Activity of a Synthetic Peptide Targeting MgtC on Pseudomonas aeruginosa Intramacrophage Survival and Biofilm Formation

Antivirulence strategies aim to target pathogenicity factors while bypassing the pressure on the bacterium to develop resistance. The MgtC membrane protein has been proposed as an attractive target that is involved in the ability of several major bacterial pathogens, including Pseudomonas aeruginosa, to survive inside macrophages. In liquid culture, P. aeruginosa MgtC acts negatively on biofilm formation. However, a putative link between these two functions of MgtC in P. aeruginosa has not been experimentally addressed. In the present study, we first investigated the contribution of exopolysaccharides (EPS) in the intramacrophage survival defect and biofilm increase of mgtC mutant. Within infected macrophages, expression of EPS genes psl and alg was increased in a P. aeruginosa mgtC mutant strain comparatively to wild-type strain. However, the intramacrophage survival defect of mgtC mutant was not rescued upon introduction of psl or alg mutation, suggesting that MgtC intramacrophage role is unrelated to EPS production, whereas the increased biofilm formation of mgtC mutant was partially suppressed by introduction of psl mutation. We aimed to develop an antivirulence strategy targeting MgtC, by taking advantage of a natural antagonistic peptide, MgtR. Heterologous expression of mgtR in P. aeruginosa PAO1 was shown to reduce its ability to survive within macrophages. We investigated for the first time the biological effect of a synthetic MgtR peptide on P. aeruginosa. Exogenously added synthetic MgtR peptide lowered the intramacrophage survival of wild-type P. aeruginosa PAO1, thus mimicking the phenotype of an mgtC mutant as well as the effect of endogenously produced MgtR peptide. In correlation with this finding, addition of MgtR peptide to bacterial culture strongly reduced MgtC protein level, without reducing bacterial growth or viability, thus differing from classical antimicrobial peptides. On the other hand, the addition of exogenous MgtR peptide did not affect significantly biofilm formation, indicating an action toward EPS-independent phenotype rather than EPS-related phenotype. Cumulatively, our results show an antivirulence action of synthetic MgtR peptide, which may be more potent against acute infection, and provide a proof of concept for further exploitation of anti-Pseudomonas strategies

Activity of a Synthetic Peptide Targeting MgtC on Pseudomonas aeruginosa Intramacrophage Survival and Biofilm Formation

International audienceAntivirulence strategies aim to target pathogenicity factors while bypassing the pressure on the bacterium to develop resistance. The MgtC membrane protein has been proposed as an attractive target that is involved in the ability of several major bacterial pathogens, including Pseudomonas aeruginosa, to survive inside macrophages. In liquid culture, P. aeruginosa MgtC acts negatively on biofilm formation. However, a putative link between these two functions of MgtC in P. aeruginosa has not been experimentally addressed. In the present study, we first investigated the contribution of exopolysaccharides (EPS) in the intramacrophage survival defect and biofilm increase of mgtC mutant. Within infected macrophages, expression of EPS genes psl and alg was increased in a P. aeruginosa mgtC mutant strain comparatively to wild-type strain. However, the intramacrophage survival defect of mgtC mutant was not rescued upon introduction of psl or alg mutation, suggesting that MgtC intramacrophage role is unrelated to EPS production, whereas the increased biofilm formation of mgtC mutant was partially suppressed by introduction of psl mutation. We aimed to develop an antivirulence strategy targeting MgtC, by taking advantage of a natural antagonistic peptide, MgtR. Heterologous expression of mgtR in P. aeruginosa PAO1 was shown to reduce its ability to survive within macrophages. We investigated for the first time the biological effect of a synthetic MgtR peptide on P. aeruginosa. Exogenously added synthetic MgtR peptide lowered the intramacrophage survival of wild-type P. aeruginosa PAO1, thus mimicking the phenotype of an mgtC mutant as well as the effect of endogenously produced MgtR peptide. In correlation with this finding, addition of MgtR peptide to bacterial culture strongly reduced MgtC protein level, without reducing bacterial growth or viability, thus differing from classical antimicrobial peptides. On the other hand, the addition of exogenous MgtR peptide did not affect significantly biofilm formation, indicating an action toward EPS-independent phenotype rather than EPS-related phenotype. Cumulatively, our results show an antivirulence action of synthetic MgtR peptide, which may be more potent against acute infection, and provide a proof of concept for further exploitation of anti-Pseudomonas strategies

Prise en charge des carcinomes canalaires in situ du sein (CCIS): Expérience de l'Institut Bordet

info:eu-repo/semantics/publishe

Structure‐based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase

International audienceMulti-drug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+-competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µM. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model

Structure-based design of NAD+ analogues targeting bacterial NAD kinases, promising targets for new antibiotics

International audienceMulti-drug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a drug design approach based on substrate-derived fragments, we have recently developed NAD+-competitive inhibitors of NADKs, which displayed in vivo activity against Staphylococcus aureus or Pseudomonas aeruginosa in animal models of infection [1-3].Funding and supports: Agence Nationale de la Recherche (ANR-17-CE18-0011-02), Institut Pasteur, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), University of Montpellier.References: [1] Clément DA, Leseigneur C, Gelin M, Coelho D, Huteau V, Lionne C, Labesse G, Dussurget O, Pochet S (2020) New chemical probe targeting bacterial NAD kinase. Molecules. doi: 10.3390/molecules25214893.[2] Gelin M, Paoletti J, Nahori MA, Huteau V, Leseigneur C, Jouvion G, Dugué L, Clément D, Pons JL, Assairi L, Pochet S, Labesse G, Dussurget O (2020) From substrate to fragments to inhibitor active in vivo against Staphylococcus aureus. ACS Infect Dis. doi: 10.1021/acsinfecdis.9b00368.[3] Rahimova R, Nogaret P, HuteauV, Gelin M, Clément D, Labesse G, Pochet S, Blanc-Potard A, Lionne C (2022) Structure-based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase. In Preparation