Newton001: Identification of host and pathogen glucose metabolism modulation during Mycobacterium leprae infection of human macrophages and Schwann ce
Lead Research Organisation:
University of Surrey
Department Name: Microbial & Cellular Sciences
Abstract
According to World Health Organization, the number of leprosy new cases in 2012 stood at 189,018 around the world. Although not fatal, leprosy is responsible for more than 12 thousands cases of irreversible motor disability and deformity per year in the world. The disease is caused by the Mycobacterium leprae, an obligatory intracellular pathogen that preferentially infects skin macrophages and Schwann cells causing nerve damage and scarring, which results in the disfigurement and the classic hand and foot deformities of leprosy. Brazil has 33,303 cases/year of leprosy, being the most affected country in Latin America, representing 14 percent of the global number of cases and is surpassed only by India, which has approximately 134,752 new cases per year.
Intracellular bacteria must obtain nutrients from their host cell and grow by either adapting their own metabolic pathways or manipulating the metabolism of the host. Understanding how they obtain nutrients from their host and how they utilize them will lead to better understanding the infection and may inspire novel ways to grow the bacteria for vaccine or drug studies; which is particularly relevant to the leprosy bacillus which cannot be grown in pure culture in the laboratory.
In this project we will use a newly-developed developed technique called 13C flux spectral analysis to investigate the metabolism of M. leprae in its host cell. The M. leprae cells will be grown in mouse footpads, and then incubated in media containing a mixture of 12C and 13C-labelled glucose and metabolites extracted for analysis. We will use another technique called mass spectrometry to determine the distribution of 12C and 13C carbon in these metabolites and computer analysis of the data will reveal clues to both the nutrients used by M. leprae and how it utilises them. The work will be published in a peer-reviewed journal and may lead to novel approaches to growing the leprosy bacillus and developing new drugs.
Intracellular bacteria must obtain nutrients from their host cell and grow by either adapting their own metabolic pathways or manipulating the metabolism of the host. Understanding how they obtain nutrients from their host and how they utilize them will lead to better understanding the infection and may inspire novel ways to grow the bacteria for vaccine or drug studies; which is particularly relevant to the leprosy bacillus which cannot be grown in pure culture in the laboratory.
In this project we will use a newly-developed developed technique called 13C flux spectral analysis to investigate the metabolism of M. leprae in its host cell. The M. leprae cells will be grown in mouse footpads, and then incubated in media containing a mixture of 12C and 13C-labelled glucose and metabolites extracted for analysis. We will use another technique called mass spectrometry to determine the distribution of 12C and 13C carbon in these metabolites and computer analysis of the data will reveal clues to both the nutrients used by M. leprae and how it utilises them. The work will be published in a peer-reviewed journal and may lead to novel approaches to growing the leprosy bacillus and developing new drugs.
Technical Summary
Live M. leprae will be prepared from the footpads of athymic nu/nu mice after six-nine months of infection and homenized. THP-1 human monocytes and ST8814 human Schwann cell lines will be kept at 37C or 33C (when infected) within a humidified 5% CO2 atmosphere and differentiated. Cultures will be infected with a MOI of 50:1 for 48hs. After the infection, cells will be washed with ice-cold PBS before being lysed with 0.1% Triton X-100. The supernatant containing the intracellular M. leprae and the soluble host material will be centrifuged at 4,000 x g for 20 min at 40C to pellet the bacteria, metabolites extracted and sent for mass spectrometry analysis.
An isotopomer network model of the central metabolism of M. leprae will be constructed, and carbon exchange between the macrophage or Schwann cell environment and M. leprae will be modelled. A biomass formula will be generated to constrain metabolic flux rates. To reveal potential carbon sources (as isotopomers) for the intracellular mycobacterial cell, a multistage sampling-based C13-MFA-type approach will be implemented. The measured labelling patterns of macrophage and Schwann cells amino acids will be deconvoluted by generating random isotopomer labeling patterns for each potential substrate imitating the measured isotopologues and used to constrain the fluxes in each reaction of the model. We will therefore determine likely substrates that the leprosy bacillus obtains from its host tcell and the metabolic pathways utilized.
An isotopomer network model of the central metabolism of M. leprae will be constructed, and carbon exchange between the macrophage or Schwann cell environment and M. leprae will be modelled. A biomass formula will be generated to constrain metabolic flux rates. To reveal potential carbon sources (as isotopomers) for the intracellular mycobacterial cell, a multistage sampling-based C13-MFA-type approach will be implemented. The measured labelling patterns of macrophage and Schwann cells amino acids will be deconvoluted by generating random isotopomer labeling patterns for each potential substrate imitating the measured isotopologues and used to constrain the fluxes in each reaction of the model. We will therefore determine likely substrates that the leprosy bacillus obtains from its host tcell and the metabolic pathways utilized.
Planned Impact
N/A
Publications
Borah K
(2020)
GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae.
in PLoS neglected tropical diseases
| Description | Leprosy is an ancient infectious disease that remains a major cause of chronic morbidity in vulnerable populations in developing countries, despite the availability of effective, though very lengthy, treatment. Victims continue to be stigmatized such that the disease has recently been described as "the world's oldest human-rights issue". New approaches are needed to control the disease, such as effective vaccines and shorter treatment regimens. However, research into leprosy, is severely hampered by our inability, for unknown reasons, to grow the causative agent, Mycobacterium leprae, in the laboratory. For example, we do not even know what the leprosy bacillus eats when it grows inside its host. In this study, we, for the first time, directly investigated the metabolism of the leprosy bacillus whilst it is growing inside its host cells. The project was in collaboration with colleagues working at the Fiocruz in Rio de Janeiro in Brazil. Our Brazilian colleagues first grew human cells infected with the leprosy bacillus in glucose labelled with a heavy isotope of carbon. They then extracted metabolites and sent the material to our laboratory to track where the label had gone in both the host cell and the leprosy bacillus growing inside the host cell. Similar experiments that we performed with the related pathogen, Mycobacterium tuberculosis, established that this pathogen does not consume glucose from its host cell, but instead eats fatty acids and amino acids made from that glucose. However, our experiments demonstrated that, in contrast to the TB bacillus, the leprosy bacillus eats host glucose. Our analysis highlighted particular areas of metabolism involved in consumption of glucose that could be targeted with new anti-leprosy drugs. The results also provide new insights into the problem of growing the leprosy bacillus in the laboratory and highlights new approaches that we will investigate in attempts to grow the leprosy bacillus to make a vaccine. |
| Exploitation Route | The findings have been published in a leading international journal and are available on open access. The data may be used to identify new anti-leprosy drug targets or stimulate new ways to grow the leprosy bacillus. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Systems-based screen of compounds that target nitrogen metabolism of Mycobacterium tuberculosis. |
| Amount | £1,072,000 (GBP) |
| Funding ID | BB/V010611/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 04/2021 |
| End | 03/2024 |
| Description | Fiocruz, Brazil |
| Organisation | Oswaldo Cruz Foundation (Fiocruz) |
| Country | Brazil |
| Sector | Public |
| PI Contribution | Our partner in Brazil has prepared leprosy bacillus homogenates which we have analyzed by mass spectrometry to determine metabolic fluxes in the pathogen. |
| Collaborator Contribution | Our partner grew the leprosy bacillus in the athymic nu/nu mouse model. They are one of the few laboratories in the world that have this model available. They purified the leprosy bacilli and lyzed the cells and, after confiming microbiological safety, they sent the cell extracts for analysis in our laboratory. |
| Impact | multidiciplinary involving: microbiology systems biology |
| Start Year | 2015 |