Integrative biology of persister cell formation: molecular circuitry, phenotypic diversification and fitness effects

Abstract

Microbial populations often contain persister cells, which reduce the extinction risk upon sudden stresses. Persister cell formation is deeply intertwined with physiology. Due to this complexity, it cannot be satisfactorily understood by focusing only on mechanistic, physiological or evolutionary aspects. In this review, we take an integrative biology perspective to identify common principles of persister cell formation, which might be applicable across evolutionary-distinct microbes. Persister cells probably evolved to cope with a fundamental trade-off between cellular stress and growth tasks, as any biosynthetic resource investment in growth-supporting proteins is at the expense of stress tasks and vice versa. Natural selection probably favours persister cell subpopulation formation over a single-phenotype strategy, where each cell is prepared for growth and stress to a suboptimal extent, since persister cells can withstand harsher environments and their coexistence with growing cells leads to a higher fitness. The formation of coexisting phenotypes requires bistable molecular circuitry. Bistability probably emerges from growth-modulated, positive feedback loops in the cell's growth versus stress control network, involving interactions between sigma factors, guanosine pentaphosphate and toxin-antitoxin (TA) systems. We conclude that persister cell formation is most likely a response to a sudden reduction in growth rate, which can be achieved by antibiotic addition, nutrient starvation, sudden stresses, nutrient transitions or activation of a TA system.

Keywords: mathematical models; microbial fitness and bet hedging; persister cell microbiology; systems biology.

Figure 1.

The middle-way approach to understand persister cell formation in an integrative manner. An overview of the three different scales (evolutionary, physiological and molecular) that we aim to integrate to achieve an improved molecular–physiological–evolutionary understanding of persister cell formation.

Figure 2.

Graphical description of differences in a genotype able to make persisters and one that is not. (a) A visual representation of a genotype I (green) which is able to form persister cell (PC) (orange), and genotype 2 (purple) which is not. The fraction of persister (ϕ) in genotype 2 is therefore always 0, which means that its growth rate during unstressed conditions is higher than that of genotype 1 with ϕ greater than 0. However, a severe stressful condition (dark grey block) would be able to kill all cells of genotype 2 (b,c), leading to extinction. Different shades of grey represent different environmental conditions. See electronic supplementary material, B–D and G for a coarse-grained mathematical description of short- and long-term fitness effects of forming a persister fraction as depicted here.

Figure 3.

Mode of action of type II TA systems, which appears inherently bistable. A schematic overview of the mode of action of a type II TA system, which appears inherently bistable. It shows several interlocked, self-perpetuating positive feedbacks that are in principle capable of giving rise to bistability. All the sigma factor dependencies were identified in EcoCyc (www.ecocyc.org). Note that the influence of the toxin on σ^H and σ^E is toxin dependent and is exerted through σ^S [76]. Some toxins also directly influence protein synthesis. (Note that for ribosome targeting antibiotics, like tetracycline, the relation between growth rate and ppGpp is opposite from depicted here [43].)

Figure 4.

How different toxins and external stresses all reduce slow growth, providing a general trigger for persister cell formation. Different toxins (their antitoxin counterparts not visualized) and stresses (e.g. antibiotics) inhibit transcription, translation and replication through different mechanisms. Regardless of their causal mechanisms, these all lead to a reduction of cellular growth rate. Slow growth or growth halt seems a general stimulus for increasing the chance that an initially growing cell switches to a persister phenotype. Toxins are shown in orange, sigma factors in dark blue, antibiotics in light blue, DksA in pink, RNAP in green, mRNA in white, a ribosome in dark green and ppGpp in purple.