CellTagging: combinatorial indexing to simultaneously map lineage and identity at single-cell resolution.

Single-cell technologies are offering unparalleled insight into complex biology, revealing the behavior of rare cell populations that are masked in bulk population analyses. One current limitation of single-cell approaches is that lineage relationships are typically lost as a result of cell processing. We recently established a method, CellTagging, permitting the parallel capture of lineage information and cell identity via a combinatorial cell indexing approach. CellTagging integrates with high-throughput single-cell RNA sequencing, where sequential rounds of cell labeling enable the construction of multi-level lineage trees. Here, we provide a detailed protocol to (i) generate complex plasmid and lentivirus CellTag libraries for labeling of cells; (ii) sequentially CellTag cells over the course of a biological process; (iii) profile single-cell transcriptomes via high-throughput droplet-based platforms; and (iv) generate a CellTag expression matrix, followed by clone calling and lineage reconstruction. This lentiviral-labeling approach can be deployed in any organism or in vitro culture system that is amenable to viral transduction to simultaneously profile lineage and identity at single-cell resolution.

Discovery and Characterization of a Nitroreductase Capable of Conferring Bacterial Resistance to Chloramphenicol.

Widespread antibiotic resistance has led to the reappraisal of abandoned antibiotics including chloramphenicol. However, enzyme(s) underlying one form of chloramphenicol resistance, nitroreduction, have eluded identification. Here we demonstrate that expression of the Haemophilus influenzae nitroreductase gene nfsB confers chloramphenicol resistance in Escherichia coli. We characterized the enzymatic product of H. influenzae NfsB acting on chloramphenicol and found it to be amino-chloramphenicol. Kinetic analysis revealed reduction of diverse substrates including the incomplete reduction of 5-nitro antibiotics metronidazole and nitrofurantoin, likely resulting in activation of these antibiotic pro-drugs to their cytotoxic forms. We observed that expression of the H. influenzae nfsB gene in E. coli results in significantly increased susceptibility to metronidazole. Finally, we found that in this strain metronidazole attenuates chloramphenicol resistance synergistically, and in vitro metronidazole weakly inhibits chloramphenicol reduction by NfsB. Our findings reveal the underpinnings of a chloramphenicol resistance mechanism nearly 70 years after its description.

Single-cell mapping of lineage and identity in direct reprogramming.

Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for 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 that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a 'dead-end' state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.

Synergistic, collaterally sensitive ß-lactam combinations suppress resistance in MRSA.

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 ß-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other 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-ß-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to ß-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older ß-lactam antibiotics could be effective against MRSA infections in humans.

Evidence for an ABC-type riboflavin transporter system in pathogenic spirochetes.

UNLABELLED: 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 B(2)). 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. IMPORTANCE: Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum's cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum's cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism's survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system.