alpha-Mannan hydrolysing enzymes as drivers of mycobacterial cell surface diversity.
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
PURPOSE OF RESEARCH:
The on-going tuberculosis pandemic is the second greatest killer of people amongst infectious diseases. Globally, mass migration and worsening living conditions caused by conflict and climate change are putting incredible strain on efforts to contain this pathogen. At a practical level, clinicians are caught between increasingly ineffective medicines and poor diagnostic options. There is an urgent and unmet need for development of innovative technologies in both areas. An effective vaccine against tuberculosis is also not available.
A key to the success of mycobacteria is their unusual cell wall. This bacterial "skin" protects the organism from the environment, but also plays a pivotal role in how it interacts with its host. The chemically distinct nature of the lipids and sugars that form this structure mean that our immune systems have been tuned to sense its presence. The bacteria, however, do not present a static cell wall to the host but instead modulate it to evade or control immunity. This intricate interplay provides an opportunity for the development of new therapeutics, diagnostics, and vaccines. To achieve this potential, the key data we are missing is an understanding of the molecular tool kit that mycobacteria use to modulate their cell wall.
We have identified a set of enzymes produced by the bacteria that degrade mannose containing structures as a critical component of this process. These enzymes cleave the cell wall, thereby sculpting the immunogenic molecules that drive the host-pathogen interaction. In this grant proposal we aim to:
1) Identify key mechanisms driving mycobacterial cell surface variability.
2) Characterise new enzymes that can be used to generate immune-active molecules.
3) Generate new mycobacterial strains with altered mannosylated glycolipid presentation and understand the impact this has on their biology.
TIMELINESS
The question of how a key mannosylated glycolipid, lipoarabinomannan, is processed and secreted is an important and long-standing problem of fundamental importance to mycobacterial biology. Now, for the first time, we have assembled the preliminary data and expertise to answer this question. The outcomes of this work are urgently required to help address the on-going and worsening tuberculosis pandemic.
VALUE FOR MONEY
This project will benefit from prior investment of the BBSRC in capacity building in the Moynihan laboratory. Our expertise in peptidoglycan recycling and techniques pertaining to the mycobacterial cell wall when combined with our wealth of preliminary data mean that we are uniquely primed to address this topic. The grant has no large equipment requests and PI time is partially covered due to overlap with Dr. Moynihan's BBSRC David Phillips Fellowship. This grant will also benefit from Dr. Moynihan's experience commercialising enzymes active against mycobacterial cell walls.
OUTCOMES
1) Fundamental understanding of mycobacterial cell wall biology. This proposal will answer a long-standing question in the mycobacterial field.
2) Likely applications in mycobacterial diagnostics or vaccine development. The data generated in this grant will provide a toolkit to either modify LAM in vitro to improve its detection or generate bacterial strains with altered immunological properties which will have impacts on vaccine development.
The on-going tuberculosis pandemic is the second greatest killer of people amongst infectious diseases. Globally, mass migration and worsening living conditions caused by conflict and climate change are putting incredible strain on efforts to contain this pathogen. At a practical level, clinicians are caught between increasingly ineffective medicines and poor diagnostic options. There is an urgent and unmet need for development of innovative technologies in both areas. An effective vaccine against tuberculosis is also not available.
A key to the success of mycobacteria is their unusual cell wall. This bacterial "skin" protects the organism from the environment, but also plays a pivotal role in how it interacts with its host. The chemically distinct nature of the lipids and sugars that form this structure mean that our immune systems have been tuned to sense its presence. The bacteria, however, do not present a static cell wall to the host but instead modulate it to evade or control immunity. This intricate interplay provides an opportunity for the development of new therapeutics, diagnostics, and vaccines. To achieve this potential, the key data we are missing is an understanding of the molecular tool kit that mycobacteria use to modulate their cell wall.
We have identified a set of enzymes produced by the bacteria that degrade mannose containing structures as a critical component of this process. These enzymes cleave the cell wall, thereby sculpting the immunogenic molecules that drive the host-pathogen interaction. In this grant proposal we aim to:
1) Identify key mechanisms driving mycobacterial cell surface variability.
2) Characterise new enzymes that can be used to generate immune-active molecules.
3) Generate new mycobacterial strains with altered mannosylated glycolipid presentation and understand the impact this has on their biology.
TIMELINESS
The question of how a key mannosylated glycolipid, lipoarabinomannan, is processed and secreted is an important and long-standing problem of fundamental importance to mycobacterial biology. Now, for the first time, we have assembled the preliminary data and expertise to answer this question. The outcomes of this work are urgently required to help address the on-going and worsening tuberculosis pandemic.
VALUE FOR MONEY
This project will benefit from prior investment of the BBSRC in capacity building in the Moynihan laboratory. Our expertise in peptidoglycan recycling and techniques pertaining to the mycobacterial cell wall when combined with our wealth of preliminary data mean that we are uniquely primed to address this topic. The grant has no large equipment requests and PI time is partially covered due to overlap with Dr. Moynihan's BBSRC David Phillips Fellowship. This grant will also benefit from Dr. Moynihan's experience commercialising enzymes active against mycobacterial cell walls.
OUTCOMES
1) Fundamental understanding of mycobacterial cell wall biology. This proposal will answer a long-standing question in the mycobacterial field.
2) Likely applications in mycobacterial diagnostics or vaccine development. The data generated in this grant will provide a toolkit to either modify LAM in vitro to improve its detection or generate bacterial strains with altered immunological properties which will have impacts on vaccine development.
Technical Summary
The mycobacterial cell wall is rich in antigenic molecules. A particularly important class of these are the mannosylated glycolipids which includes lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylmyo-insitol mannosides (PIMs). Decades of research has focused on the biosynthesis of this family of molecules, with very little understanding of their post-synthetic fate. This lack of data is an important problem because the break-down or modification of these molecules has significant biological implications. For example, LAM is known to be anchored to the cytoplasmic membrane and yet its carbohydrate domain is sensed by the host and makes up to 20% of the mycobacterial capsule. How it gets there is completely unknown. Capping and side-chain modifications of LAM and LM are also thought to be important for immune modulation, but the degree to which these vary in purified LM and LAM suggests that the bacteria use enzymes to tailor these structures. These two examples point towards a glycoside hydrolase-driven dynamically modulated cell surface.
In this proposal we will investigate the function of three alpha-mannan degrading enzymes that are likely to modify PIMs, LM and LAM. These belong to two distinct classes. First, we will study an endo-mannanase which we have shown will cleave within the mannan backbone of LM and LAM and we hypothesise is responsible for arabinomannan release to the cell surface. Second, we will investigate two exo-mannosidases which are likely involved in trimming capping and side chain motifs within LAM. We will characterise these enzymes both biochemically, and biologically to understand what they cut, where they cut it and what the consequences are for this activity. Together these data will provide a new view of mycobacterial cell surface variation and could lead to new diagnostics, therapeutics or vaccines against mycobacterial diseases such as tuberculosis.
In this proposal we will investigate the function of three alpha-mannan degrading enzymes that are likely to modify PIMs, LM and LAM. These belong to two distinct classes. First, we will study an endo-mannanase which we have shown will cleave within the mannan backbone of LM and LAM and we hypothesise is responsible for arabinomannan release to the cell surface. Second, we will investigate two exo-mannosidases which are likely involved in trimming capping and side chain motifs within LAM. We will characterise these enzymes both biochemically, and biologically to understand what they cut, where they cut it and what the consequences are for this activity. Together these data will provide a new view of mycobacterial cell surface variation and could lead to new diagnostics, therapeutics or vaccines against mycobacterial diseases such as tuberculosis.
