Defining the molecular basis of host-pathogen interaction in bovine TB

The sustainable control of Bovine tuberculosis (bTB) is a major challenge for UK agriculture. bTB is caused by a bacterium, Mycobacterium bovis, that can infect a range of wild and domesticated animals, as well as man. Control of bTB is therefore important for public health as well as animal health and welfare. However, current bTB control measures have been ineffective in the UK. The number of herds newly infected with bTB has doubled every nine years; in 2013 over 6.2 million bTB tests were performed in England leading to the slaughter of over 26,000 cattle at a cost to the taxpayer of almost £100Million. New approaches to halt the spread of infection are desperately needed.
Mycobacterium bovis (Mbv) is a close relative of Mycobacterium tuberculosis (Mtb), the causative agent of TB in humans. While some of the mechanisms used by Mtb to cause disease in humans are well characterised, not much is known about the strategies used by Mbv to cause disease in cattle. DNA sequencing of the genomes of Mtb and Mbv have revealed that they share more than 99.9% genetic identity. While both Mbv and Mtb can infect humans, Mbv rarely transmits between humans and is instead spread by contaminated dairy products. On the other hand, Mtb can only cause a mild form of the disease in cattle. The distinct host preference of Mbv and Mtb indicates that the two mycobacterial species have evolved distinct strategies to infect and cause disease in their respective hosts. Through previous studies and our own work, a range of discrete molecular variations have been identified between Mtb and Mbv; these include differences in the expression of proteins, termed MPB70 and MPB83, and lipids, such as 'sulfolipid', between these bacteria. The role of these proteins and lipids in the virulence of Mbv for cattle has however not been defined; in this study we will explore the hypothesis that these molecular differences are among the key reasons why Mbv causes disease in cattle. We will test our hypothesis by first generating genetically-engineered Mbv strains that are 'Mtb-like' with regards to the expression of these lipids and proteins. These strains will then be tested along with their parental Mtb and Mbv strains in cellular infection models. These models will focus on laboratory grown cell cultures of bovine macrophages, cells of the bovine immune system that are the first to encounter and engulf invading Mbv cells. We have initial data to show that these cells respond differently to infection with Mbv and Mtb, and will use the genetically engineered strains of 'Mtb-like' Mbv strains to elucidate the mechanisms used by Mbv to establish infection in cattle. To achieve this we will develop an array of tools and new methodologies, such as the use of fluorescently-labelled Mbv and genetic-knockdown techniques, to study the infected macrophages. These tools will help us monitor critical intracellular components of the macrophage machinery, as well as providing tools for the wider bovine immunology research community. Our findings will shed new light on the molecular components that Mbv uses to cause disease in cattle, information that will help in the long term development of vaccines and diagnostics against this devastating disease for UK agriculture.

Our knowledge of the virulence mechanisms employed by Mycobacterium bovis (Mbv) to establish infection and ultimately disease in cattle is severely lacking. In this application we will directly address this knowledge gap in bTB host-pathogen interaction by identifying Mbv-specific virulence adaptations. To achieve this we will use a 'One Health' approach to build on comparative analyses between Mbv and Mycobacterium tuberculosis (Mtb). These analyses have shown Mtb to be attenuated in the bovine host and revealed differential Type I IFN and autophagy responses in bovine macrophages infected with Mtb and Mbv. Using a combination of molecular genetics, lipidomics, transcriptomics and cellular microbiology, we will explore the molecular basis of virulence and pathoadaptive mutations in Mbv during interactions with its natural host cell, the bovine macrophage.
The overarching aim of this study is to define host-tropic virulence factors of Mbv for cattle, and to determine how these factors drive the interaction of Mbv with bovine macrophages. Specifically, we hypothesise that Mbv virulence is driven by two key molecular differences between Mbv and Mtb:
1. The loss of the glycolipids SL-1 and PATs in Mbv resulting from a mutation of the PhoPR two component regulon.
2. Sigma factor SigK-dependant increased expression of two antigens MPB70 and MPB83 in Mbv.
These aims will be achieved by the fulfilling the following objectives:
1. Construct and characterise recombinant 'Mtb-like'-Mbv strains that recapitulate intermediate steps in the evolution of Mbv, to be eventually tested in our bovine macrophage model: this will be achieved by generating recombinant strains of Mbv carrying Mtb alleles of phoPR and rskA-sigK into Mbv.
2. Develop cell biology tools to study the bovine macrophage response to infection with Mbv.
3. Outline the interaction of Mbv recombinant strains with the bovine macrophage, focussing in particular on the type I IFN and autophagy pathways.

1) Direct Impact: The proposed research will have a direct impact on tuberculosis (TB) research and the immediate beneficiaries will be the PI's research group in particular, and on a wider scale the TB research community.
(i) PDRAs: The PDRAs employed on this project will be joining Institutions that host large microbiology research clusters in the U.K and ROI giving them exposure to world class research. The PDRAs will also gain training in a wide range of methodologies via collaborations between the three groups, with the PDRAs facilitating cross-fertilisation of ideas contribute new ideas to this partnership.

(ii) The PI's research groups at the three institutions: The research groups based at UoB, UCD and NIMR (from April 2015, The Francis Crick Institute) will also benefit from this work as new avenues of research are opened, expanding the themes of each laboratory.
(iii) The Research Community: The outcomes from our proposed project will have the potential to further our understanding of a number of aspects of bovine TB including the study of evolution of MTB complex members, the immunology of bovine and human TB, vaccine research and cell biology of bovine and human TB.

2) Future Beneficiaries beyond the timescale of the project:
(i) Industry: The UK farming industry suffers considerable losses due to the bTB. In 2014 bTB controls cost the taxpayer nearly £100 million, while costs to farmers are estimated to have run to tens of millions of pounds (Defra). New approaches to disease control are therefore urgently needed, and the research in this project offers new avenues to the development of both improved vaccines (such as live-attenuated vaccines based on modified versions of Mbv), as well as improved diagnostics (through the identification of potential biomarkers of infection at the cellular level that could then be explored in peripheral blood). Improved diagnostics in particular would have obvious attraction for the pharmaceutical industry; in 2014 over 6 million bTB diagnostic test were performed in the UK alone, offering a considerable market for new bTB diagnostics.
(ii) Global Health and the General Public:
bTB is a paradigm for the One Health concept with the integration of human, animal and environmental health. Zoonotic transmission of Mbv infection from infected animals to humans is one of the central reasons for the control of TB in cattle. The role of wildlife, such as the badger, in acting as reservoir hosts of infection speak to the need for an integrated approach to bTB control that incorporates environmental and animal health and welfare. This is true not just in the UK but across the globe. Indeed in many developing countries the threat of bTB to public health and indigenous wildlife is well recognised, with bTB control policies in multiple stages of development. Our project takes a unique approach in attempting to define Mbv virulence factors from a One Health perspective, using comparative analyses with the human pathogen Mtb as a route to defining virulence in Mbv. As such we will deliver novel insight into not only what makes Mbv a highly successful animal pathogen, but also into what makes Mtb the world's most deadly human bacterial pathogen.