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Lipopolysaccharide Transport to the Cell Surface: New Insights in Assembly into the Outer Membrane

2016; Elsevier BV; Volume: 24; Issue: 6 Linguagem: Inglês

10.1016/j.str.2016.05.005

1878-4186

Paola Sperandeo, Alessandra Polissi,

Bacterial Genetics and Biotechnology

In this issue of Structure, Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar report crystal structures of the outer membrane LptDE translocon from three relevant Gram-negative pathogens. This study provides new details into the molecular mechanism of lipopolysaccharide assembly at the cell surface. In this issue of Structure, Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar report crystal structures of the outer membrane LptDE translocon from three relevant Gram-negative pathogens. This study provides new details into the molecular mechanism of lipopolysaccharide assembly at the cell surface. In addition to the cytoplasmic inner membrane, Gram-negative “diderm” bacteria are surrounded by the outer membrane, an asymmetric lipid bilayer containing lipopolysaccharide (LPS) in its outer leaflet (Figure 1). The presence of the tightly packed LPS makes outer membrane fairly impermeable and protects the bacteria from harmful compounds such as detergents and lipophilic antibiotics. Most of the LPS biosynthesis and assembly pathways are essential for bacteria survival; moreover, this molecule is also a virulence factor and a potent stimulant of the mammalian immune response, making its biogenesis an excellent target for the development of antibacterial and anti-inflammatory therapies. In this issue of Structure, Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar provide new insights into the peculiar mechanisms by which the amphipathic LPS molecule is delivered to its final destination and is assembled at the outer membrane (Okuda et al., 2016Okuda S. Sherman D.J. Silhavy T.J. Ruiz N. Kahne D. Nat. Rev. Microbiol. 2016; 14: 337-345Crossref PubMed Scopus (234) Google Scholar). The LPS molecule is made of the three covalently linked moieties: the lipid A, or endotoxin, which is the hydrophobic anchor in the membrane; a core oligosaccharide; and a variable O-antigen sugar chain (Whitfield and Trent, 2014Whitfield C. Trent M.S. Annu. Rev. Biochem. 2014; 83: 99-128Crossref PubMed Scopus (433) Google Scholar). Being an amphipathic molecule, LPS faces several challenges during its transport to the cell surface. Indeed, LPS needs to cross three different cellular compartments, i.e., the inner membrane, the periplasm, and the outer membrane, unidirectionally without compromising the integrity of the cellular envelope. Efficient LPS export relies on the peculiar structure and functioning of the Lpt molecular machine (Okuda et al., 2016Okuda S. Sherman D.J. Silhavy T.J. Ruiz N. Kahne D. Nat. Rev. Microbiol. 2016; 14: 337-345Crossref PubMed Scopus (234) Google Scholar) (Figure 1). LPS is extracted from the inner membrane via an energy-consuming process mediated by the LptB2FG ABC transporter and delivered to the hydrophobic channel formed by the LptC and LptA proteins. In vivo photo-cross-linking experiments have shown that LPS crosses the periplasm with its lipid portion inside the grove made up by LptC and LptA, while the large hydrophilic LPS moiety remains exposed in the aqueous periplasm (Okuda et al., 2012Okuda S. Freinkman E. Kahne D. Science. 2012; 338: 1214-1217Crossref PubMed Scopus (147) Google Scholar). LPS is then delivered to the outer membrane translocon made by two membrane proteins, the β-barrel protein LptD and lipoprotein LptE. The LptDE complex, characterized by a unique plug and barrel architecture (Okuda et al., 2016Okuda S. Sherman D.J. Silhavy T.J. Ruiz N. Kahne D. Nat. Rev. Microbiol. 2016; 14: 337-345Crossref PubMed Scopus (234) Google Scholar), is responsible for the insertion of LPS on the outer membrane and its assembly in the outer leaflet. One important open question is how the outer membrane Lpt components facilitate LPS insertion and assembly at the cell surface. Our understanding of this process has been greatly aided by the availability of the crystal structure of the LptDE complex from Shigella flexneri (Sf) and Salmonella thyphimurium (Qiao et al., 2014Qiao S. Luo Q. Zhao Y. Zhang X.C. Huang Y. Nature. 2014; 511: 108-111Crossref PubMed Scopus (182) Google Scholar, Dong et al., 2014Dong H. Xiang Q. Gu Y. Wang Z. Paterson N.G. Stansfeld P.J. He C. Zhang Y. Wang W. Dong C. Nature. 2014; 511: 52-56Crossref PubMed Scopus (186) Google Scholar). LptD is built primarily by β strands that fold in two domains: an N-terminal β-jellyroll domain that extends in the periplasm, and a C-terminal β-barrel domain inserted in the outer membrane. The lipoprotein LptE appears to act as a plug, as it is almost entirely inserted in the LptD C-terminal β-barrel domain (Figure 1). Importantly, the N-terminal domain of LptD displays the same β-jellyroll structure of LptA and LptC (Suits et al., 2008Suits M.D. Sperandeo P. Dehò G. Polissi A. Jia Z. J. Mol. Biol. 2008; 380: 476-488Crossref PubMed Scopus (119) Google Scholar, Tran et al., 2010Tran A.X. Dong C. Whitfield C. J. Biol. Chem. 2010; 285: 33529-33539Crossref PubMed Scopus (100) Google Scholar, Bollati et al., 2015Bollati M. Villa R. Gourlay L.J. Benedet M. Dehò G. Polissi A. Barbiroli A. Martorana A.M. Sperandeo P. Bolognesi M. Nardini M. FEBS J. 2015; 282: 1980-1997Crossref PubMed Scopus (27) Google Scholar). This fold appears to play a crucial role in the LPS export system: it is the “basic module” used to assemble the Lpt machinery (Villa et al., 2013Villa R. Martorana A.M. Okuda S. Gourlay L.J. Nardini M. Sperandeo P. Dehò G. Bolognesi M. Kahne D. Polissi A. J. Bacteriol. 2013; 195: 1100-1108Crossref PubMed Scopus (78) Google Scholar) and its hydrophobic interior is used to shield the hydrophobic LPS moiety during transport across the periplasm (Okuda et al., 2012Okuda S. Freinkman E. Kahne D. Science. 2012; 338: 1214-1217Crossref PubMed Scopus (147) Google Scholar). The picture that emerged from molecular dynamics simulations, mutational analyses, and cross-linking studies predicts that the N-terminal domain of LptD receives the LPS molecules while keeping the lipid A moiety within the β-jellyroll, whereas the sugar moieties are oriented by LptE inside the hydrophilic β-barrel lumen. LPS is then inserted in the outer leaflet of the outer membrane via a lateral gate formed by two β strands (β1 and β26) that are scarcely hydrogen-bonded in the β-barrel domain. The work by Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar extends and complements our current knowledge by providing additional structures of the core LptDE complex, which lacks the LptD N-terminal domain, from three medically relevant pathogens, Yersinia pestis, Klebsiella pneumonia (Kp) and Pseudomonas aeruginosa (Pa), as well as a low-resolution structure of K. pneumoniae full-length LptDE complex, thus providing a unique contribution to the understanding of LptDE structure-function relationships. First, Botos et al. show that the basic plug and barrel architecture of the LptDE protein complex is conserved across the three different pathogens. The mechanisms of assembly of the LPS substrate in the outer membrane also appears to be conserved: indeed, the authors identify two highly conserved proline residues in the β1 and β2 strands (P231 and P246) which, by perturbing the secondary structure of the strands, prevent the formation of the typical β sheet hydrogen bonds, and generate a local gap that provides gateways for the lateral migration of the LPS molecules between the LptDE complex and the membrane. A second important contribution to our understanding of the LPS export mechanisms comes from the comparison of the full-length KpLptDE and SfLptDE structures, which reveals a substantial rotation of the N-terminal domain with respect to the β–barrel barrel domain. The position of the LptD N-terminal domain relative to the C-terminal barrel is crucial to allow the insertion of the LPS lipid A moiety into the membrane, while maintaining the saccharide portion within the LptD lumen (Dong et al., 2014Dong H. Xiang Q. Gu Y. Wang Z. Paterson N.G. Stansfeld P.J. He C. Zhang Y. Wang W. Dong C. Nature. 2014; 511: 52-56Crossref PubMed Scopus (186) Google Scholar), and for the interaction with LptA (Okuda et al., 2016Okuda S. Sherman D.J. Silhavy T.J. Ruiz N. Kahne D. Nat. Rev. Microbiol. 2016; 14: 337-345Crossref PubMed Scopus (234) Google Scholar). It is known that the biogenesis of a functional LptDE translocon requires the formation of intramolecular disulfide bonds between non-consecutive cysteine residues, which connect the C-terminal and N-terminal domains of LptD (Chng et al., 2012Chng S.S. Xue M. Garner R.A. Kadokura H. Boyd D. Beckwith J. Kahne D. Science. 2012; 337: 1665-1668Crossref PubMed Scopus (76) Google Scholar); this is also is a prerequisite for the N-terminal domain of LptD to interact with LptA. The two full-length LptDE structures indeed differ in that the disulfides are fully formed in KpLptDE (Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), whereas in SfLptDE only one disulfide is observed (Dong et al., 2014Dong H. Xiang Q. Gu Y. Wang Z. Paterson N.G. Stansfeld P.J. He C. Zhang Y. Wang W. Dong C. Nature. 2014; 511: 52-56Crossref PubMed Scopus (186) Google Scholar). Notably, formation of either one of the two disulfide bridges is sufficient to build a functional LptDE complex (Chng et al., 2012Chng S.S. Xue M. Garner R.A. Kadokura H. Boyd D. Beckwith J. Kahne D. Science. 2012; 337: 1665-1668Crossref PubMed Scopus (76) Google Scholar). Therefore, it appears that disulfide bond formation conveys to the system the degree of flexibility between the periplasmic and the β-barrel domains that is required to maintain integrity of the transenvelope bridge during LPS transport. PaLptD is the most divergent of the three proteins presented, in terms of amino acid sequence and shows structural differences relative to the other structures. PaLptD displays the largest luminal volume, its lumen being composed of two cavities in contrast to the one present in the other LptD structures. This observation may reflect the peculiar P. aeruginosa LPS O-antigen composition and structure (Di Lorenzo et al., 2015Di Lorenzo F. De Castro C. Lanzetta R. Parrilli M. Silipo A. Molinaro A. Lipopolysaccharides as microbe-associated molecular patterns: a structural perspective.in: Cañada F.J. Martín-Santamaría S. Carbohydrates in Drug Design and Discovery: RSC Drug Discovery Series 43, Jiménez-Barbero. The Royal Society of Chemistry, Cambridge, UK2015: 36-63Crossref Google Scholar), which needs to be accommodated in PaLptD lumen for insertion in the outer membrane. PaLptD is also the binding target of POL7080, a new peptidomimetic currently in phase II clinical development (Srinivas et al., 2010Srinivas N. Jetter P. Ueberbacher B.J. Werneburg M. Zerbe K. Steinmann J. Van der Meijden B. Bernardini F. Lederer A. Dias R.L. et al.Science. 2010; 327: 1010-1013Crossref PubMed Scopus (417) Google Scholar), which specifically inhibits LPS transport in P. aeruginosa but not in other Gram-negative bacteria, including E. coli. The structural differences observed in PaLptDE, which are concentrated in its loop regions, may in part explain the specificity of POL7080 action. The discovery of POL7080 validates the Lpt machinery as an excellent target for novel antibiotic agents. LptD is an especially suited drug target being an essential and surface-exposed factor, thus readily accessible by drugs. The Lpt machinery is a modular system that originates from the assembly of homologous domains, suggesting that a drug lead targeting a specific protein-protein interaction within the machinery may indeed turn effective in perturbing the assembly of the transenvelope complex at any point. In conclusion, the work by Botos et al., 2016Botos I. Majdalani N. Mayclin S.J. McCarthy J.G. Lundquist K. Wojtowicz D. Barnard T.J. Gumbart J.C. Buchanan S.K. Structure. 2016; 24 (this issue): 965-976Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar reveals molecular details of the LptDE complex that can be exploited for the rational development of novel LptDE-specific antibacterial molecules. Structural and Functional Characterization of the LPS Transporter LptDE from Gram-Negative PathogensBotos et al.StructureMay 5, 2016In BriefCrystal structures of the lipopolysaccharide (LPS) transporter LptDE from three bacterial pathogens reveal new features of the LPS transport mechanism. The N-terminal domain of LptD, which accepts transported LPS from the periplasmic protein LptA, undergoes a large rotation that may facilitate assembly of the LptCAD scaffold. Full-Text PDF Open Archive