Dataset Information

Proteomics Analysis of Escherichia coli Persisters

ABSTRACT: Persisters, a dormant and multi-drug tolerant subpopulation that are able to resuscitate after antibiotic treatment, have recently received considerable attentions as the major risk of the relapse of various infectious diseases in clinics. However, due to their low abundance and inherent mutability, it is extremely difficult to study them by proteomics. Here, we developed a magnetic beads-based separation approach to enrich Escherichia coli persisters and then subject them to Filter-Aided Sample Perparation (FASP) followed by LC-MS/MS analysis. We applied spectral counting-based quantitative proteomics to study the proteomic changes of E. coli persisters under high concentration of ampicillin treatment.

INSTRUMENT(S): LTQ

ORGANISM(S): Escherichia Coli

SUBMITTER: Jordy Evan Sulaiman

LAB HEAD: Henry Lam

PROVIDER: PXD010590 | Pride | 2018-10-16

REPOSITORIES: Pride

ACCESS DATA

Publications

Specific Enrichment and Proteomics Analysis of Escherichia coli Persisters from Rifampin Pretreatment.

Sulaiman Jordy Evan JE Hao Chunlin C Lam Henry H

Journal of proteome research 20181018 11

Bacterial persisters, a dormant and multidrug tolerant subpopulation that are able to resuscitate after antibiotic treatment, have recently received considerable attention as a major cause of relapse of various infectious diseases in the clinic. However, because of their low abundance and inherent transience, it is extremely difficult to study them by proteomics. Here we developed a magnetic-beads-based separation approach to enrich Escherichia coli persisters and then subjected them to shotgun ...[more]

PMID: 30336045

Similar Datasets

Project description:Several groups have shown that through evolution experiments, tolerance evolved rapidly under cyclic antibiotic treatment which then promote the emergence of resistance. In other words, intermittent antibiotic exposure will “train” the bacteria to become tolerant to the drug, and then boost the chances for them to become fully resistant. This evolutionary trajectory of evolving tolerance and resistance not only occurs in vitro, but also in clinical patients with MRSA blood infections. Despite the fact that repetitive antibiotic treatment will lead to tolerance and resistance development in bacterial populations, the difference on the evolutionary path between those treated with a single drug and drug combination remains unaddressed. In this study, we monitored the development of tolerance in MRSA by treating them with either daptomycin alone for 2 weeks (Scheme 1), or daptomycin combined with rifampin for two weeks (Scheme 2), in a cyclic manner. In addition, we also investigate another scenario where we treat MRSA cells with daptomycin alone for one week, and subsequently treat them with daptomycin and rifampin combination in the second week (Scheme 3). At the end of the evolution experiment, we observed that the population from Scheme 1 developed tolerance towards daptomycin, the population from Scheme 2 developed both tolerance and resistance towards daptomycin, while the population from Scheme 3 developed tolerance after a week of treatment, but then the tolerance phenotype diminished at the end of the second week due to the drug combination treatment. We subjected them to whole genome sequencing and identified single point mutations that are responsible for the observed phenotype. Through proteomics, we compared the proteome profile of the population evolved from scheme 1 (S1D14), scheme 2 (S2D14) and scheme 3 (S3D14), and we observed that these three populations employ distinct mechanisms to survive antibiotic treatment, suggesting that the treatment strategy for these populations should be tailored depending on the treatment conditions.

Project description:Single cell RNA sequencing (scRNAseq) has emerged as an essential technique in biology. Given the complexity and cost of experiments it is critical that they are well planned and executed. An essential component to scRNAseq is the ability to have biological replicates. This adds to the potential cost and complexity of the experiment, but is essential as it has been shown that false discoveries are possible when lacking information pertaining to cell origin. One option to overcome the financial and experimental constraints of biological replicates in scRNAseq data is to pool samples. Upon pooling it is essential to then understand the sample origin of each cell. Experiments in humans have shown that when pooling multiple genetically distinct individuals into one sample the genetic diversity (i.e., single nucleotide polymorphisms (SNP)) can be used to assign cells to their sample origin. This approach is called SNP-based demultiplexing. The question of whether lab species, like Pleurodeles waltl, and various other non-traditional model species harbor enough genetic diversity to enable such approaches is unclear. To formally test the whether SNP-based demultiplexing is possible across various species we designed this experiment in which three spleens from three different Pleurodeles waltl from animals expressing unique fluorescent genes (one female tgTol2(CAG:Nucbow CAG:Cytbow)Simon (5.67g weight, 10.8cm snout-to-tail length), one male tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon (5.36g, 11.1cm), and one female tgTol2(CAG:loxP-Cherry-loxP-H2B::YFP)Simon (6.25g, 10.8cm)) were collected. Spleens were filtered through 70um nylon filters followed by FACS, gating on fluorescent positive cells and excluding erythrocytes. After FACS cells were pooled into one tube and subjected to 10x Genomics 3' v3.1 scRNAseq aiming for 10,000 cells. Spleens were filtered through 70um nylon filters followed by FACS gating on fluorescent positive cells and excluding erythrocytes. Samples were prepared and sequenced according to 10x Genomics recommendations. After sequencing, using reads mapping to fluorescent genes to identify animal origin we then compared the ability of SNP-based demultiplexing to properly assign cells to the correct animal.

Dataset Information

Proteomics Analysis of Escherichia coli Persisters

Publications

Specific Enrichment and Proteomics Analysis of Escherichia coli Persisters from Rifampin Pretreatment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure