Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic infections and antibiotic treatment failure1–3. In Escherichia coli, toxin/antitoxin (TA) modules have been linked to persister formation4–6. The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance into stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers is associated with a 100–1000 fold increase in the likelihood of survival to antibiotic challenge. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ~1,600 proteins with median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2,300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.

The pathogen Bacillus cereus causes diarrheal disease in humans. In the small intestine, B. cereus has to deal with anaerobiosis, low oxidoreduction potential, and carbohydrate limitation conditions. To gain insight into the virulence potential of low density B. cereus cells in such an environment, we cultured bacteria in low and high oxidoreduction potential anoxic conditions and in fully oxic conditions and compared their full secretomes. A unique pattern of proteins assigned to virulence factors was revealed. Among the 57 virulence-related factors, 31 were found for the first time in the B. cereus secretome. The putative fourth component of hemolysin BL (HblB), enterotoxin FM, hemolysin II, and three new putative conserved enterotoxins were uncovered. Cross-comparison of the relative abundance of secreted proteins reveals that a restricted set comprising 19 proteins showed significant changes in response to redox condition changes. We complemented these results with transcriptomics data and confirmed the cytotoxicity of the B. cereus secretome toward Caco-2 human epithelial cells. Our data suggest that (i) the redox-dependent regulatory pathway may modulate the expression of a subset of virulence factors to ensure an appropriate response in a specific redox environment, and (ii) an early growth phase-dependent pathway could regulate the expression of several virulence factors, allowing B. cereus to infect a host whatever the redox conditions. This early growth phase-dependent pathway may function, at least partially, independently of the pleiotropic virulence gene regulator PlcR and may therefore be more specific to the B. cereus group.