Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria

Imdahl, Fabian; Vafadarnejad, Ehsan; Homberger, Christina; Saliba, Antoine‐Emmanuel; Vogel, Jörg

doi:10.1038/s41564-020-0774-1

Cited by 137 publications

(121 citation statements)

References 31 publications

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“…Perhaps the surviving population after six hours of ampicillin treatment are a subpopulation present after three hours, but the expression profile is overshadowed by other subpopulations that are killed later. Understanding transcriptomic heterogeneity in the tolerant population would necessitate further experiments, such as single-cell transcriptomics, which has only recently been demonstrated in bacteria 95 . Regardless, future experiments (ribosome profiling, mass spectrometry, single-cell transcriptomics etc.)…”

Section: Resultsmentioning

confidence: 99%

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Deter

Hossain

Butzin

2020

Preprint

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In a given bacterial population, antibiotic treatment will kill a large portion of the population, while a small, tolerant subpopulation will survive. Tolerant cells disrupt the efficacy of antibiotic treatment and increase the likelihood that a population gains antibiotic resistance. When a population becomes resistant, antibiotic treatment fails, which is a major health concern. Since antibiotic tolerance often leads to resistance, we have taken a systems biology approach to examine how transcriptional networks respond to antibiotic stress so that cells can survive and recover after antibiotic treatment. We have compared gene expression with and without ampicillin in E. coli. While many previous studies have identified aspects of the antibiotic stress response at either the protein or transcriptional level, our work leverages existing knowledge of transcriptional regulation to link the dynamics of the two allowing for a more holistic approach. Here, we are the first to develop a whole-cell, transcriptional regulatory network (TRN) of antibiotic tolerant subpopulations and examine how cells respond at the transcriptional level to bactericidal concentrations of an antibiotic. Using TRN analysis, we show how certain sigma and transcription factors are affecting transcriptional regulation under antibiotic stress, and that changes in gene expression specific to ampicillin treatment can be directly linked to cell survival and recovery. The resulting network demonstrates that cells are mounting an active and coordinated response to the antibiotic that spans multiple systems and pathways. The redundancy and interconnectivity of the networks suggest that accounting for whole-cell dynamics may be crucial when modeling and studying antibiotic tolerance in the future.

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Section: Resultsmentioning

confidence: 99%

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Deter

Hossain

Butzin

2020

Preprint

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“…However, this method did not deplete rRNA, resulting in mRNA detection efficiencies of~2.5-10% (or~200 mRNA per exponential phase E. coli cell). As an alternate approach, Imdahl et al used MATQ-seq to generate sufficient cDNA from individually isolated bacterial cells; however, similar to the previous work, this method also did not deplete rRNA prior to sequencing [43]. In another study, Kuchina et al combined rRNA depletion with combinatorial barcoding to achieve~5-10% mRNA detection efficiencies in B. subtilis [14].…”

Section: Discussionmentioning

confidence: 99%

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

et al. 2020

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Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.

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“…Single cell analyses that are capable of addressing small subpopulations of intracellular STM, such as the approaches applied here, provide a more precise insight into the physiological state of persisters. Novel single cell transcriptomics approaches for bacteria are emerging (Imdahl et al, 2020) and may provide a future option for analyses of persisters.…”

Section: Discussionmentioning

confidence: 99%

The protected physiological status of intracellularSalmonella entericapersisters reduces host cell-imposed stress

Schulte

Olschewski

Hensel

2020

Preprint

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Today, we are faced with increasingly occurring bacterial infections that are hard to treat and often tend to relapse. These recurrent infections can occur possibly due to antibiotic-tolerant persister cells. Antibiotic persistent bacteria represent a small part of a bacterial population that enters a non-replicating (NR) state arising from phenotypic switching. Intracellularly, after uptake by phagocytic cells, Salmonella enterica serovar Typhimurium (STM) forms persister cells that are able to subvert immune defenses of the host. However, the clear physiological state and perceptual properties are still poorly understood and many questions remain unanswered. Here we describe further development of fluorescent protein-based reporter plasmids that were used to detect intracellular NR persister cells and monitor the expression of stress response genes via extensive flow cytometric analyses. Moreover, we performed extensive measurements of the metabolic properties of NR STM at the early course of infection. Our studies demonstrate that NR STM persister cells perceive their environment and are capable respond to stress factors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not a consequence of a lower metabolic capacity, the persistent status of STM serves as protective niche. Furthermore, up to 95% of NR STM were metabolically active at the beginning of infection additionally showing no difference in the metabolic capacity compared to R STM. The accessory capability of NR STM persisters to sense and to react to stress with constant metabolic activity may supports the pathogen to create a more permissive environment for recurrent infections.

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Single-cell RNA-sequencing reports growth-condition-specific global transcriptomes of individual bacteria

Cited by 137 publications

References 31 publications

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

The protected physiological status of intracellularSalmonella entericapersisters reduces host cell-imposed stress

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