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A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus

Synthesis scheme.(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.
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ppat.1005071.g002: Synthesis scheme.(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.

Mentions: The synthesis strategy for the production of agrocinopine A (Fig 2) allowed us to produce derivatives, which are L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and agrocinopine-3’-O-benzoate (S1 Fig). A D-glucose-2-phosphate was also synthetized (Fig 2 and S1 Fig).


A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

El Sahili A, Li SZ, Lang J, Virus C, Planamente S, Ahmar M, Guimaraes BG, Aumont-Nicaise M, Vigouroux A, Soulère L, Reader J, Queneau Y, Faure D, Moréra S - PLoS Pathog. (2015)

Synthesis scheme.(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526662&req=5

ppat.1005071.g002: Synthesis scheme.(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.
Mentions: The synthesis strategy for the production of agrocinopine A (Fig 2) allowed us to produce derivatives, which are L-arabinose-2-phosphate, L-arabinose-2-isopropylphosphate and agrocinopine-3’-O-benzoate (S1 Fig). A D-glucose-2-phosphate was also synthetized (Fig 2 and S1 Fig).

Bottom Line: This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif.It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), Department of Biophysics, Biochemistry and Structural Biology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France; Institute for Integrative Biology of the Cell (I2BC), Department of Microbiology, CNRS CEA University Paris-Sud, Gif-sur-Yvette, France.

ABSTRACT
Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

No MeSH data available.


Related in: MedlinePlus