Persisters are a small subpopulation of cells that are tolerant to antibiotic treatment and could resuscitate once the antibiotic concentration has dropped. It has recently received substantial attention in clinics as the major risk of the relapse of various infectious diseases. Different from the more well-understood mechanisms of resistance that occurs through genetic alterations, persistence is more of a phenotypic state arising from a perturbed biological network of numerous interacting molecules. Thus, proteomics is expected to be useful in providing fresh insights on bacterial persistence. However, the inherent transience and low abundance of persisters makes it technically challenging to study them by proteomics. This is why it has only been used to study persisters in some biofi...[ Read more ]

Persisters are a small subpopulation of cells that are tolerant to antibiotic treatment and could resuscitate once the antibiotic concentration has dropped. It has recently received substantial attention in clinics as the major risk of the relapse of various infectious diseases. Different from the more well-understood mechanisms of resistance that occurs through genetic alterations, persistence is more of a phenotypic state arising from a perturbed biological network of numerous interacting molecules. Thus, proteomics is expected to be useful in providing fresh insights on bacterial persistence. However, the inherent transience and low abundance of persisters makes it technically challenging to study them by proteomics. This is why it has only been used to study persisters in some biofilm-forming bacteria as they are more abundant and easier to isolate in biofilms. In planktonic culture such as those in Escherichia coli, which is the model species on which most of non-proteomics studies of persisters were based, the amount of persisters is too low for proteomics analysis and they are mixed together with the normal growing cells.

Here, we adopt the chemical pretreatment method using rifampin to increase the persister population in E. coli for proteomics analysis, and then used ampicillin to kill the non-persisters. To eliminate the effect of dead cells accumulation from antibiotic treatment, we developed a magnetic beads-based separation approach to enrich the persisters and then subjected them to shotgun proteomics. By spectral counting-based quantification, we observed that 105 proteins (70 down-regulated, 35 up-regulated) were differentially expressed compared to normal cells. Through comparison of the differentially expressed proteins between the enriched persisters and nonenriched persisters, we showed that persister isolation is important to minimize the cumulative effect of dead cells. Some proteins involved in carbohydrate metabolism, fatty acid and amino acid biosynthesis 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, which is one of the most up-regulated protein detected in persisters, led to 10-fold reduced persister formation under ampicillin treatment.

Keywords: Proteomics, persistence, persisters, tolerance, antibiotics