Heterogeneous Flagellar Expression in Single Salmonella Cells Promotes Diversity in Antibiotic Tolerance

Lyu, Zhihui; Yang, Angela; Villanueva, Patricia; Singh, Abhyudai; Ling, Jiqiang

doi:10.1128/mbio.02374-21

Cited by 27 publications

(15 citation statements)

References 54 publications

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“…While we do not detect distinct subpopulations, we do see a gradient in flagellar gene expression, with a wide range of expression of flagella genes within each growth phase. This observation is in accordance with previous findings describing heterogeneous expression of fliC in Salmonella directly associated with cell motility and the potential to evade host inflammatory responses (2, 34).…”

Section: Resultssupporting

confidence: 93%

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Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads

Homberger

Hayward

Barquist

et al. 2022

Preprint

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Bulk RNA-sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA-sequencing protocol that is based on MATQ-seq, achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based ribosomal RNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over growth revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regards to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria.

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Section: Resultssupporting

confidence: 93%

“…This will allow the exploration of the regulatory functions of sRNA on the single-cell level in future studies. Moreover, our data confirm previously described heterogeneity within the same cell population, especially regarding Salmonella pathogenicity genes and genes encoding components of the flagellum (32, 34).…”

Section: Discussionsupporting

confidence: 90%

Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads

Homberger

Hayward

Barquist

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…This latent variation in phenotypic plasticity reveals in unfavorable environmental condition 4 . Noise in gene expression can drive this heterogeneity, and heterogeneity is hypothesized to be a key player that regulates bet-hedging strategies to endure harsh environmental uctuation, such as bacterial persistence 2,42 . Persisters have reduced e cacy to antibiotic treatment and are a key contributor to the In this study, we used a powerful uctuation test framework to infer transient cell states that arise via reversible and non-genetic mechanisms, recently employed in probing cancer persistence 13,16,17 , to nd hidden features of bacterial persistence.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli cells are primed for survival before lethal antibiotic stress

Hossain

Singh

Butzin

2022

Preprint

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Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria–Delbrück fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from ~ 60–100 and ~ 40–70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for ~ 5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (> 7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

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Section: Discussionmentioning

confidence: 99%

Escherichia colicells are primed for survival before lethal antibiotic stress

Hossain

Singh

Butzin

2022

Preprint

Self Cite

View full text Add to dashboard Cite

Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbruck fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from ~60-100 and ~40-70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for ~5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

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Heterogeneous Flagellar Expression in Single Salmonella Cells Promotes Diversity in Antibiotic Tolerance

Abstract: Antibiotic resistance and tolerance pose a severe threat to human health. How bacterial pathogens acquire antibiotic tolerance is not clear.

Cited by 27 publications

References 54 publications

Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads

Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads

Escherichia coli cells are primed for survival before lethal antibiotic stress

Escherichia colicells are primed for survival before lethal antibiotic stress

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