A Cationic Lumen in the Wzx Flippase Mediates Anionic O-antigen Subunit Translocation in Pseudomonas Aeruginosa PAO1

Overview

Journal Mol Microbiol

Specialties Microbiology
Molecular Biology

Date 2012 May 5

PMID 22554073

Citations 24

Authors

Salim T Islam

Robert J Fieldhouse

Erin M Anderson

Veronique L Taylor

Robert A B Keates

Robert C Ford

Joseph S Lam

Affiliations

Soon will be listed here.

Abstract

Heteropolymeric B-band O-antigen (O-Ag) biosynthesis in Pseudomonas aeruginosa PAO1 follows the Wzy-dependent pathway, beginning with translocation of undecaprenyl pyrophosphate-linked anionic O-Ag subunits (O units) from the inner to the outer leaflets of the inner membrane (IM). This translocation is mediated by the integral IM flippase Wzx. Through experimentally based and unbiased topological mapping, our group previously observed that Wzx possesses many charged and aromatic amino acid residues within its 12 transmembrane segments (TMS). Herein, site-directed mutagenesis targeting 102 residues was carried out on the TMS and loops of Wzx, followed by assessment of each construct's ability to restore B-band O-Ag production, identifying eight residues important for flippase function. The importance of various charged and aromatic residues was highlighted, predominantly within the TMS of the protein, revealing functional 'hotspots' within the flippase, particularly within TMS2 and TMS8. Construction of a tertiary structure homology model for Wzx indicated that TMS2 and TMS8 line a central cationic lumen. This is the first report to describe a charged flippase lumen for mediating anionic O-unit translocation across the hydrophobic IM.

Citing Articles

Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide.

Le Bas A, Clarke B, Teelucksingh T, Lee M, El Omari K, Giltrap A Open Biol. 2025; 15(1):240310.

PMID: 39772807 PMC: 11706664. DOI: 10.1098/rsob.240310.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective β-Barrel Porin Requirements for Polymer Secretion.

Saidi F, Mahanta U, Panda A, Kezzo A, Jolivet N, Bitazar R Microbiol Spectr. 2022; 10(5):e0129022.

PMID: 36200915 PMC: 9603273. DOI: 10.1128/spectrum.01290-22.

Bacterial Glycocalyx Integrity Impacts Tolerance of to Antibiotics and Oxidative-Stress Agents.

Saidi F, Bitazar R, Bradette N, Islam S Biomolecules. 2022; 12(4).

PMID: 35454160 PMC: 9029694. DOI: 10.3390/biom12040571.

Structure and Mechanism of the Lipid Flippase MurJ.

Kuk A, Hao A, Lee S Annu Rev Biochem. 2022; 91:705-729.

PMID: 35320686 PMC: 10108830. DOI: 10.1146/annurev-biochem-040320-105145.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

Islam S, Vergara Alvarez I, Saidi F, Guiseppi A, Vinogradov E, Sharma G PLoS Biol. 2020; 18(6):e3000728.

PMID: 32516311 PMC: 7310880. DOI: 10.1371/journal.pbio.3000728.

References

He X, Szewczyk P, Karyakin A, Evin M, Hong W, Zhang Q . Structure of a cation-bound multidrug and toxic compound extrusion transporter. Nature. 2010; 467(7318):991-4. PMC: 3152480. DOI: 10.1038/nature09408. View

Whitfield C . Biosynthesis of lipopolysaccharide O antigens. Trends Microbiol. 1995; 3(5):178-85. DOI: 10.1016/s0966-842x(00)88917-9. View

Henzler-Wildman K . Analyzing conformational changes in the transport cycle of EmrE. Curr Opin Struct Biol. 2011; 22(1):38-43. PMC: 3288627. DOI: 10.1016/j.sbi.2011.10.004. View

Cuthbertson L, Mainprize I, Naismith J, Whitfield C . Pivotal roles of the outer membrane polysaccharide export and polysaccharide copolymerase protein families in export of extracellular polysaccharides in gram-negative bacteria. Microbiol Mol Biol Rev. 2009; 73(1):155-77. PMC: 2650888. DOI: 10.1128/MMBR.00024-08. View

Islam S, Taylor V, Qi M, Lam J . Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures. mBio. 2010; 1(3). PMC: 2932511. DOI: 10.1128/mBio.00189-10. View

Khafizov K, Staritzbichler R, Stamm M, Forrest L . A study of the evolution of inverted-topology repeats from LeuT-fold transporters using AlignMe. Biochemistry. 2010; 49(50):10702-13. DOI: 10.1021/bi101256x. View

Robbins P, Bray D, Dankert B, Wright A . Direction of chain growth in polysaccharide synthesis. Science. 1967; 158(3808):1536-42. DOI: 10.1126/science.158.3808.1536. View

Frank C, Sanyal S, Rush J, Waechter C, Menon A . Does Rft1 flip an N-glycan lipid precursor?. Nature. 2008; 454(7204):E3-4. DOI: 10.1038/nature07165. View

Forrest L, Rudnick G . The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters. Physiology (Bethesda). 2009; 24:377-86. PMC: 3012352. DOI: 10.1152/physiol.00030.2009. View

10.

Ravna A, Sylte I . Homology modeling of transporter proteins (carriers and ion channels). Methods Mol Biol. 2012; 857:281-99. DOI: 10.1007/978-1-61779-588-6_12. View

11.

Elumalai P, Rajasekaran M, Liu H, Chen C . Investigation of cation-π interactions in sugar-binding proteins. Protoplasma. 2010; 247(1-2):13-24. DOI: 10.1007/s00709-010-0132-x. View

12.

Feldman M, Marolda C, Monteiro M, Perry M, Parodi A, Valvano M . The activity of a putative polyisoprenol-linked sugar translocase (Wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J Biol Chem. 1999; 274(49):35129-38. DOI: 10.1074/jbc.274.49.35129. View

13.

Schneider G, Fechner U . Advances in the prediction of protein targeting signals. Proteomics. 2004; 4(6):1571-80. DOI: 10.1002/pmic.200300786. View

14.

Malik A, Ahmad S . Sequence and structural features of carbohydrate binding in proteins and assessment of predictability using a neural network. BMC Struct Biol. 2007; 7:1. PMC: 1780050. DOI: 10.1186/1472-6807-7-1. View

15.

Guan L, Mirza O, Verner G, Iwata S, Kaback H . Structural determination of wild-type lactose permease. Proc Natl Acad Sci U S A. 2007; 104(39):15294-8. PMC: 2000551. DOI: 10.1073/pnas.0707688104. View

16.

Cunneen M, Reeves P . Membrane topology of the Salmonella enterica serovar Typhimurium Group B O-antigen translocase Wzx. FEMS Microbiol Lett. 2008; 287(1):76-84. DOI: 10.1111/j.1574-6968.2008.01295.x. View

17.

Silhavy T, Kahne D, Walker S . The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2010; 2(5):a000414. PMC: 2857177. DOI: 10.1101/cshperspect.a000414. View

18.

Lo A, Chiu Y, Rodland E, Lyu P, Sung T, Hsu W . Predicting helix-helix interactions from residue contacts in membrane proteins. Bioinformatics. 2009; 25(8):996-1003. PMC: 2666818. DOI: 10.1093/bioinformatics/btp114. View

19.

Huda M, Morita Y, Kuroda T, Mizushima T, Tsuchiya T . Na+-driven multidrug efflux pump VcmA from Vibrio cholerae non-O1, a non-halophilic bacterium. FEMS Microbiol Lett. 2001; 203(2):235-9. DOI: 10.1111/j.1574-6968.2001.tb10847.x. View

20.

Abeyrathne P, Daniels C, Poon K, Matewish M, Lam J . Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide. J Bacteriol. 2005; 187(9):3002-12. PMC: 1082828. DOI: 10.1128/JB.187.9.3002-3012.2005. View