Decrypting Mycobacterium cytochrome P450 (CYP) physiological functions by testing hypotheses emitted form large-scale comparative genomics analysis

Project Information

deCrYPtion

Grant agreement ID: 839116

DOI

10.3030/839116

Project closed

EC signature date 27 March 2019

Start date 1 October 2019

End date 30 March 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

SWANSEA UNIVERSITY
United Kingdom

Periodic Reporting for period 1 - deCrYPtion (Decrypting Mycobacterium cytochrome P450 (CYP) physiological functions by testing hypotheses emitted form large-scale comparative genomics analysis)

Reporting period: 2019-10-01 to 2021-09-30

Summary of the context and overall objectives of the project

The project number titled: “Decrypting Mycobacterium cytochrome P450 (CYP) physiological functions by testing hypotheses emitted from large-scale comparative genomics analysis” was proposed and carried out by Dr Rémi Zallot, under the supervision of Prof. Steven L. Kelly. The work described in the deCrYPtion action took place at the Institute of Life Science, within Swansea University Medical School (SUMS), in Wales, United Kingdom.

More than 190 species belong to the Mycobacterium genus. Among those, several are pathogenic to human and animals. The high number of human lives lost and the economic consequences of livestock infection are important incentives in the control and eradication of the responsible organisms. M. tuberculosis, one of the causative agents of tuberculosis (TB), is the most problematic representative: in 2016, 1.7 million deaths worldwide have been attributed to TB. Like for other infectious organisms, antibiotic resistances have appeared in Mycobacterium. Thus, there is a fundamental need to develop new antimycobacterial drugs.

The inhibition of enzymes belonging to the Cytochrome P450 (CYP) protein family has been shown to suppresses the growth of several Mycobacterium species, making CYPs targets for drug development. Unfortunately, most CYPs are considered orphan: proteins for which no physiological function is known. The deCrYPtion action aims to define the physiological function of a selected set of mycobacterial CYPs. It will prove determinant in the development of new antibiotics.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Bacteria from the Mycobacteriaceae family pose a threat that needs to be tackled. While the rapid expansion of genome sequencing with genome and pathway annotations has greatly improved the progress of drug discovery, it has also pointed out how much our understanding of metabolism is limited. Within the gap of knowledge constituted by genes of unknown or imprecise function are some that encode for metabolic enzymes defining uncharted pathways, potential targets for the development of new fighting methods against those pathogens. Among uncharacterized enzymes that represent exciting opportunities are members of the cytochrome P450 (CYP) protein family. This is because azoles, known CYPs specific inhibitors, have been demonstrated to prohibit M. tuberculosis growth. Logically, CYPs, their required redox partners, and the pathways they are involved in, mostly uncharacterized, have been proposed to be drug targets.

The work carried out for the “deCrYPtion” action consisted in performing a large-scale comparative genomics analysis of the members of the Cytochrome P450 (CYP) protein family present in the genomes of Mycobacterium bacterial species, to propose and then experimentally test functions for some selected targets.

Experimental results obtained regarding the function of previously uncharacterized genes are fundamental in nature. They increase the understanding of pathogens that impact human and animal health. The newly obtained understanding can be leveraged to inform intervention development, i.e. newly obtained knowledge serves the development of new drugs.

At a personal level, the results obtained have been and will be leveraged for the development of proposals aiming at obtaining funding to further the research needed in the context of developing new antitubercular drugs. Research work will continue on the main topic of the deCrYPtion proposal, and it is expected it will lead to publication in international journals.

Dissemination activities:

Open Access data
The scientific information obtained for WP1 and 2 has been made available using the Zenodo open repository and can be accessed at https://doi.org/10.5281/zenodo.6583999.

Publication
A manuscript describing the comparative genomics approach and its usefulness has been published.
Rémi Zallot, Nils Oberg, John A Gerlt, Discovery of new enzymatic functions and metabolic pathways using genomic enzymology web tools, Current Opinion in Biotechnology, Volume 69, 2021, Pages 77-90, ISSN 0958-1669, https://doi.org/10.1016/j.copbio.2020.12.004.
(https://www.sciencedirect.com/science/article/pii/S0958166920301907 and https://cronfa.swan.ac.uk/Record/cronfa60085)

Invited presentations
Dr Rémi Zallot was invited to present some of work performed during the deCrYPtion action:
- Understanding antimicrobial resistance requires characterising unknown genes identified involved MIB Fellowship days at the Manchester Institute of Biotechnology, The University of Manchester, January 26, 2022
- Similarity Networks (SSNs) and Genome Neighbourhood Networks (GNNs) for function assignment, School of Chemistry, Cardiff University, April 3, 2021

Conference attended (online)
The Microbiology Society: WHY MICROBIOLOGISTS MATTER?
23 – 26 November 2020

Outreach activities
Demonstration of how genomic DNA can be extracted from fruits, allowing to cover what DNA is and to show the public that DNA is something that can be extracted, seen, and worked on by scientists to gain a better understanding of living organisms. The public mostly consisted of a non-scientific audience. The demonstrations were aimed towards families and children.
List of events I was a demonstrator for
- November 2021: St Tydfil’s Old Parish Church, Merthyr Tydfil, in the context of UK Science Festivals Network, Making Connections project grant to Dr. Claire Price.
- July 2021: Merthyr Tydfil Science Festival
- May 2021: Wales Gene Park Genomics Showcase
- February 2021: Cardiff Science Festival
- October 2020: Swansea Science Festival

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

A set of experiments was performed to explore the involvement of a specific previously uncharacterised Cytochrome P450 in an area of metabolism that could be targeted for the development on new anti-tubercular drugs.

The enzyme focused on can be specifically inhibited with azoles. It is active on a metabolite originating from the host, presumably available during infection by Mycobacterium species.
A crystal structure of the purified enzyme, with an inhibitor in the active site was obtained. L

This series of experiments validate the potential of this particular as a target in the future development of anti-tubercular drugs.
In vivo validation of the proposed hypothesis has been initiated. At this time, this experiment has not been yet completed, and no conclusion can be made.

SSN for Cytochrome P450 enzymes present in Mycobacterium species