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Détection de la résistance à la vancomycine par mise en évidence d'une activité D-Ala-D-Ala dipeptidase

Abstract : Glycopeptide antibiotics have been used intensively in humans and animals since they have been approved. Emergence of glycopeptide resistant Enterococcus faecium has been ascribed to a selective pressure due to the increasing use of vancomycine in hospitals and avoparcin as a growth promoter in intensive livestock. Vancomycin resistant Enterococcus faecium represents a serious and increasing threat for world public health according to WHO. Glycopeptides resistance detection methods do exist, however they are slow and/or expensive. The present study aims at discovering a much faster method that would allow early medical care and isolation of the patient, thus limiting the dissemination of the resistance. The concept of the proposed method is based on the detection of vancomycin resistance through the evidencing of a D-Alanine-D-Alanine dipeptidase activity of VanX by releasing a chromophore. To demonstrate the concept of this method, ethyl 2-chloronicotinate and tert-butyl 2-chloronicotinate were functionalized with an N-protected D-alanine. This functionalization has proven to be complex due to a strong tendency towards cyclization of the nicotinate system, which moreover proves the effectiveness of the self-immolation of the system that we offer. Following the same functionalization protocol with tert-butyl 2-chloronicotinate, a first furopyridinone derivative with a D-Ala-D-Ala sequence was obtained. After deprotection, this molecule can be used to detect VanX activity. It can also serve as a pivot for synthesis of self-immolable pro-chromophoric molecules. Cloning and over-expression of the VanX protein was carried out in two types of vectors giving respectively a recombinant protein 6xHis-VanX and a recombinant native protein VanX which both possess D-Ala-D-Ala dipeptidase activity.
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Submitted on : Thursday, June 25, 2020 - 9:04:10 PM
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  • HAL Id : tel-02881618, version 1

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Windbedema Prisca Ouedraogo. Détection de la résistance à la vancomycine par mise en évidence d'une activité D-Ala-D-Ala dipeptidase. Médecine humaine et pathologie. Normandie Université, 2020. Français. ⟨NNT : 2020NORMC404⟩. ⟨tel-02881618⟩

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