Structural Disorder within Henipavirus Nucleoprotein and Phosphoprotein: From Predictions to Experimental Assessment

Henipaviruses are newly emerged viruses within the Paramyxoviridae family. Their negative-strand RNA genome is packaged by the nucleoprotein (N) within α-helical nucleocapsid that recruits the polymerase complex made of the L protein and the phosphoprotein (P). To date structural data on Henipaviruses are scarce, and their N and P proteins have never been characterized so far. Using both computational and experimental approaches we herein show that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we show that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. We then report the cloning, the bacterial expression, purification and characterization of Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we show that both NTAIL and PNT belong to the premolten globule sub-family within the class of intrinsically disordered proteins. This study is the first reported experimental characterization of Henipavirus P and N proteins. The evidence that their respective N-terminal and C-terminal domains are highly disordered under native conditions is expected to be invaluable for future structural studies by helping to delineate N and P protein domains amenable to crystallization. In addition, following previous hints establishing a relationship between structural disorder and protein interactivity, the present results suggest that Henipavirus PNT and NTAIL domains could be involved in manifold protein-protein interactions

New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors

International audienceBACKGROUND : Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS : To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the latest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step CONCLUSIONS/SIGNIFICANCE : Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound

Rôle d'un mécanisme d'efflux et d'une nouvelle porine dans le transport membranaire chez Campylobacter

Le transport de molécules à travers l enveloppe des bactéries à Gram négatif dépend de canaux pour l entrée et de pompes d efflux pour la sortie. J ai mis en évidence un système d efflux actif chez Campylobacter, sensible à un inhibiteur, le PA N et sélectif des macrolides. L inhibiteur restaure une concentration intracellulaire active d antibiotique. Ce système est différent des transporteurs déjà décrit, et est impliqué dans la résistance de bas niveau chez des isolats humains et animaux. La résistance de haut niveau aux macrolides est attribuée à des mutations dans le gène cible qui diminuent l efficacité de fixation de l antibiotique. J ai étudié les propriétés de la protéine CadF. J ai cloné, produit, et purifié le domaine transmembranaire puis la forme entière de la protéine. J ai mis en évidence une activité canal des deux formes de la protéine dans un système de reconstitution en bicouches lipidiques artificielles. Puis j ai caractérisé l interaction de la protéine avec le peptidoglycan bactérien. L ensemble de mes travaux rendent compte de la capacité d adaptation de Campylobacter.The transport of molecules through the envelope of Gram negative bacteria depends on channels for the entry and pumps for the exit. I highlighted an active efflux system in Campylobacter sensitive to an inhibitor, the PA N and selective to macrolides. The inhibitor restores an efficient intracellular antibiotic concentration. This system different from the transporters already described, is involved in low-level resistant of human and animal isolates. High-level resistance to macrolides is explained by target gene mutations that decrease the effectiveness of binding of antibiotic. I studied the properties of the CadF protein. I cloned, produced, and purified the transmembrane domain and the whole protein. I highlighted a channel activity of the two shapes of the protein in a system of reconstitution into artificial lipid bilayers. Then I characterized the interaction of CadF with the bacterial peptidoglycan. The whole of my work account for properties of Campylobacter adaptation.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Structures of PDF-Is used.

<p>Structures of PDF-Is used.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Effect of efflux pump inhibitors and membrane permeabilizers.

<p>Values are means of four independent assays.</p>a<p>PMBN was used at 1/5 MIC.</p>b<p>PAβN was used at 20 µg/ml.</p

Activity of PDF-Is on MDR Gram-negative isolates.

<p>Values are means of four independent assays listed in µg/ml.</p><p>CM, chloramphenicol; NFX, norfloxacin; CAZ, ceftazidime; ERY, erythromycin; TC, tetracycline.</p>a<p>PMBN was used at 1/5 MIC.</p>b<p>in bracket the results obtained with 1/5 PMBN are indicated.</p

Effect of EDTA and detergents on PDF-Is activity.

<p>Values are means of four independent assays.</p><p>The different compounds were used at concentration for which no direct antibacterial effect was observed. EDTA was used at 1 mM ; SDS at 100 µg/ml; Tx-100 at 1000 µg/ml; DOC at 1000 µg/ml.</p