An integrative 'omics approach to define functional variation between the human and bovine tubercle bacilli

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Title: An integrative 'omics approach to define functional variation between the human and bovine tubercle bacilli
Authors: Malone, Kerri M.
Permanent link: http://hdl.handle.net/10197/7539
Date: Jan-2016
Abstract: Tuberculosis in both man and animal is caused by pathogenic mycobacterial species of the Mycobacterium tuberculosis complex (MTBC). Human tuberculosis, caused by infection with M. tuberculosis, ranks as the world’s leading cause of death from an infectious disease with over 1.5 million deaths and a further estimated 9.6 million new cases reported in 2014 (WHO 2015). The emergence of multidrug resistant and extensively drug resistant M. tuberculosis strains along with a rise in the number of individuals co-­‐infected with human immunodeficiency virus (HIV) complicates the ultimate goal of eliminating tuberculosis worldwide by 2050 and also places emphasis on the need to develop novel therapeutics for treatment. Also, there is a need to improve current tuberculosis diagnostic tools to differentiate individuals with active disease from those who are latently infected (StopTB 2015). While M. tuberculosis is human host restricted, M. bovis infects a wide range of mammals including humans and zoonotic transmission represents a further human health risk and a One Health concern (Sternberg Lewerin 2015). Bovine tuberculosis amongst Irish herds is estimated to cost the economy ~€70 million per annum and eradication of the disease is proving difficult with the interplay of maintenance species (such as the badger) complicating efforts (EU-­‐Commission 2014). As Ireland tends towards obtaining official bovine tuberculosis free status, efforts must focus on improving current diagnostic tests. Widening our understanding of how both of these pathogens have evolved, cause disease and persist within their respective hosts will facilitate future rational therapeutic and diagnostic design. As members of the MTBC, M. tuberculosis and M. bovis are over 99% identical at the nucleotide level with no unique genes per se found in the M. bovis AF2122/97 genome, yet the two species exhibit distinct host preference (Garnier et al. 2003). Owing to this, the question as to what drives host specificity in these highly related organisms is of particular interest. We hypothesized that relatively minor variation in nucleotide sequence identity could result in functional variation between the two species. Comparative in silico analyses reveals differences in the number of annotated coding sequences between the two organisms (3,951 versus 4,031 coding sequences respectively) despite similar nucleotide identity (Cole et al. 1998; Garnier et al. 2003). Whether these discrepancies are of evolutionary consequence, or are simply cases of misannotation in the M. bovis AF2122/97 sequence assembly that remains as per the original genome publication, had yet to be demonstrated. Thus, prior to inter-­‐species comparative ‘omic analyses we sought to update the M. bovis AF2122/97 reference genome. Through a combination of DNA-­‐sequencing and proteogenomics, we revealed the coding capacity of the M. bovis AF2122/97 reference genome and disclosed the presence of the supposedly deleted RD900 genomic region, resulting in an up-­‐to-­‐date reference genome assembly that will serve as a standard for the scientific community (Chapter 3). Owing to the fact that the genomic sequences of M. tuberculosis and M. bovis are so similar, the basis of the exhibited host specificity between the two species may be explained by differential expression profiles as a result of minor nucleotide alterations between the two species. Through a combination of RNA-­‐sequencing and SWATH mass spectrometry we demonstrate for the first time the varying adaptive responses of the human and bovine tubercle bacilli during both exponential growth (Chapter 4) and in response to in vivo like conditions (Chapter 5) in vitro on a global transcriptional and translational scale. An in vitro model mimicking the metallic ion concentrations relevant to the mycobacterial-­‐containing phagosome 24 hr after infection at low pH was developed (Wagner et al. 2005a) and initiated a robust –and divergent-­‐ response in M. tuberculosis H37Rv and M. bovis AF2122/97 that resembles those found in the presence of stresses such as nitric oxide and hypoxia, nutrient starvation and the macrophage environment itself (Chan et al. 2001; Schnappinger et al. 2003; Voskuil et al. 2003; Kendall et al. 2004). Furthermore, the exposure model results highlighted divergent expression and activity of transcriptional regulators such as PhoP, DosR and WhiB3, governing the control of virulence associated pathways involved in processes like dormancy (DosR regulon), the production of virulent lipids sulfolipid-­‐1 (SL-­‐1) and phthiocerol dimycocerosate (PDIM) and the secretion of ESX-­‐1-­‐associated virulence factors which may be responsible for the exhibited host preference between the two species in vivo (Converse et al. 2009; Singh et al. 2009; Gonzalo-­‐Asensio et al. 2014). In Chapter 6 we highlight the application of ‘omics data to novel antigen discovery; a high priority list of potential T-­‐cell epitopes was constructed through a combination of RNA and protein expression profiles and in silico MHC-­‐II binding predictions for bovine tuberculosis diagnosis (Farrell & Gordon 2015). Finally we also present data supporting the novel concept of translation from regions of antisense transcriptional activity in M. tuberculosis and M. bovis. Therefore, the data presented in this thesis highlights the power granted by combinatorial profiling of both the transcriptome and proteome in revealing the functional variation between the human and bovine adapted M. tuberculosis and M. bovis towards understanding the host preference of these two highly important pathogens.
Type of material: Doctoral Thesis
Publisher: University College Dublin. School of Veterinary Medicine
Advisor: Ph.D.
Copyright (published version): 2016 the author
Keywords: Bovine tuberculosis;Mycobacterium tuberculosis;Mycobacterium bovis;RNA-sequencing;Mass spectrometry;SWATH;DNA-sequencing;Differential gene expression;Bovine tuberculosis diagnosis;Antisense translation
Language: en
Status of Item: Peer reviewed
Appears in Collections:Veterinary Medicine Theses

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