RNA REGULATION................................................................................................................................ 31 5.1 CIS -ACTING RNA S .............................................................................................................................. 32 5.1.1 Nutritional depravation.............................................................................................................. 32 5.1.2 pH .............................................................................................................................................. 34 5.1.3 Thermosensors ........................................................................................................................... 34 5.1.4 Riboswitches .............................................................................................................................. 35 5.1.4.1 Molecular recognition of SAM................................................................................................ 37 5.1.4.2 Structure of a SAM riboswitch ................................................................................................ 38 5.1.4.3 SAM riboswitch-mediated gene regulation............................................................................. 38 5.1.4.4 Other types of riboswitches..................................................................................................... 39 5.2 TRANS -ACTING RNA S......................................................................................................................... 40 5.2.1 ncRNAs that sequester proteins ................................................................................................. 40 5 5.2.1.1 CsrB and CsrC........................................................................................................................ 40 5.2.1.2 6S RNA.................................................................................................................................... 41 5.2.2 ncRNAs that bind to target mRNAs............................................................................................ 42 5.2.3 DsrA........................................................................................................................................... 43 5.2.4 RyhB........................................................................................................................................... 45 5.3 Protein factors important for the function of trans-encoded ncRNAs........................................... 47 5.3.1 Hfq ............................................................................................................................................. 47 5.3.2 RNase E and RNase III .............................................................................................................. 48 5.4 RNA-MEDIATED GENE REGULATION IN PATHOGENS ........................................................................... 49 5.4.1 RNAIII in Staphylococcus aureus .............................................................................................. 50 5.4.2 VrrA in Vibrio cholerae ............................................................................................................. 51 5.4.3 LhrA-C in Listeria monocytogenes ............................................................................................ 52 AIMS OF THIS THESIS ........................................................................................................................... 53 RESULTS AND DISCUSSION................................................................................................................