Ex vivo tissue perturbations coupled to single-cell RNA-seq reveal multilineage cell circuit dynamics in human lung fibrogenesis.

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing and induced a multilineage circuit of fibrogenic cell states in hPCLS. We showed that these cell states were highly similar to the in vivo cell circuit in a multicohort lung cell atlas from patients with pulmonary fibrosis. Using micro-CT-staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state, and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in the hPCLS model provided evidence that the basaloid cell state was derived from alveolar type 2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in hPCLS, and antifibrotic drug treatments showed highly cell type-specific effects. Our work provides an experimental framework for perturbational single-cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration, and pathology. We further demonstrate that hPCLS offer an avenue for scalable, high-resolution drug testing to accelerate antifibrotic drug development and translation.

To kill or to be killed: pangenome analysis of Escherichia coli strains reveals a tailocin specific for pandemic ST131.

BACKGROUND: Escherichia coli (E. coli) has been one of the most studied model organisms in the history of life sciences. Initially thought just to be commensal bacteria, E. coli has shown wide phenotypic diversity including pathogenic isolates with great relevance to public health. Though pangenome analysis has been attempted several times, there is no systematic functional characterization of the E. coli subgroups according to the gene profile. RESULTS: Systematically scanning for optimal parametrization, we have built the E. coli pangenome from 1324 complete genomes. The pangenome size is estimated to be ~25,000 gene families (GFs). Whereas the core genome diminishes as more genomes are added, the softcore genome (≥95% of strains) is stable with ~3000 GFs regardless of the total number of genomes. Apparently, the softcore genome (with a 92% or 95% generation threshold) can define the genome of a bacterial species listing the critically relevant, evolutionarily most conserved or important classes of GFs. Unsupervised clustering of common E. coli sequence types using the presence/absence GF matrix reveals distinct characteristics of E. coli phylogroups B1, B2, and E. We highlight the bi-lineage nature of B1, the variation of the secretion and of the iron acquisition systems in ST11 (E), and the incorporation of a highly conserved prophage into the genome of ST131 (B2). The tail structure of the prophage is evolutionarily related to R2-pyocin (a tailocin) from Pseudomonas aeruginosa PAO1. We hypothesize that this molecular machinery is highly likely to play an important role in protecting its own colonies; thus, contributing towards the rapid rise of pandemic E. coli ST131. CONCLUSIONS: This study has explored the optimized pangenome development in E. coli. We provide complete GF lists and the pangenome matrix as supplementary data for further studies. We identified biological characteristics of different E. coli subtypes, specifically for phylogroups B1, B2, and E. We found an operon-like genome region coding for a tailocin specific for ST131 strains. The latter is a potential killer weapon providing pandemic E. coli ST131 with an advantage in inter-bacterial competition and, suggestively, explains their dominance as human pathogen among E. coli strains.

Conserved sequence motifs in human TMTC1, TMTC2, TMTC3, and TMTC4, new O-mannosyltransferases from the GT-C/PMT clan, are rationalized as ligand binding sites.

BACKGROUND: The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). RESULTS: Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifs rationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. CONCLUSIONS: With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases.