IDENTIFICATION OF VIRULENCE DETERMINANTS OF MYCOBACTERIUM TUBERCULOSIS VIA GENETIC COMPARISONS OF A VIRULENT AND AN ATTENUATED STRAIN OF MYCOBACTERIUM TUBERCULOSIS. by ALICE HOY LAM LI B.Sc., The University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIRMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Pathology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) MARCH 2008 Alice Hoy Lam Li, 2008 i ABSTRACT Candidate virulence genes were sought through the genetic analyses of two strains of Mycobacterium tuberculosis, one virulent, H37Rv, one attenuated, H37Ra. Derived from the same parent, H37, genomic differences between strains were first examined via two-dimensional DNA technologies: two-dimensional bacterial genome display, and bacterial comparative genomic hybridisation. The two-dimensional technologies were optimised for mycobacterial use, but failed to yield reproducible genomic differences between the two strains. Expression differences between strains during their infection of murine bone-marrow-derived macrophages were then assessed using Bacterial Artificial Chromosome Fingerprint Arrays. This technique successfully identified expression differences between intracellular M. tuberculosis H37Ra and H37Rv, and six candidate genes were confirmed via quantitative real-time PCR for their differential expression at 168 hours post-infection. Genes identified to be upregulated in the attenuated H37Ra were frdB, frdC, and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE, and Rv1571. Further qPCR analysis of these genes at 4 and 96h post-infection revealed that the frd operon (encoding for the fumarate reductase enzyme complex or FRD) was expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 was higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of culture when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Furthermore, inhibition of the fumarate reductase complex in intracellular bacteria resulted in a significant reduction of intracellular growth. Microarray technology was also applied in the expression analysis of intracellular bacteria at 168h post-infection. Forty-eight genes were revealed to be ii differentially expressed between the H37Ra and H37Rv strains, and a subset were further analysed via qPCR to confirm and validate the microarray data. phoP was expressed at a lower level in the attenuated M. tuberculosis H37Ra, whereas members of the phoPR regulon were up- regulated in the virulent H37Rv. Additionally, a group of genes (Rv3616c-Rv3613c) that may associate with the region of difference 1 were also up-regulated in the virulent H37Rv. iii TABLE OF CONTENTS ABSTRACT ................................................................................................................................. ii TABLE OF CONTENTS...............................................................................................................iv LIST OF TABLES.......................................................................................................................viii LIST OF FIGURES ....................................................................................................................... ix ABBREVIATIONS ....................................................................................................................... xi ACKNOWLEDGEMENTS......................................................................................................... xiv CHAPTER 1: Introduction............................................................................................................. 1 1.1 Background..................................................................................................................... 1 1.2 Mycobacterium tuberculosis........................................................................................... 3 1.2.1 Genomic organisation in M. tuberculosis............................................................... 3 1.2.2 Lipid metabolism .................................................................................................... 4 1.2.3 PE and PPE gene family ......................................................................................... 5 1.3 Intracellular life for M. tuberculosis ............................................................................... 6 1.3.1 Encountering the host ............................................................................................. 7 1.3.2 Adaptations of M. tuberculosis to host defences. ................................................... 8 1.4 Bacterial model of interest: M. tuberculosis H37Ra and H37Rv ................................... 9 1.5 Difference analyses of M. tuberculosis H37Ra and H37Rv......................................... 10 1.5.1 Genomic analyses of M. tuberculosis H37Ra versus H37Rv............................... 11 1.5.2 Expression analyses examining differences between M. tuberculosis H37Ra and H37Rv ............................................................................................................................... 12 1.6 Project goals.................................................................................................................. 14 1.6.1 Hypothesis............................................................................................................. 15 1.6.2 Objective and specific aims of project.................................................................. 15 CHAPTER 2: Materials and methods .......................................................................................... 16 2.1 Growth of mycobacteria............................................................................................... 16 2.1.1 Aerobic Growth of Mycobacterium tuberculosis H37Ra and H37Rv.................. 16 2.1.2 Growth of Mycobacterium tuberculosis H37Ra and H37Rv for fumarate reductase studies. ............................................................................................................................... 18 2.2 Isolation of nucleic acids from broth culture ................................................................ 19 iv 2.2.1 Isolation of genomic DNA from axenic broth cultures of Mycobacterium tuberculosis. .......................................................................................................................... 19 2.2.2 Isolation of RNA from axenic broth cultures of Mycobacterium tuberculosis. ... 20 2.2.3 Purification of mycobacterial RNA...................................................................... 20 2.3 Derivation, culture, and infection of murine bone marrow-derived macrophages (BM- MΦ). ....................................................................................................................................... 21 2.3.1 Derivation and culture of BM-MΦ ....................................................................... 21 2.3.2 Infection of BM-MΦ with Mycobacterium tuberculosis H37Ra and H37Rv ...... 22 2.3.3 Extraction of RNA from macrophage-associated Mycobacterium tuberculosis .. 23 2.4 Two-dimensional DNA displays................................................................................... 23 2.4.1 Generation of two-dimensional bacterial genome displays (2DBGD)................. 23 2.4.2 Generation of bacterial comparative genomic hybridisation (BCGH) profiles.... 24 2.4.3 Generation and hybridisation of DIG-labelled probes with BCGH profiles ........ 25 2.5 Bacterial artificial chromosome fingerprint arrays (BACFAs) .................................... 27 2.5.1 Growth and harvest of bacterial artificial chromosome (BAC) DNA .................. 27 2.5.2 Generation of BACFAs......................................................................................... 28 2.5.3 cDNA synthesis and DIG-labelling of cDNA probes for BACFA analysis......... 29 2.5.4 Hybridisation of BACFAs .................................................................................... 31 2.5.5 Analysis of BACFAs ............................................................................................ 32 2.5.6 Quantitative real-time PCR analysis of expression differences observed with BACFA ............................................................................................................................... 33 2.5.7 Extraction of whole cell lysate for fumarate reductase enzyme assessments in cultures mimicking microaerophilic conditions.................................................................... 34 2.5.8 Fumarate reductase enzyme assay ........................................................................ 34 2.5.9 Western blot detection of fumarate reductase in whole-cell-lysates of M. tuberculosis. .......................................................................................................................... 35 2.5.10 Evaluation of mercaptopyridine-N-oxide (MPNO) effects on BM-MΦ and on growth of intracellular M. tuberculosis................................................................................. 36 2.5.11 Statistical analysis................................................................................................. 37 2.6 DNA microarrays.........................................................................................................