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1.
J Biol Chem ; 299(11): 105314, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37797696

RESUMO

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.


Assuntos
Alginatos , Pseudomonas aeruginosa , Acetilação , Alginatos/química , Proteínas de Bactérias/metabolismo , Biofilmes , Periplasma/metabolismo , Processamento de Proteína Pós-Traducional , Pseudomonas aeruginosa/metabolismo
2.
Nat Microbiol ; 9(3): 763-775, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38336881

RESUMO

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway.


Assuntos
Bactérias , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Retroalimentação , Parede Celular/metabolismo , Lipídeos
3.
Science ; 381(6654): eadg9091, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37440661

RESUMO

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.


Assuntos
Antibacterianos , Bacteriólise , Bacteriófago phi X 174 , Proteínas de Escherichia coli , Escherichia coli , Proteínas Virais , Antibacterianos/metabolismo , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Imagem Individual de Molécula , Microscopia Crioeletrônica
4.
bioRxiv ; 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-37577621

RESUMO

Many bacterial surface glycans such as the peptidoglycan (PG) cell wall, O-antigens, and capsules are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting lipid carrier is effectively distributed among competing pathways has remained unclear for some time. Here, we describe the isolation and characterization of hyperactive variants of Pseudomonas aeruginosa MraY, the essential and conserved enzyme catalyzing the formation of the first lipid-linked PG precursor called lipid I. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in a purified system. Amino acid substitutions within the activated MraY variants unexpectedly map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural evidence and molecular dynamics simulations suggest that the cavity is a binding site for lipid II molecules that have been transported to the outer leaflet of the membrane. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism to prevent the sequestration of lipid carrier in the PG biogenesis pathway. MraY belongs to the broadly distributed polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase (PNPT) superfamily of enzymes. We therefore propose that similar feedback mechanisms may be widely employed to coordinate precursor supply with demand by polymerases, thereby optimizing the partitioning of lipid carriers between competing glycan biogenesis pathways.

5.
Nat Commun ; 13(1): 7631, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494359

RESUMO

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.


Assuntos
Alginatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Alginatos/metabolismo , Ácidos Hexurônicos/metabolismo , Proteínas de Bactérias/metabolismo , Ácido Glucurônico/metabolismo , Biofilmes , Polímeros/metabolismo
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