Lipids tailor membrane identities and function as molecular hubs in all cellular processes. However, the ways in which lipids modulate protein function and structure are poorly understood and still ...require systematic investigation. In this Innovation article, we summarize pioneering technologies, including lipid-overlay assays, lipid pull-down assays, affinity-purification lipidomics and the liposome microarray-based assay (LiMA), that will enable protein-lipid interactions to be deciphered on a systems level. We discuss how these technologies can be applied to the charting of system-wide networks and to the development of new pharmaceutical strategies.
Bacteria respond to changes in their environment with specific transcriptional programmes, but even within genetically identical populations these programmes are not homogenously expressed
. Such ...transcriptional heterogeneity between individual bacteria allows genetically clonal communities to develop a complex array of phenotypes
, examples of which include persisters that resist antibiotic treatment and metabolically specialized cells that emerge under nutrient-limiting conditions
. Fluorescent reporter constructs have played a pivotal role in deciphering heterogeneous gene expression within bacterial populations
but have been limited to recording the activity of single genes in a few genetically tractable model species, whereas the vast majority of bacteria remain difficult to engineer and/or even to cultivate. Single-cell transcriptomics is revolutionizing the analysis of phenotypic cell-to-cell variation in eukaryotes, but technical hurdles have prevented its robust application to prokaryotes. Here, using an improved poly(A)-independent single-cell RNA-sequencing protocol, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria across all RNA classes and genomic regions. These transcriptomes provide important reference points for single-cell RNA-sequencing of other bacterial species, mixed microbial communities and host-pathogen interactions.
The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It ...remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.
Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of
...species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that
persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the
pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing
cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
•Many new methods for performing single-microbe transcriptomics have been developed.•A new generation of microfluidics is streamlining microbial culture, manipulation, and sorting.•Smart-seq, ...YscRNA-seq, and SCRB-seq allow multiplexed single-cell analysis of protozoa and yeast.•MATQ-seq, CEL-seq2, PETRI-seq, and microSPLiT, PatH-Cap are emerging technologies for performing single-bacteria RNA-seq.•Single-microbe genomics paves the way to designing precise antimicrobial treatments.
Microbes have developed complex strategies to respond to their environment and escape the immune system by individualizing their behavior. While single-cell RNA sequencing has become instrumental for studying mammalian cells, its use with fungi, protozoa and bacteria is still in its infancy. In this review, we highlight the major progress towards mapping the molecular states of microbes at the single-cell level using genome-wide transcriptomics and describe how these technologies can be extended to probe thousands of species at high throughput. We envision that mammalian and microbial single-cell profiling could soon be integrated for the study of microbial communities in health and disease.
Abstract
Aims
Accumulation of mononuclear phagocytes monocytes, macrophages, and dendritic cells (DCs) in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of ...mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease.
Methods and results
We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a ‘mreg-DC’ signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans.
Conclusions
We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.
For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally ...released bacterial bitter “taste” substances are most probably initiated by tracheal brush cells (BC). Our single‐cell RNA‐seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in Ca2+i in BC and subsequent ACh‐release. ACh‐release is regulated in an autocrine manner. While the muscarinic ACh‐receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased Ca2+i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5‐ and M3R‐mediated. We show that ACh‐release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
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