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PTMyco: from mycoloylation of bacterial envelope proteins to the possible genesis of new antibiotics

Research Article published on 01 December 2023 , Updated on 16 January 2024

The aim of the PTMyco project is to better understand the role of mycoloylation in bacterial envelope proteins, a chemical modification of these molecules after their synthesis, consisting of the addition of a specific fatty acid, mycolic acid. Led by scientists from the Institute for Integrative Cell Biology (I2BC – Univ Paris-Saclay, French Alternative Energies and Atomic Energy Commission CEA, National Centre for Scientific Research CNRS), the Institute of Molecular Chemistry and Materials of Orsay (ICMMO – Univ. Paris-Saclay, CNRS) and the Institute for Structural and Functional Glycobiology (UGSF – Univ. de Lille, CNRS), this project will eventually lead to the identification of new antibiotics active against bacteria related to Mycobacterium tuberculosis, responsible for human tuberculosis.

Some infectious diseases, thought to have been eradicated, are making a comeback, while others have never disappeared. Such is the case for tuberculosis, contracted by over ten million people worldwide in 2021, or leprosy, with 216,000 sufferers in 2022, mainly in Brazil and India. Closer to home, 92 cases of diphtheria were reported across seven European countries at the end of 2022, including 30 in mainland France. The pathogens that cause these illnesses are bacteria belonging to the order Corynebacteriales. They are also responsible for opportunistic infections affecting, for example, the lungs (Mycobacterium abscessus) or bone marrow (Corynebacterium jeikeium).

Bacteria are single-celled microorganisms without a nucleus whose genome takes the form of a single circular chromosomal DNA "bathing" in the cell's cytoplasm. To combat these pathogens, medicine uses antibiotics that either limit their growth or kill them. They act by preventing certain chemical reactions essential to the bacterium's metabolism from taking place, by interfering with the translation of genetic material into proteins, or by blocking the formation of the bacterium's cell envelope. Today, with the emergence of resistant bacterial strains, there are fewer antibiotics available on the market that are still effective against Corynebacteriales, and their use is limited.

The PTMyco project, launched at the end of 2022, brings together scientists from the Institute for Integrative Cell Biology (I2BC – Univ Paris-Saclay, CEA, CNRS), specialists in bacteria related to tuberculosis, the Institute of Molecular Chemistry and Materials of Orsay (ICMMO – Univ. Paris-Saclay, CNRS), experts in complex sugars, and the Institute for Structural and Functional Glycobiology (UGSF – Univ. de Lille, CNRS), leaders in the chemical biology (chemo-biology) of lipids. It aims to shed light on the molecular mechanisms at work in the biogenesis of bacterial envelopes. By targeting this complex structure, the project aims to eventually identify new antibiotics active against pathogenic bacteria, particularly those responsible for tuberculosis.


The importance of bacterial envelopes

It all started in 2010, when a team from Toulouse, in collaboration with Nicolas Bayan's team at I2BC, discovered a new type of post-translational modification of proteins present in the envelope of the model organism Corynebacterium glutamicum. Derived from the translation of messenger RNAs, certain proteins undergo chemical modifications after their synthesis by the ribosome. These post-translational modifications (PTMs) influence the final three-dimensional structure of proteins or their localisation, in other words their function. "The addition of a particular chemical group to a protein, usually with the help of an enzyme, can modify its function or its original purpose. It will, for example, become more active or be anchored in a specific site of the cell to enable it to perform its function effectively," says Nicolas Bayan.

There are two main types of PTM in Corynebacteriales: by sugars or by fatty acids. The former stabilise the protein, while the latter, with their hydrophobic carbon chain, anchor it in the cell membrane. In their work, the two scientific teams have demonstrated that certain Corynebacteriales proteins can be modified by a specific fatty acid: mycolic acid, found in the envelope of bacteria related to M. tuberculosis. This is the first known example of PTM by the addition of a mycolic acid: mycoloylation. Following their discovery, the teams began studying atypical post-translational modification using biochemical and genetic approaches, and more recently a chemo-biological approach developed at ICMMO.


Questions galore and the launch of the PTMyco project

Bacteria are classified according to various criteria, such as their morphology, respiratory requirements and mobility. Their Gram stain result is another criteria, and comes from the structure of their cell envelope. "Gram staining is used to distinguish between bacteria capable of fixing gentian violet (Gram-positive) and those that are not (Gram-negative). The cell envelope of Gram-positive bacteria is characterised by a single membrane and a thick wall, and that of the Gram-negative bacteria by a double membrane," explains Nicolas Bayan. The C. glutamicum bacterium is known to be Gram-positive. Although surprisingly, C. glutamicum has two membranes, one of which is made up of atypical lipids derived from mycolic acids. This is a unique case in the living world as it is limited to bacteria related to M. tuberculosis! Numerous questions arise in the face of this analysis. "How did such a mycoloylation system come about in the course of evolution? What advantages does it bring for the bacteria? Does this system affect protein sorting between membranes?" asks Nicolas Bayan. And what about the potential impact of mycoloylation on pathogen virulence in human health?

In 2014, a chance meeting between members of I2BC and ICMMO led to a new collaboration, later joined by Lille's chemical centre, UGSF. The scientists involved applied to a call for projects from the French National Research Agency (ANR) to fund their PTMyco project, designed to provide answers to their questions. At the same time, Nicolas Bayan and his team identified and crystallised, in its intermediate state, the enzyme responsible for mycoloylation in C. glutamicum. In December 2022, the PTMyco project received funding of €385,000 from the ANR, spread over 36 months. The amount is divided equally, with each team getting one third. "This money is used primarily for running the laboratory, purchasing consumables and chemical products, and maintaining small pieces of equipment. We are currently writing a post-doctoral proposal, which should be filled in January 2024," explains Nicolas Bayan.


A widespread cellular process and interactions yet to be defined

Launched almost a year ago, PTMyco is producing its first results. "To find how common mycoloylation was in C. glutanicum, we sought to estimate the number of proteins modified by the addition of a mycolic acid. Thanks to ICMMO's production of a rather unusual synthetic acid and mass spectrometry, we were able to identify all the cellular proteins that had potentially incorporated this acid," explains Nicolas Bayan. At the end of the study, around fifty proteins were found to be candidates, suggesting a process that is widely used by cells. To confirm this lead, the scientists are now tackling the cloning of the identified genes and purification of the corresponding proteins. They also have to check that each protein is individually modified by a mycolic acid.

Next, Nicolas Bayan and his colleagues plan to study the role of PTM. "To do this, we'll work in vivo and analyse the impact of the absence of mycoloylation on the function of certain proteins. These analyses will be carried out in Lille, at UGSF, and will take same time. We're excited to see the results. On our side, we'll be welcoming a new recruit by the beginning of 2024 to attempt to purify and crystallise the mycoloylation enzyme in interaction with its protein substrate," announces the researcher. This work promises a detailed observation of the key interactions involved in the process, and will potentially lead to finding molecules capable of interfering with them. Indeed, blocking mycoloylation could prevent the biogenesis of the bacterial envelope, the Achilles heel of these microorganisms, and provide a new way of fighting these pathogens.


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