Direct lineage reprogramming involves the remarkable conversion of cellular identity. Single-cell technologies aid in deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here, we present ‘CellTagging’, a combinatorial cell indexing methodology, permitting the parallel capture of clonal history and cell identity, where sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor (iEP) reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a ‘dead-end’ state, paths determined in the earliest reprogramming stages. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory, where its addition to the reprogramming cocktail increases the yield of iEPs. Together, these results demonstrate the utility of our lineage tracing method to reveal dynamics of direct reprogramming.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple β-lactam combination meropenem/piperacillin/tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA N315 and 72 clinical MRSA isolates in vitro, and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem/penicillin/β-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to β-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein 2a (PBP2a) action via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing similar in vivo activity to linezolid, ME/PI/TZ demonstrates that combinations of older β-lactam antibiotics could be effective against MRSA infections in humans.

Highlights d Bacteria can resist chloramphenicol via reduction, but the causal genes are unknown d nfsB nitroreductase expression can confer chloramphenicol resistance in E. coli d H. influenzae NfsB quickly reduces chloramphenicol to amino-chloramphenicol in vitro d Metronidazole potentiates chloramphenicol activity against nitro reducing E. coli

Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host.

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