Peptidoglycan release and recycling in pathogenic mycobacteria.

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

The UN and WHO have both recognised tuberculosis (TB) as a major driver of poverty with drug-resistant forms of the disease causing high mortality rates. Drug-resistant strains have poor treatment options, and TB infection is compounded by frequent co-infection with HIV. These factors contributed to TB killing approximately 1.8 million people last year. Despite global efforts, this problem is only getting worse. The percentage of TB cases reported to be resistant to rifampicin, a front-line antibiotic rose from 31% in 2015 to 41% in 2016. This is an antibiotic resistance crisis and new therapeutic options are urgently needed. One of the most efficient ways to address this crisis is to rehabilitate existing medicines. For years beta-lactam antibiotics have not been used in TB treatment due to its endogenous broad-spectrum beta-lactamase. Recent studies, including a clinical trial have changed this view. When combined with a beta-lactamase inhibitor, beta-lactam antibiotics perform as well as existing therapeutics. Despite this, we know relatively little about the metabolism of their either eventual target, peptidoglycan in Mycobacterium tuberculosis, or how M. tuberculosis uses PG to modulate the host immune system.

M. tuberculosis is the causative agent of the TB and generates one of the most complex cell walls amongst bacteria. The mycobacterial cell wall is comprised of three main layers including peptidoglycan, arabinogalactan and mycolic acids. A complex cell wall creates constraints on growth and division, which must be overcome through careful remodelling of the cell wall during division or the insertion of envelope spanning structures. This process necessarily leads to the production of small molecule metabolites, which are known to be recognised by the immune system.

Peptidoglycan is a combination of long polysaccharides with a repeating disaccharide unit cross-linked through short peptides. This mesh-like structure gives the organism shape and protects it from numerous stresses. During my BBSRC Future Leader Fellowship, I have shown that mycobacteria generate two distinct forms of peptidoglycan fragments during growth as a consequence of a novel peptidoglycan recycling system. Immunostimulatory disaccharide-peptide conjugates activate host NOD receptors, while disaccharides stripped of their stem peptide serve as signals to the bacterium itself driving growth and antibiotic resistance.

Peptidoglycan metabolism can be thought of as a cycle, with synthesis, remodelling, degradation and recycling intimately associated. In this BBSRC David Phillips Fellowship I will genetically and biochemically dissect these pathways using a multi-disciplinary approach. By employing unbiased genetic screens and our body's ability to sense peptidoglycan fragments I will uncover for the first time the genetic and mechanistic basis of peptidoglycan stem-peptide recycling in pathogenic mycobacteria. I will go on to dissect the molecular basis of peptidoglycan biosynthesis, remodelling and degradation so that we might better understand this important and druggable pathway in mycobacteria, with the long-term aim of reducing the burden of antimicrobial resistance.

Technical Summary

Most bacteria wrap themselves in a cell wall which protects them from a wide range of stresses. Textbooks present this wall as a static, un-changing structure. This is completely false. To grow, divide and carry out metabolic operations the cell wall must be a dynamic and plastic structure. This is true of all walled bacteria but represents a unique challenge to Mycobacterium tuberculosis (Mtb) which has a complex, tripartite cell wall. The peptidoglycan (PG) layer of mycobacteria is covalently bound to a large polysaccharide called arabinogalactan. This layer is then esterified by mycolic acids which along with free glycolipids form the outer membrane of Mtb. This means that at each stage of growth and division the bacteria need to manipulate all of these layers by cutting and remodelling. This process generates a diverse array of small molecules including fragments of PG.

I have previously shown that pathogenic mycobacteria, including Mtb recycle these PG fragments and that this process is important for antimicrobial resistance and growth in macrophages. During this David Phillips Fellowship I will elucidate the mechanism by which mycobacteria recycle their stem peptides by harnessing our bodies' PG sentinels, the NOD-receptors. To complete this pathway, I will uncover the molecular basis of the cleavage of a key bond in PG, the D-lactyl-ether of MurNAc. Next, I will determine the molecular basis of PG degradation in Mtb, which will allow us to identify the fragments produced by the bacterium that feed PG-fragment recycling. Finally, to complete the cycle I will establish the biosynthetic and inhibitory potential of the penicillin binding protein complement from Mtb. Together, these data will provide an unprecedented window into the details of PG biosynthesis, remodelling, degradation and recycling in this global pathogen. This will support future drug discovery efforts and help us understand how these bacteria modulate or evade sensing by the immune system.

Planned Impact

Tuberculosis (TB) is a major driver of poverty, afflicting young people disproportionately in their most productive years. This burden is increasing with the rise of antimicrobial resistance, including multi- and extensively-drug resistant strains of Mycobacterium tuberculosis. Any efforts to alleviate this disease could have long term health and economic impacts, especially in developing nations. Peptidoglycan (PG) metabolism in mycobacteria is an important field of research that is gaining in profile. By developing a molecular understanding of this crucial pathway, we will be able to inform future drug discovery efforts. As such, the single greatest impact of this work would be felt with the development of a novel antimycobacterial drug. A key consideration here is that the timeline of bringing a drug to market from an initial hit is on the scale of 10-20 years. Despite this lag, one of the substantial benefits of this Fellowship is that I propose to characterise a system for which drug scaffolds already exist. This can accelerate the use of beta-lactam antibiotics in mycobacterial disease treatment which could have a much more immediate impact.

Another aspect of this project is in non-tuberculosis mycobacterial diseases. For example, Mycobacterium avium subspecies paratuberculosis (MAP) is a major health threat in the farming industry causing an estimated £13 million in losses each year. It is possible that treatments that are less effective at treating TB, or have poor side-effects in humans will be useful in farming. Intriguingly, MAP is also associated with human Crohn's disease in a manner that is thought to be NOD-dependent. Insights into PG fragment recycling in TB may be applicable in this disease as well, which could have substantial impacts in human healthcare.

The second group for which a significant impact would be felt is the UK research community. This will be felt in two ways aside from those listed in the academic beneficiaries statement. First by awarding me this David Phillips Fellowship the BBSRC will be further investing in a centre of research excellence at the University of Birmingham. This investment will help to enhance Birmingham's research-driven teaching programmes which will develop the next generation of UK bioscience innovators. Secondly, within the immediate timescale of this Fellowship I will be involved in the training of a research technician and a PhD student (provided by the School of Biosciences) who will gain or enhance their expertise in a variety of techniques which will position them to contribute in meaningful ways to the growing bioeconomy regardless of their career aspirations.

The final group of beneficiaries is the public. I am currently engaged with several outreach activities including the Access 2 Birmingham program, the IMI Summer School program, and University Open Days. In these capacities I have sought to share my research in an accessible way so that people have an understanding of how we are using their money to tackle complex and important problems. As another means of public engagement, I am active on social media in promoting science and good science practises as well as my own research outputs.

This David Phillips Fellowship will deliver excellent short-term impacts and has the potential to make exceptional long-term impacts on human health by addressing the BBSRC priority area of antimicrobial resistance. Through this David Phillips Fellowship the BBSRC will be able to improve our fundamental understanding of an important human pathogen, combat antimicrobial resistance and contribute to the bioeconomy.

Publications

10 25 50
 
Description While this award has only been active for one year, we have already made a couple of important advances. First, during the initial phase of this award we finished the work of my previous Fellowship which enabled its publication. This is described in more detail in the "Nothing wasted: Peptidoglycan recycling in mycobacteria" award. Briefly, we found that mycobacteria recycle their peptidoglycan and that a hydrolase, LpqI, makes this possible. The second major finding so far is the discovery of a group of enzymes which collectively degrade mycobacterial arabinogalactan. We have not yet published this work, but it is a major finding and will dramatically enhance the field of mycobacterial cell wall biology.
Exploitation Route The recycling work has been previously described, however it will lead to a better understanding of how the organism interfaces with the host and from there will enable the development of host-directed therapeutics. The novel cell wall enzymes will be used first by the research community as tools, especially given that similar proteins have never been described. Secondly, we are leveraging their activity for potential translational impacts such as developing a reagent to efficiently lyse mycobacteria for sequencing efforts, something which is not currently possible without significant equipment investment.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Exploration of the immunological impact of peptidoglycan recycling in mycobacteria 
Organisation Institute of Molecular and Cell Biology
Country Portugal 
Sector Academic/University 
PI Contribution During the course of my research I have generated several new strains of mycobacteria lacking specific cell wall metabolism genes. I have provided these strains to the group of Margarida Saraiva at IBMC so that they can test them for altered immunological properties.
Collaborator Contribution The group of Margarida Saraiva at IBMC will be evaluating the immunological impact of strains provided by me in the context of macrophage infection.
Impact The group at IBMC have identified significant changes in the survival (more than a log increase in growth) of PG- recycling deficient M. bovis BCG mutants within bone marrow-derived macrophages. They are now following up with additional studies to try and pinpoint the cause and effect of this change.
Start Year 2016
 
Description Identification of enzymes involved in the degradation of mycobacterial cell walls by human gut bacteria. 
Organisation Newcastle University
Department Institute for Cell and Molecular Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution In this project I have been providing substrates for the growth of gut bacteria and study of specific enzymes related to the degradation of mycobacterial cell walls. I have also been providing expertise on mycobacterial cell wall components and study design for enzyme characterisation. In later stages of the collaboration I will be characterising some enzymes using difficult to obtain and ship substrates.
Collaborator Contribution My collaborators at Newcastle (lead by Dr. Elisabeth Lowe) have been testing gut bacteria for their ability to degrade polymers derived from the mycobacterial cell wall. This work identified a few bacteria, and subsequently specific enzymes required for this function.
Impact This has only just started. The collaboration involves biochemistry, structural biology and microbiology.
Start Year 2017
 
Description Investigation of dormancy responses by mutant strains of mycobacteria 
Organisation University of Leicester
Department Department of Physics & Astronomy
Country United Kingdom 
Sector Academic/University 
PI Contribution We are generating mutant strains of mycobacteria to be tested by the group of Galina Mukmalova in Leicester for their capacity to resuscitate from dormancy.
Collaborator Contribution The group of Galina Mukmalova in Leicester will be taking our strains into assays they are proficient in to determine their ability to resuscitate from dormancy.
Impact It is still too early for outcomes/outputs to be generated.
Start Year 2016
 
Description Systems biology of mycobacteria 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Dr. Manuel Banzhaf and I are currently engaged on a project involving a chemical genomics screen of mycobacteria. He has obtained some funding (which I helped to write the applications for) to pursue this project. Briefly, we will be taking our ordered, single-gene-deletion library of M. bovis BCG and subjecting it to thousands of perturbations so that we might identify functions for the large proportion of unknown function genes in the mycobacterial genome.
Collaborator Contribution Dr. Banzhaf is an expert in chemical genomics, and I am an expert in mycobacteriology. We are combining our expertise in this project as such. We have a co-supervised PhD student working on this project (MIBTP - BBSRC DTP).
Impact It is too early to have outputs, this project will take some time to develop and ideally more funding after we have generated some preliminary data.
Start Year 2019