The Dynamic Transition of Persistence toward the Viable but Nonculturable State during Stationary Phase Is Driven by Protein Aggregation

Abstract

Decades of research into bacterial persistence has been unable to fully characterize this antibiotic-tolerant phenotype, thereby hampering the development of therapies effective against chronic infections. Although some active persister mechanisms have been identified, the prevailing view is that cells become persistent because they enter a dormant state. We therefore characterized starvation-induced dormancy in Escherichia coli. Our findings indicate that dormancy develops gradually; persistence strongly increases during stationary phase and decreases again as persisters enter the viable but nonculturable (VBNC) state. Importantly, we show that dormancy development is tightly associated with progressive protein aggregation, which occurs concomitantly with ATP depletion during starvation. Persisters contain protein aggregates in an early developmental stage, while VBNC cells carry more mature aggregates. Finally, we show that at least one persister protein, ObgE, works by triggering aggregation, even at endogenous levels, and thereby changing the dynamics of persistence and dormancy development. These findings provide evidence for a genetically controlled, gradual development of persisters and VBNC cells through protein aggregation. IMPORTANCE While persistence and the viable but nonculturable (VBNC) state are currently investigated in isolation, our results strongly indicate that these phenotypes represent different stages of the same dormancy program and that they should therefore be studied within the same conceptual framework. Moreover, we show here for the first time that the dynamics of protein aggregation perfectly match the onset and further development of bacterial dormancy and that different dormant phenotypes are linked to different stages of protein aggregation. Our results thereby strongly hint at a causal relationship between both. Because many conditions known to trigger persistence are also known to influence aggregation, it is tempting to speculate that a variety of different persister pathways converge at the level of protein aggregation. If so, aggregation could emerge as a general principle that underlies the development of persistence which could be exploited for the design of antipersister therapies.

Keywords: ObgE; VBNC; antibiotic resistance; antibiotic tolerance; chronic infection; dormancy; persistence; protein aggregation; stationary phase.

FIG 1

The VBNC state, persistence, and protein aggregation are natural phenomena that occur in stationary phase. (A) At different time points, the number of CFU/ml of an E. coli culture was measured, as well as the number of viable cells, by counting SYTOX green-negative cells with flow cytometry. The difference between the two curves is the number of VBNC cells present in the culture. (B) The absolute number of persister cells after treatment with ofloxacin was followed over time (error bars are too small to be visible). (C) Microscopy images (phase contrast and GFP channels) of E. coli ibpA-msfGFP at different time points show the development of protein aggregates over time. Arrowheads point to aggregates that are visible in the phase contrast channel. Scale bar, 2 μm. (D) Quantitative analysis of microscopy images was performed to determine the percentage of cells that carry protein aggregates. Aggregation was evaluated by the presence of fluorescent IbpA-msfGFP foci and phase-bright structures. For every repeat and every time point, at least 50 cells were analyzed. Data are represented as averages ± SEM; n ≥ 3. For statistical analysis of the difference between the IbpA-msfGFP and Ph curves, individual time points were compared using paired t tests with the Holm-Sidak method for multiple comparisons; *, P < 0.05; ***, P < 0.001. Ph, phase bright.

FIG 2

Persistence is correlated with early-stage IbpA-msfGFP aggregates, while the deeply dormant VBNC state is correlated with late-stage Ph aggregates at the single-cell level. (A and B) E. coli ibpA-msfGFP was grown for 24 h and treated with ofloxacin to kill all antibiotic-sensitive cells. Regrowth of persisters on fresh medium was followed by time-lapse microscopy. Cells were classified into two groups, persister or non-persister, based on whether or not they were able to resume cell division within 16 h. Violin plots of the maximal IbpA-msfGFP fluorescence intensity (A) and maximal intensity in the phase contrast channel (B) are shown. (C and D) To sample cells at different stages of dormancy, E. coli ibpA-msfGFP was grown for 24, 40, or 72 h, and regrowth on fresh medium was monitored by time-lapse microscopy. Cells were classified into two groups, culturable or VBNC, based on whether or not they were able to resume cell division within 16 h. Violin plots of the maximal IbpA-msfGFP fluorescence intensity (C) and maximal intensity in the phase contrast channel (D) are shown. Black lines in the violin plots indicate median values. n is the number of cells measured. ***, P < 0.001; ****, P < 0.0001.

FIG 3

The persister protein ObgE accelerates protein aggregation, the development of persistence, and entry into the VBNC state. E. coli ibpA-msfGFP with pBAD33Gm (vector) or pBAD33Gm-obgE (ObgE) was grown for 72 h in the presence of the inducer arabinose. (A) At different time points, microscopy pictures were taken. Scale bar, 2 μm. (B and C) Quantitative analysis of microscopy images was performed to determine the percentage of cells that carry protein aggregates. Aggregation was evaluated by the presence of fluorescent IbpA-msfGFP foci (B) and phase-bright structures (C). For every repeat and every time point, at least 50 cells were analyzed. (D) At different time points, the number of CFU/ml was determined. (E) The absolute number of persister cells after treatment with ofloxacin was followed over time. Data are represented as averages ± SEM; n ≥ 3. Ph, phase bright.

FIG 4

Wild-type levels of ObgE correlate with the amount of protein aggregation. The fluorescence of E. coli cells containing the genomic markers ibpA-msfGFP, obgE-mCherry, or both was measured by flow cytometry after 24 h of growth. Four different biological repeats were performed. (A) Representative flow cytometry plots and corresponding Pearson’s correlation coefficients are shown. (B) The correlation coefficients for all four repeats are plotted. Statistical analysis indicates that the Pearson’s R values obtained for the sample that contains both ibpA-msfGFP and obgE-mCherry are significantly higher in all repeats (P < 0.0001).

FIG 5

ATP depletion associated with entry into stationary phase is required for ObgE to induce protein aggregation. (A) E. coli overexpressing obgE was grown normally (nonbalanced growth, NBG) or maintained in balanced growth (BG). At different time points, the percentage of cells that carry IbpA aggregates was determined. (B and C) The percentage of cells of a wild-type and ΔrelA ΔspoT culture that carry protein aggregates upon obgE overexpression was determined by quantitative analysis of microscopy images. Aggregation was evaluated by the presence of fluorescent IbpA-msfGFP foci (B) and phase-bright structures (C). (D and E) There is a correlation between cellular ATP levels and the tendency to develop either IbpA (D) or Ph (E) protein aggregates. Both correlations are highly significant (P < 0.0001). (F) E. coli ibpA-msfGFP with pBAD33Gm (vector) or pBAD33Gm-obgE (ObgE) was maintained in balanced growth (BG) with 0.5 mM arsenate (ars) to lower ATP levels. At different time points, the percentage of cells that carry IbpA aggregates was determined. The ObgE NBG curve from panel A is also shown to allow easy comparison.

FIG 6

Biochemical analysis of protein aggregates reveals their time-dependent development and nonrandom composition. (A and B) After isolation of protein aggregates from E. coli carrying an empty expression vector (A) or overexpressing obgE (B) at several different time points, Coomassie staining on protein gels was performed. Quantitative interpretation of these Coomassie-stained gels is shown as “protein mass.” Data are represented as averages ± SEM; n = 5. (C) COG functional categories were assigned to all aggregated proteins identified by MS, and an enrichment analysis was performed. The amount of enrichment of all identified categories at the time point where Ph aggregation reaches its plateau is shown (vector = 48 h, ObgE = 24 h). Significantly enriched COG categories (C, F, J, O; P < 0.05) are highlighted by including the description of the category inside the figure. All descriptions: A, RNA processing and modification; C, energy production and conversion; D, cell cycle control, cell division, and chromosome partitioning; E, Amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; q, secondary metabolites biosynthesis, transport, and catabolism; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms. (D) The amount of GyrA detected in the soluble protein fraction by Western blotting with anti-GyrA antibodies is shown. The detected signals were normalized to the first time point of the vector control (8 h).

FIG 7

Model for the development of bacterial dormancy through protein aggregation. Upon nutrient starvation in stationary phase, cellular ATP concentrations decrease. As a consequence, proteostasis cannot be maintained, and protein aggregates develop. Protein aggregation occurs progressively. In an early stage of development, protein aggregates are marked by the small chaperone IbpA. Later on, they develop further into phase-bright structures. These different stages of protein aggregation are tightly coupled to different stages of bacterial dormancy. Persister cells are characterized by early-stage IbpA aggregates, while deeply dormant VBNC cells contain more developed aggregates that can be seen in phase contrast microscopy.