Imperial College London School of Public Health The evolution of Cryptococcus neoformans var. grubii in the context of clinical disease using multilocus sequence typing Sitali P. Simwami September 2011 Submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Public Health of Imperial College London and the Diploma of Imperial College London Declaration I herewith certify that all material in this dissertation which is not my own work has been properly acknowledged. Sitali P. Simwami 3 Abstract The global burden of HIV-associated cryptococcal meningitis (CM) is es- timated at one million cases per year, causing up to a third of all AIDS- related deaths. Cryptococcus neoformans variety grubii (Cng) is the most ubiquitous cause of cryptococcal meningitis worldwide, however patterns of molecular diversity are understudied across some geographical regions ex- periencing significant burdens of disease. Cryptococcus species are notable in the degree that virulence differs amongst lineages, and highly virulent emerging lineages are changing patterns of human disease both temporally and spatially. Molecular epidemiology constitutes the main methodology for understanding the factors underpinning the emergence of this under- studied, yet increasingly important, group of pathogenic fungi. A multilo- cus sequence typing (MLST) scheme was used to characterise a genetically depauperate Cng population in Thailand, and a contrastingly highly di- verse Cng population in Cape Town, South Africa. Sequence types (STs) from these populations were integrated into a dataset comprising global STs of Cng and patterns of range expansion were traced. Evidence from haplotypic networks and coalescent analyses revealed an ancestral African population of molecular type VNB, from which emerged a VNI lineage. This VNI lineage expanded globally out of Africa and led to the introduction of a limited number of genotypes in novel regions, including Asia. Bayesian inference estimated this spread of VNI to have occurred between 1 600 and 70 500 years ago, putatively vectored by the anthropogenically mediated spread of domesticated pigeons, historically native African birds. Clinical data collected from patients presenting with AIDS-associated CM showed that infecting African Cng isolates were associated with poorer long-term survival compared to Asian isolates. As mortality rates reported for these patients in Sub-Saharan Africa are higher than those seen in Asia, sup- ported by a Galleria mellonella virulence model, this apparently elevated virulence is postulated as being the result of recombinant progeny with di- verse phenotypes being created through frequent meiotic recombination. 5 Acknowledgments First, I would like to thank my principal supervisor, Dr. Matthew Fisher, for his vision for this project, guidance, and confidence in my abilities. I could not have asked for a better mentor. Thank you to the members of the Fisher Lab, past and present, and especially to Daniel who somehow knew the answers to all my questions. I also thank my second supervisor, Prof. Christl Donnelly, Dr. Jombart Thibaut and Dr. Miltiadis Mavrakakis for their valuable statistical advice, and the BBSRC for funding this work. Thank you to members of the CBS (Ferry and Ning), St. George's and Hammersmith Hospitals (Tom, Tihana, Jo, Anne-Marie and Michael), and Duke University (Ana), whose valuable collaborations made this work pos- sible. Thank you to the the University of Cape Town's Institute of Infectious Disease and Molecular Medicine for hosting me during my fieldwork in South Africa (Widaad, Robert, Graeme). I also extend an especially warm thank you to those who made my stay so memorable (Tsungai, Vee, Slee, Joanna, Banda and Lee) | I will be back! Thank you to my friends whose support never wavered, despite me reg- ularly disappearing for long periods of time. Bee and Shmi, knowing our relationships remained unchanged made this process a lot easier. I also thank my little sister Moono; our relationship kept me grounded and means more to me than you know. Thank you to my fiance, Milt, for being everything to me. As a wise Greek once said, 'A hungry bear does not dance'. Finally, I would like to thank my parents for their constant love, support and wisdom. I owe you everything. 6 To my parents, Jonathan and Sharon, and to Milt. Trust in the Lord with all your mind, in all your ways acknowledge him, and he shall direct your path. | Proverbs 3:5 7 Contents 1. Introduction 17 1.1. Cryptococcus ........................... 17 1.2. Species classification and distribution . 18 1.3. Cryptococcus neoformans var grubii .............. 21 1.4. Life cycle and sexual cycle of Cn ................ 22 1.5. Infection and disease caused by Cng .............. 26 1.6. Cryptococcosis and HIV/AIDS . 28 1.6.1. AIDS in Thailand . 30 1.6.2. AIDS in South Africa . 31 1.7. Classical virulence phenotypes of Cn . 32 1.8. Animal Host Models . 36 1.8.1. Cn { Galleria mellonella pathosystem . 37 1.9. Rationale and importance of study . 38 1.10. Thesis overview . 42 1.11. Aims of the project . 43 2. Methodology and data analysis 44 2.1. Molecular sequencing of Cn ................... 44 2.1.1. Yeast cultivation and DNA extraction . 44 2.1.2. Polymerase Chain Reaction amplification (PCR) . 45 2.1.3. PCR cleanup . 45 2.1.4. Sequencing . 47 2.1.5. Precipitation of sequencing cycling products . 47 2.2. MLST determination . 48 2.3. Mating-type and serotype analyses . 48 2.4. Molecular data analyses . 49 2.4.1. Diversity indices . 49 2.4.2. Analysis of genetic structure based on allelic profiles . 51 2.4.3. Linkage disequilibrium and recombination . 53 9 2.4.4. Genetic differentiation between populations . 53 2.5. Phylogenetic analyses and molecular type determination . 54 2.6. Estimates of times of divergence and haplotype networks . 55 2.7. MLST website eBURST tool . 55 2.8. Clinical data and analysis . 56 2.9. Environmental fieldwork . 56 2.9.1. Sampling of Cn ...................... 56 2.9.2. Preparation of niger seed agar . 56 2.9.3. Isolation and identification . 57 2.10. Galleria mellonella wax moth model . 58 3. Multilocus Sequence Typing 60 3.1. Introduction . 60 3.2. History of the identification of fungi . 61 3.3. Rationale for the standardisation of the typing of Cn ..... 65 3.4. ISHAM, our aims . 66 3.4.1. Consensus genotype nomenclature . 67 3.4.2. Consensus standard strains . 67 3.4.3. Consensus typing scheme MLST . 72 3.5. MLST . 72 3.5.1. MLST loci . 73 3.5.2. MLST website and Cng Database . 75 3.6. Discussion . 77 4. Cng in Thailand 79 4.1. Introduction . 79 4.1.1. Cn in Thailand . 80 4.2. Aim . 81 4.3. Materials and Methods . 81 4.3.1. Isolates . 81 4.4. Ethics Statement . 87 4.5. Results . 88 4.5.1. Mating type, variety and MLST sequence type . 88 4.6. Discussion . 95 5. Cng in Cape Town 99 5.1. Introduction . 99 10 5.2. Aims . 100 5.3. Materials and Methods . 100 5.3.1. Isolates . 100 5.3.2. Molecular methods . 101 5.4. Ethics Statement . 103 5.5. Results . 103 5.5.1. Mating, molecular, and sequence types of isolates . 103 5.5.2. Molecular analyses . 108 5.6. Discussion . 113 6. Global Cng 119 6.1. Introduction . 119 6.2. Aim . 120 6.3. Materials and Methods . 120 6.3.1. Global Cng isolates . 120 6.3.2. Methods . 125 6.4. Results . 125 6.4.1. Structure of the global population of Cng isolates based on allelic profiles . 125 6.4.2. Phylogenetic analyses revealed three molecular groups within the global Cng population . 126 6.4.3. The African population exhibits the most genetic di- versity of the global Cng subpopulations . 130 6.4.4. Predominant clonality detected within the global Cng populations . 134 6.4.5. Divergence time estimates and haplotype networks support a hypothesis of African ancestry for global Cng isolates . 138 6.5. Discussion . 142 7. Clinical analysis 149 7.1. Introduction . 149 7.2. Aims . 150 7.3. Materials and methods . 150 7.4. Results . 153 7.4.1. Study population according to ST . 153 11 7.4.2. ST and baseline variables . 155 7.4.3. Death by weeks 2 and 10 . 157 7.4.4. Early fungicidal activity and treatment . 157 7.4.5. Genotypic effect on EFA at the allelic profile level but not ST level . 160 7.4.6. Survival analysis . 161 7.5. Discussion . 167 8. Environmental Cape Town isolates 175 8.1. Introduction . 175 8.2. Materials and Methods . 176 8.3. Results . 176 8.4. Discussion . 179 9. Galleria mellonella - Cng virulence assay 182 9.1. Introduction . 182 9.2. Aims . 182 9.3. Materials and Method . 183 9.3.1. Isolates . 183 9.4. Result . 183 9.5. Discussion . 188 10.Final Discussion 193 A. Published work 202 A.1. Meyer et al., 2009 . 203 A.2. Simwami et al., 2011 . 217 B. Additional tables and figures 239 C. R Code 251 C.1. PCA commands . 251 C.2. Inter-class PCA commands . 251 C.3. Clinical analysis . 252 C.4. Galleria mellonella assay anaylsis . 255 D. Glossary 257 12 List of Tables 1.1. Genes linked to the major virulence phenotypes of Cn..... 35 2.1. Mating-type primer sequences. 49 3.1. Relationships among different molecular groups in Cn var grubii................................ 66 3.2. Concordance of different molecular typing methods used for Cryptococcus ........................... 69 3.3. Standard reference strains for Cryptococcus neoformans typing. 70 3.4. Standard reference strains for Cryptococcus gattii typing. 71 3.5. Seven Multilocus Sequence Typing Loci. 75 4.1. Allelic profiles of the Thai Cng isolates. 83 4.2. Diversity indices, Thailand.