Specific Enrichment and Proteomics Analysis ofEscherichia coliPersisters from Rifampin Pretreatment

Abstract

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 proteomics. Rifampin pretreatment was employed to increase persister formation, and the resulting cells were exposed to a high concentration of ampicillin (10× MIC) to remove nonpersisters. The survivors were analyzed by spectral counting-based quantitative proteomics. On average, 710 proteins were identified at a false discovery rate of 0.01 for enriched E. coli persisters. By spectral counting-based quantification, 105 proteins (70 down-regulated, 35 up-regulated) were shown to be differentially expressed compared with normal cells. A comparison of the differentially expressed proteins between the magnetic beads-enriched persisters and nonenriched persisters (a mixture of persisters and intact dead cells) shows only around half (∼58%) overlap and different protein–protein interaction networks. This suggest that persister enrichment is important to eliminate the cumulative effect of dead cells that will obscure the proteome of persisters. As expected, proteins involved in carbohydrate metabolism, fatty acid and amino acid biosynthesis, and bacterial chemotaxis were found to be down-regulated in the persisters. Interestingly, membrane proteins including some transport proteins were up-regulated, indicating that they might be important for the drug tolerance of persisters. Knockout of the pal gene expressing peptidoglycan-associated lipoprotein, one of the most up-regulated proteins detected in persisters, led to 10-fold reduced persister formation under ampicillin treatment.

ppGpp Ribosome Dimerization Model for Bacterial Persister Formation and Resuscitation

“…By inhibiting the activity of ribosomes, we have developed means to convert E. coli cells into a 20 population that consists solely of persister cells (6) so that insights may be made about persister cell resuscitation. Using this approach, which has been verified by six independent labs to produce bona fide persister cells (7)(8)(9)(10)(11)(12), we have shown that persister cells resuscitate rapidly primarily in response to external signals (13), such as fresh nutrients, rather than stochastically (14). Critically, persister cell resuscitation is heterogeneous and depends on the number of active ribosomes; once a threshold of active ribosomes is reached, persister cells elongate and divide (13).…”

Persister Cells Resuscitate via Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD

“…The persister cells generated in this way have been (i) confirmed eight ways (19), (ii) used to determine that persister cells wake via ribosome activation (19) and chemotaxis (20), (iii) used to show that the cells capable of resuscitation in a viable but not culturable 95 population are equivalent to persister cells (15), (iv) used to identify compounds that kill persister cells (27), and (v) used to show that the alarmone ppGpp directly creates persister cells by stimulating ribosome dimerization (16). In addition, our method to generate a high population of persister cells has been utilized by at least six independent groups (28)(29)(30)(31)(32)(33).…”

A Novel LysR-Type Global Regulator RpvA Controls Persister Formation and Virulence inStaphylococcus aureus

“…Faced with environmental challenges such as antibiotics, stresses and nutrient deficiency, bacteria can adjust the cellular metabolism in a timely manner to form dormant persister cells, which can tolerate high stress levels and bactericidal effect of antibiotics (5, 6, 65). Previous studies have shown that many metabolic pathways such as carbohydrate metabolism, lipid, nucleotide and amino acid metabolism are involved in the formation of persisters (66, 67). Based on the transcriptomics and metabolomics analyses, we systematically analyzed the pathways by which RpvA could regulate the metabolism of S. aureus (Figure 14).…”