Metagenomic heterogeneity explains dual immune effects of endotoxins Susanne Brix, PhD, Carsten Eriksen, BSc, Jeppe Madura Larsen, PhD, Hans Bisgaard, DMSci  Journal of Allergy and Clinical Immunology  Volume 135, Issue 1, Pages 277-280 (January 2015) DOI: 10.1016/j.jaci.2014.09.036 Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Cataloguing of penta-acylated and hexa-acylated endotoxin–producing bacteria. The endotoxin biosynthesis pathway identifies the enzymes responsible for addition of the fifth acyl chain to tetra-acylated endotoxin (LpxL), which generates penta-acylated endotoxin, and addition of the sixth acyl chain (LpxM), which enables production of hexa-acylated endotoxin. A bioinformatics analysis for these bacterial genes allows classification of bacteria carrying the LpxL gene, resulting in production of penta-acylated endotoxin (blue), and bacteria carrying LpxL and LpxM, producing hexa-acylated endotoxin (red). The box displays the individual bacterial phyla, in which the gram-positive phyla Firmicutes represents Veillonella species only, and “Other” represents Chrysiogenetes, Elusimicrobia, Fibrobacteres, Fusobacteria, Gemmatimonadetes, Planctomycetes, Synergistetes, and Verrucomicrobia. Bacterial species were annotated based on presence of all genes required to produce tetra-acylated endotoxin. These bacteria were then clustered based on presence of LpxL and/or LpxM by extracting the reference genes LpxL and LpxM per bacterium from the KEGG Organism database (Release 61.1) and aligning them to the fully sequenced genomes available at the National Center for Biotechnology Information database by using the BLAST algorithm. Journal of Allergy and Clinical Immunology 2015 135, 277-280DOI: (10.1016/j.jaci.2014.09.036) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Prevalence of penta-acylated and hexa-acylated endotoxins in lungs from healthy subjects and asthmatic patients. Distributions of bacteria with penta- and hexa-acylated endotoxin variants in BAL fluid of children based on data from Hilty et al3 were stratified according to control subjects (n = 7, A) and patients with difficult asthma (n = 13, B). The gram-positive, non–endotoxin-producing bacteria (gray color) were clustered in one group, whereas the categorization of the gram-negative bacteria (divided into hexa-acylated endotoxin–producers [red] and penta-acylated endotoxin [blue] through our database of LpxL and LpxL plus LpxM–expressing abilities) is otherwise identical to the original data, for which statistics are also available.3 The category Gammaproteobacteria is made up of Haemophilus species only, whereas the classification “Proteobacteria” (purple color) was not classified in the original data set to a level enabling separation into endotoxin variants and can include both penta-acylated and hexa-acylated endotoxin–producing bacteria. Journal of Allergy and Clinical Immunology 2015 135, 277-280DOI: (10.1016/j.jaci.2014.09.036) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions