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A biomimetic electrostatic assistance for guiding and promoting N-terminal protein chemical modification

The modification of protein electrostatics by phosphorylation is a mechanism used by cells to promote the association of proteins with other biomolecules. In this work, we show that introducing negatively charged phosphoserines in a reactant is a powerful means for directing and accelerating the chemical modification of proteins equipped with oppositely charged arginines. While the extra charged amino acid residues induce no detectable affinity between the reactants, they bring site-selectivity to a reaction that is otherwise devoid of such a property. They also enable rate accelerations of four orders of magnitude in some cases, thereby permitting chemical processes to proceed at the protein level in the low micromolar range, using reactions that are normally too slow to be useful in such dilute conditions.

 

A biomimetic electrostatic assistance for guiding and promoting N-terminal protein chemical modification
Ollivier N, Sénéchal M, Desmet R, Snella B, Agouridas V, Melnyk O.
Nat Commun. 2022 Nov 5;13(1):6667


Evolutionary history of extensive antibiotic resistance in Mycobacterium tuberculosis

Phylogenetic tree based on the sequencing of the genomes of 720 W148 strains and 12 related external strains. The different colored circles show, from the inside out, the presence (full box) or absence (empty box) of mutations conferring resistance to different drugs (INH isoniazid, SM streptomycin, RIF rifampicin, EMB ethambutol, PZA pyrazinamide, FQ fluoroquinolones, KM kanamycin, ETH ethionamide and PAS para-aminosalicylic acid), the associated antibiotic resistance profiles (XDR extensive resistance and pre-XDR pre- extensive resistance), and the presence of compensatory mutations. Groups of epidemic variants particularly localized in Belarus and Estonia are indicated.


At the origin of 1.5 million deaths each year, tuberculosis is a leading cause of death due to an infectious disease, only surpassed by COVID in 2020-2021. A study based on genomic sequencing has determined the bacterial factors and certain historical events involved in the emergence and international spread of multidrug-resistant strains. These results, published in Nature Communications, highlight the need to deploy new means of detecting extended resistance.

Multi-/extensively drug resistant (MDR/XDR) tuberculosis, mostly driven by human-to-human transmission, is the leading cause of human death due to antimicrobial resistance. Particular branches of a lineage of Mycobacterium tuberculosis strains are responsible for the high prevalence of multidrug resistance in Eurasia, and show exceptional transnational distributions. Dr Philip Supply’s research group at the CIIL, together with other French and German scientists, have determined the factors underlying the emergence and epidemic spread of the branch called W148 by analyzing the genomes of more than 700 strains from 23 countries. The results date a common ancestor already resistant to isoniazid and streptomycin to the early 1960s, consistent with the introduction of these antibiotics at that time. Successive epidemic expansions followed in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet era. These strains evolved to (pre-)XDR levels in less than two decades, accumulating resistance mutations to up to 11 drugs. Transmission of the most resistant strains is associated with the acquisition of mutations compensating the metabolic cost due to resistance mutations. All W148 strains also possess a hypervirulence mutation, and some of them have acquired additional mutations that may further promote the development of additional resistance.

This exceptional genetic arsenal and the wide geographical distribution of these strains constitute a "perfect storm", jeopardizing the success of the introduction of new treatments for MDR tuberculosis. The deployment on a larger scale of new tests with an extended diagnostic spectrum, such as the Deeplex Myc-TB test recently developed by GenoScreen with the assistance of Dr Philip Supply, will be essential to combat the spread of such highly resistant strains.

Reference:

Transcontinental spread and evolution of Mycobacterium tuberculosis W148 European/Russian clade toward extensively drug resistant tuberculosis.

Merker M, Rasigade JP, Barbier M, Cox H, Feuerriegel S, Kohl TA, Shitikov E, Klaos K, Gaudin C, Antoine R, Diel R, Borrell S, Gagneux S, Nikolayevskyy V, Andres S, Crudu V, Supply P, Niemann S, Wirth T.

Nat Commun. 2022 13:5105. doi: 10.1038/s41467-022-32455-1


An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell

Hepatitis E virus (HEV) is the major cause of acute viral hepatitis worldwide. Although infection with HEV is usually self-resolving, it can cause up to 30% mortality in pregnant women and leads to a chronic infection in immunocompromised patients. There is no specific treatment nor universal vaccine to fight against this virus.

Few years ago, we developed an efficient cell culture system for HEV which allows for studying the molecular and cellular mechanisms of the HEV lifecycle. This model notably enabled the pioneering demonstration that, during its lifecycle, HEV produces at least 3 forms of its ORF2 capsid protein: infectious ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). These ORF2 forms perform distinct functions in the HEV lifecycle. The ORF2i protein is the structural component of infectious particles whereas the ORF2g/c proteins are not associated with infectious material but likely act as a humoral immune decoy that inhibits antibody-mediated neutralization. The ORF2g/c proteins are secreted in large amounts in culture supernatant and are the most abundant antigens detected in patient sera.

In the study of Hervouet, Ferrié, Ankavay et al., we deciphered the molecular determinants of ORF2 multifunctionality. We identified an arginine-rich motif (ARM) located in the ORF2 N-terminal region that controls the subcellular localization, the fate, the maturation and functions of ORF2 forms. It also promotes ORF2-host cell interactions. Our observations highlighted the ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle. Thus, our study enabled the pioneering demonstration that HEV developed a master strategy to condense multiple information into only five amino acid residues of its capsid protein. This strategy is likely also exploited by other pathogens.

 

Hervouet, K.*, Ferrié, M.*, Ankavay, M.*, et al. PLoS Pathog. 2022 Aug 25;18(8):e1010798. doi: 10.1371/journal.ppat.1010798.


Investigating drug resistance of Mycobacterium leprae in the Comoros: a deep-sequencing study

Although considered a scourge of the past, leprosy is an infectious disease that still affects several hundred thousand people each year. The emergence of resistance to antibiotic treatments is worrying. However, this resistance cannot be detected by conventional diagnostic methods, given the impossibility of growing the bacterium in vitro. In order to overcome this stalemate, a novel molecular test, called Deeplex Myc-Lep, has been developed by Genoscreen, a biotech located on the campus of the Institut Pasteur de Lille, with Dr Philip Supply, Director of CNRS Research, at the Infection and Immunity Center of Lille. This test makes it possible to deep sequence all the known genetic targets associated with the antibiotic resistance of the bacillus, directly from DNA extracted from skin biopsies of patients. In collaboration with the Institute of Tropical Medicine in Antwerp and the Damien Foundation, the use of this test has enabled us to show the absence of emergence of resistance to curative or preventive treatments in patients suffering from leprosy in the Comoros, a country heavily affected by the disease. These results provide encouraging information on the effectiveness of the control program currently deployed in the country, and open up the prospect of large-scale use of this new molecular approach for diagnosis and monitoring. These results were published in the prestigious journal Lancet Microbe.

Reference:

Braet, SM, Jouet, A, Aubry, A, Van Dyck-Lippens, M,  Lenoir, E, Assoumani, Y, Baco, A, Mzembaba, A, Cambau, E, Gonçalves Vasconcellos, SE, Rigouts, L, Suffys, PN, Hasker, E, Supply, P, de Jong, BC. Investigating drug resistance of Mycobacterium leprae in the Comoros: a deep-sequencing study. Lancet Microbe, 2022




Clofoctol as a potential treatment against COVID-19

Repurposed drugs may have a speedier path to clinical use because they have already been shown to be safe in people. A study publishing May 19th in the open access journal PLOS Pathogens by members of the CIIL suggests clofoctol may be an effective treatment for SARS-CoV-2 infections as shown in a preclinical model. While COVID-19 vaccines reduce hospitalizations and death, they do not control virus transmission, and affordable, effective therapies are needed. Previous attempts to repurpose medicines to treat COVID-19 patients have been unsuccessful. In order to identify potential antiviral therapies effective against COVID-19, the authors accessed the Apteeus drug library, a collection of 1,942 approved drugs to identify molecules that exhibit antiviral activity against SARS-CoV-2. The authors selected clofoctol based on its antiviral potency and tested their hypothesis by testing its effects in SARS-CoV-2-infected mice. The researchers found that human ACE2 transgenic mice treated with clofoctol had a decreased viral load, reduced inflammatory gene expression, and lowered pulmonary pathology. The antiviral and anti-inflammatory properties of clofoctol, associated with its safety profile and unique pharmacokinetics make a strong case for proposing clofoctol as an affordable therapeutic candidate for the treatment of COVID-19 patients. Finally, the relatively low cost of this drug suggests that it is a potential clinical option for treatment of COVID-19 patients in resource-poor settings.

Large scale screening discovers clofoctol as an inhibitor of SARS-CoV-2 replication that reduces COVID-19-like pathology.

Belouzard et al. (2022). PLoS Pathogens 18(5):e1010498


The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis

Alain Baulard, Senior Research Scientist at Inserm, and researchers from Institut Pasteur de Lille, University of Lille, and a large public-private international consortium, published this week in Science Translational Medicine their progress in developing a highly innovative drug candidate that reverses ethionamide-resistance in the deadly human pathogen Mycobacterium tuberculosis. 

French researchers at Institut Pasteur de Lille and University of Lille and their colleagues of a large public-private consortium including BioVersys and GSK are publishing today in Science Translational Medicine their progress in the development of a highly innovative drug-candidate that reverses ethionamide resistance in the deadly pathogen M. tuberculosis.

Tuberculosis (TB) has been a scourge of humankind for centuries and remains today a major infectious disease killer. Worldwide antibiotic campaigns have been essential to tackle the TB pandemic, but recently there has been a worrisome progression of antibiotic resistance that is now undermining these public health strategies.

Since the discovery of antibiotics, a continuous fight opposes humans and bacterial pathogens, the latter trying to escape the more and more sophisticated drugs developed by the former. At the origin of the fight described today in Science Translational Medicine by M. Flipo and colleagues, is ethionamide, which belongs to a peculiar family of anti-tuberculosis drugs called pro-antibiotics. Following their penetration into the bacteria, pro-antibiotics such as ethionamide are bioactivated by specific bacterial enzymes to produce derivatives that kill the bug.

In their permanent struggle to overcome the effects of antibiotics, some M. tuberculosis clinical strains are able to shut down the biochemical pathway responsible for ethionamide bioactivation.

The game could end here, but it is not the case. A first step forward in this fight was achieved by the team thanks to the identification of a small molecule called SMARt420 that up-regulates an alternative enzyme able to activate ethionamide in resistant bacteria, switching them back to being ethionamide sensitive.

In today's story, the authors describe a new version of their anti-TB weapon, with the discovery of the small molecule SMARt751 able to engage an additional, more efficient, ethionamide activation pathway.

Not only does SMARt751 overcome resistant strains, but it also drastically increases ethionamide potency on sensitive strains. SMARt751 is safe, and active in vivo against chronic TB in infected mice. Extrapolation of the results from mice to humans predicts that SMARt751 would be sufficient to lower the efficacious dose of ethionamide in humans by four-fold, which would drastically improve ethionamide’ tolerability.

Is the game over? Certainly not, as M. tuberculosis will, as always, fight back with alternative resistance mechanisms, but SMARt751 and its derivatives that are now in clinical development with our partners BioVersys and GSK are powerful weapons that can make the pathogen’s life significantly more difficult, and potentially offer patients a more efficacious and safer treatment option.

To learn more : HERE

 


Investigating the micropumps behind antibiotic resistance

Juan-Carlos Jiménez Castellanos is a researcher at the Pasteur Institute in Lille, France. Credit: Lucas Barioulet for Nature

Antibiotic-resistant bacteria, especially those that are resistant to multiple drugs, pose a major threat to health globally; tackling them is a crucial research priority of the World Health Organization. I am a protein chemist and molecular biologist investigating why bacteria often refuse to die when attacked by antibiotics.

Bacteria can evade antibiotics in clever ways. One involves ejecting them through efflux pumps embedded in the bacterium’s inner membrane. These are like tiny vacuum cleaners that constantly flush out substances that are toxic to bacteria.

I’ve been fascinated by efflux pumps ever since my undergraduate studies in Mexico more than 16 years ago. I’m particularly interested in how they can bind to and extrude dozens of chemically unrelated compounds quickly, efficiently and simultaneously. I’m working on the hypothesis that they do this using a unique group of proteins that lack stable structures — known as intrinsically disordered proteins.

Here at the Pasteur Institute in Lille, France, I’m using advanced screening technology to test the ability of different molecules to inhibit the actions of efflux pumps. I’m helped by an automated circuit, with a range of experimental equipment, and a robot that can load up to 200 assay plates into a carousel. I can program in what I want and go for a coffee while the robot gets to work.

I find it amazing that the proteins in all life forms consist of different combinations of the same 20 amino acids. I can’t really describe why, but solving problems about proteins makes me so happy.

With colleagues, I’ve also been working to develop new efflux pump inhibitors (C. Plé et al. Nature Commun. 13, 115; 2022). My hope is that these can be used both to advance our understanding of how efflux pumps work and, with existing antibiotics, to counter antibiotic and multidrug resistance.

Nature 603, 756 (2022)


Future Investments" program Call for PPR projects "Antibiotic resistance: understand, innovate, act PPR-AMR-Edition 2021

MustartMultiparametric Strategies against Antibiotic Resistance in Tuberculosis

The reduction in the global incidence of tuberculosis (TB) observed each year is too small to compensate for the worrying increase in multidrug resistance to anti-tuberculosis drugs (MDR-TB). While Europe has the lowest incidence of TB in the world, it is also where the rate of MDR-TB is the highest. The worrisome dispersion of MDR strains in a growing population of latent healthy carriers, in which resistance cannot be detected to date, constitutes a major obstacle to WHO's eradication objectives.

In comparison to other bacterial infections, tuberculosis therapy is very long, going from months for completion, to even years in the case of MDR-TB. The length of this treatment reveals that the efficiency of current therapy is certainly not optimal, which can lead to lower compliance and the gradual selection of MDR or XDR tuberculosis.

A major reason why treating TB takes so long is probably because, in the body, M. tuberculosis resides in different (stochastic) physiological and metabolic forms, some of which are tolerant to many of the antibiotics in use. In this context, the recent burden of MDR-TB adds to the urgent need for combinations of novel antibiotic classes that target the mycobacteria in its various niches.

An additional drawback to tuberculosis therapy, and indeed to the efficient development of novel tuberculosis therapies, is that it is not possible to routinely define treatment efficacy in real time, neither are there tools to monitor the early emergence of subpopulations of drug resistance.

Nine French research teams are joining their expertise to tackle these questions through concerted and integrated research actions, from the laboratory to the patient's bedside. This consortium brings together seven powerful research teams specialized in TB (including two in Hospitals) as well as two partners from other fields providing new and complementary methodological approaches, making possible this ambitious and structuring project.

The consortium will share 9 innovative experimental compounds that have been identified for their ability to inhibit M. tuberculosis in at least one of its physiological niches, including by stimulating the response of the host to the infection. To complete this first objective, partners will share innovative screening models to enrich this list of potentially synergistic compounds. Combinations will be evaluated using ex vivo and in vivo models of infection in order to identify optimized multifaceted regimens.

This consortium has skills in microbiology, cell biology, immunology, biophysics, therapeutic chemistry, mathematics, biomedical resources, and most of all, the common will to carry out these objectives.

Scientific coordinator

Alain Baulard, DR-Inserm

CIIL - Center for Infection and Immunity of Lille

Experts involved in the CIIL

Priscille Brodin

Ruben Hartkoorn

Camille Locht

François Massol

Philip Supply

Aurélie Tasiemski

Partner institution(s) involved in the project

1.   CIIL - Institut Pasteur de Lille, Lille

2.   IPBS, Toulouse

3.   Sorbonne Université, Paris

4.   U1177, Lille

5.   IP, Paris

6.   TBI, Toulouse

7.   LMGM, Toulouse

8.   CEA, Saclay

9.   CIRI-HCL, Lyon

Funding

2.400.000 €

PIA —Programme Investissements d’Avenir 2021, Gestion ANR, Coordination Scientifique Inserm

More details on the ANR-AMR Initiative and the selected projects

https://anr.fr/fr/detail/call/antibioresistance-comprendre-innover-agir-appel-a-projets-2020/

INTERNSHIPS

For all internship applications, please send your request directly to the group leader of the research area that you are interested in :
1- Bacteria, Antibiotics & Immunity : Contact
2- Biology of Apicomplex Parasites : Contact
3- Cellular Microbiology & Physics of Infection : Contact
4- Chemical Biology of Antibiotics : Contact
5- Chemical Biology of Flatworms : Contact
6- Chemogenomics of Intracellular Mycobacteria : Contact
7- Ecology & Physiopathology of Intestinal Protozoa : Contact
8- Influenza, Immunity & Metabolism : Contact
9- Lung Immunity : Contact
10- Molecular & Cellular Virology : Contact
11- Opportunistic Infections, Immunity, Environment & Lung Diseases: Contact
12- Plague & Yersinia pestis : Contact
13- Research on Mycobacteria and Bordetella: Contact
14- Tropical Biomes & Immuno-Pathophysiology : Contact

15- Mechanobiology of Host-Microbe Interactions : Contact


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