@article{c9101001b04c4fe9b18f6a0b3c5685d4,
title = "Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis",
abstract = "Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp. Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2′ and 3′ positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.",
keywords = "Evolution, Immune evasion, Lipid A, Pathogenesis, Yersinia",
author = "Chandler, {Courtney E.} and Harberts, {Erin M.} and Pelletier, {Mark R.} and Iyarit Thaipisuttikul and Jones, {Jace W.} and Hajjar, {Adeline M.} and Sahl, {Jason W.} and Goodlett, {David R.} and Pride, {Aaron C.} and Rasko, {David A.} and Trent, {M. Stephen} and Bishop, {Russell E.} and Ernst, {Robert K.}",
note = "Funding Information: 28. M. Matsuura, Structural modifications of bacterial lipopolysaccharide that facilitate Gram-negative bacteria evasion of host innate immunity. Front. Immunol. 4, 109 (2013). Materials and Methods Detailed materials and methods describing bacterial strains, culture conditions, Y. pestis PagP mutagenesis, LPS purification and lipid A isolation, mass spectrometry, membrane experiments, cell stimulations, and bioinformatics can be found in SI Appendix. Data Availability. All study data are included in the article and SI Appendix. All GenBank and protein sequence data are publicly available. All experimental data will be made available upon request. ACKNOWLEDGMENTS. We thank Francesca Gardner for review and critique of the manuscript. This work was supported in part by NIH Grants AI123820 and GM111066 (to R.K.E. and D.R.G.), U19AI110820 (to D.A.R.), AI129940 (to M.S.T.), AI150098 (to M.S.T.), and AI138576 (to M.S.T.); the International Centre for Cancer Vaccine Science project of the International Research Agendas program of the Foundation for Polish Science cofinanced by the European Union under the European Regional Development Fund (MAB/ 2017/03) at the University of Gdansk (to D.R.G.); and by Canadian Institutes of Health Research Grant MOP-125979 (to R.E.B.). 29. R. Rebeil, R. K. Ernst, B. B. Gowen, S. I. Miller, B. J. Hinnebusch, Variation in lipid A structure in the pathogenic yersiniae. Mol. Microbiol. 52, 1363–1373 (2004). 30. A. M. Hajjar et al., Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica. PLoS Pathog. 8, e1002963 (2012). 31. S. W. Montminy et al., Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response. Nat. Immunol. 7, 1066–1073 (2006). 32. R. Rebeil et al., Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation. J. Bacteriol. 188, 1381–1388 (2006). 33. J. L. Goodin et al., Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague. Protein Expr. Purif. 53, 63–79 (2007). 34. R. E. Bishop, The lipid A palmitoyltransferase PagP: Molecular mechanisms and role in bacterial pathogenesis. Mol. Microbiol. 57, 900–912 (2005). 35. S. Chalabaev et al., Biofilms formed by gram-negative bacteria undergo increased lipid a palmitoylation, enhancing in vivo survival. MBio 5, e01116-14 (2014). 36. L. Guo et al., Lipid A acylation and bacterial resistance against vertebrate antimi-crobial peptides. Cell 95, 189–198 (1998). 37. M. R. Pilione, E. J. Pishko, A. Preston, D. J. Maskell, E. T. Harvill, pagP is required for resistance to antibody-mediated complement lysis during Bordetella bronchiseptica respiratory infection. Infect. Immun. 72, 2837–2842 (2004). 38. A. Preston et al., Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract. Mol. Microbiol. 48, 725–736 (2003). 39. D. A. Benson et al., GenBank. Nucleic Acids Res. 43, D30–D35 (2015). 40. L. E. Hittle et al., Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis. Pathog. Dis. 73, ftv053 (2015). 41. M. Eppinger et al., The complete genome sequence of Yersinia pseudotuberculosis IP31758, the causative agent of Far East scarlet-like fever. PLoS Genet. 3, e142 (2007). 42. J. Parkhill et al., Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413, 523–527 (2001). 43. K. E. Holt et al., High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat. Genet. 40, 987–993 (2008). 44. R. E. Bishop, Structural biology of membrane-intrinsic beta-barrel enzymes: Sentinels of the bacterial outer membrane. Biochim. Biophys. Acta 1778, 1881–1896 (2008). 45. R. E. Bishop et al., Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria. EMBO J. 19, 5071–5080 (2000). 46. V. E. Ahn et al., A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin. EMBO J. 23, 2931–2941 (2004). 47. P. M. Hwang et al., Solution structure and dynamics of the outer membrane enzyme PagP by NMR. Proc. Natl. Acad. Sci. U.S.A. 99, 13560–13565 (2002). 48. C. P. Moon, K. G. Fleming, Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers. Proc. Natl. Acad. Sci. U.S.A. 108, 10174–10177 (2011). 49. S. H. White, How hydrogen bonds shape membrane protein structure. Adv. Protein Chem. 72, 157–172 (2005). 50. H. de Cock, S. van Blokland, J. Tommassen, In vitro insertion and assembly of outer membrane protein PhoE of Escherichia coli K-12 into the outer membrane. Role of Triton X-100. J. Biol. Chem. 271, 12885–12890 (1996). 51. M. Struyv{\'e}, M. Moons, J. Tommassen, Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J. Mol. Biol. 218, 141–148 (1991). 52. C. L. Airhart et al., Induction of innate immunity by lipid A mimetics increases survival from pneumonic plague. Microbiology 154, 2131–2138 (2008). 53. C. L. Airhart et al., Lipid A mimetics are potent adjuvants for an intranasal pneumonic plague vaccine. Vaccine 26, 5554–5561 (2008). 54. N. O. Fischer et al., Colocalized delivery of adjuvant and antigen using nano-lipoprotein particles enhances the immune response to recombinant antigens. J. Am. Chem. Soc. 135, 2044–2047 (2013). 55. K. A. Gregg et al., Rationally eesigned TLR4 ligands for vaccine adjuvant discovery. MBio 8, e00492-17 (2017). 56. B. D. Needham et al., Modulating the innate immune response by combinatorial engineering of endotoxin. Proc. Natl. Acad. Sci. U.S.A. 110, 1464–1469 (2013). MICROBIOLOGY Funding Information: ACKNOWLEDGMENTS. We thank Francesca Gardner for review and critique of the manuscript. This work was supported in part by NIH Grants AI123820 and GM111066 (to R.K.E. and D.R.G.), U19AI110820 (to D.A.R.), AI129940 (to M.S.T.), AI150098 (to M.S.T.), and AI138576 (to M.S.T.); the International Centre for Cancer Vaccine Science project of the International Research Agendas program of the Foundation for Polish Science cofinanced by the European Union under the European Regional Development Fund (MAB/ 2017/03) at the University of Gdansk (to D.R.G.); and by Canadian Institutes of Health Research Grant MOP-125979 (to R.E.B.). Publisher Copyright: {\textcopyright} 2020 National Academy of Sciences. All rights reserved.",
year = "2020",
month = sep,
day = "15",
doi = "10.1073/pnas.1917504117",
language = "English (US)",
volume = "117",
pages = "22984--22991",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "37",
}