Emergence of growth and dormancy from a kinetic model of the Escherichia coli central carbon metabolism

Physiological states of bacterial cells exhibit a wide spectrum of timescale. Under nutrient-rich conditions, most of the cells in an isogenic bacterial population grow at certain rates, while a small subpopulation sometimes stays in a dormant state where the growth rates slow down by orders of magnitude. For revealing the origins of such heterogeneity of timescales, we studied the kinetic model of Escherichia coli central carbon metabolism including the dynamics of the energy currency molecules. We found that the model robustly exhibits both the growing- and the dormant state. In order to unveil the mechanism of distinct behaviours, we developed a recursive method to simplify the model without changing the qualitative feature of the dynamics. Analytical and numerical studies of the 2-variable minimal model revealed the necessary conditions for the distinct behaviour, namely, the depletion of energy due to the futile cycle and its non-uniform impact on the kinetics because of the coexistence of the energy currency-coupled and uncoupled reactions as well as branching of the network. The result is consistent with the experimental reports that the dormant cells commonly exhibit low ATP levels, and provides a possible explanation for the appearance of dormant cells that causes antibiotic persistence.

Functional heterogeneity of cell populations increases robustness of pacemaker function in a numerical model of the sinoatrial node tissue

Each heartbeat is initiated by specialized pacemaker cells operating within the sinoatrial node (SAN). While individual cells within SAN tissue exhibit substantial heterogeneity of their electrophysiological parameters and Ca cycling, the role of this heterogeneity for cardiac pacemaker function remains mainly unknown. Here we investigated the problem numerically in a 25x25 square grid of coupled-clock Maltsev-Lakatta cell models and tested the hypothesis that functional heterogeneity of cell populations increases robustness of SAN function. The tissue models were populated by cells with different degree of heterogeneity of the two key model parameters of the coupled-clock system, maximum L-type Ca current conductance (gCaL) and sarcoplasmic reticulum Ca pumping rate (Pup). Our simulations showed that in the areas of Pup-gCaL parametric space at the edge of the system stability where action potential (AP) firing was absent or dysrhythmic in tissues populated by identical cells, rhythmic AP generation was rescued in tissues populated by cells with uniformly random distributions of gCaL or Pup (but keeping the same average values). This effect to increase robust AP generation was synergistic with respect to heterogeneity in both gCaL and Pup and was further strengthened by clustering of cells with higher gCaL or Pup. The effect of functional heterogeneity was not due to a simple summation of activity of intrinsically firing cells naturally present in SAN; rather AP firing cells locally and critically interacted with non-firing/dormant cells. When firing cells prevailed, they recruited many dormant cells to fire, strongly enhancing overall SAN function. And vice versa, prevailing dormant cells suppressed AP firing in cells with intrinsic automaticity and halted SAN automaticity.

Noise in a metabolic pathway leads to persister formation in Mycobacterium tuberculosis

Tuberculosis is difficult to treat due to dormant cells in hypoxic granulomas, and stochastically-formed persisters tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that noise in the expression of an energy-generating component will produce rare persister cells. In sorted low ATP M. tuberculosis grown on acetate there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters and suggests that the mechanism of their antibiotic tolerance is similar to that of dormant cells in a granuloma. Entrance into a low energy state driven by stochastic variation in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.

Actively growing cells are the predominant persisters in exponential phase of Escherichia coli

Bacterial persistence is a phenomenon in which a small fraction of isogenic bacterial cells survives a lethal dose of antibiotics. It is generally assumed that persistence is caused by growth-arrested dormant cells generated prior to drug exposure. However, evidence from direct observation is scarce due to extremely low frequencies of persisters, and is limited to high persistence mutants or to conditions that significantly increase persister frequencies. Here, utilizing a microfluidic device with a membrane-covered microchamber array, we visualize the responses of more than 106 individual cells of wildtype Escherichia coli to lethal doses of antibiotics, sampling cells from different growth phases and culture media. We show that preexisting dormant persisters constitute only minor fractions of persistent cell lineages in populations sampled from exponential phase, and that most persistent cell lineages grew actively before drug exposure. Actively growing persisters exhibit radical morphological changes in response to drug exposure, including L-form-like morphologies or filamentation depending on antibiotic type, and restore their rod-like shape after drug removal. Incubating cells under stationary phase conditions increases both the frequency and the probability of survival of dormant cells. While dormant cells in late stationary phase express a general stress response regulator, RpoS, at high levels, persistent cell lineages tended to show low to moderate RpoS expression among the dormant cells. These results demonstrate that heterogeneous survival pathways may coexist within bacterial populations to achieve persistence and that persistence does not necessarily require dormant cells.

The Formation of Spore-Like Akinetes: A Survival Strategy of Filamentous Cyanobacteria

Some cyanobacteria of the order Nostocales can form akinetes, spore-like dormant cells resistant to various unfavorable environmental fluctuations. Akinetes are larger than vegetative cells and contain large quantities of reserve products, mainly glycogen and the nitrogen storage polypeptide polymer cyanophycin. Akinetes are enveloped in a thick protective coat containing a multilayered structure and are able to germinate into new vegetative cells under suitable growth conditions. Here, we summarize the significant morphological and physiological changes that occur during akinete differentiation and germination and present our investigation of the physiological function of the storage polymer cyanophycin in these cellular processes. We show that the cyanophycin production is not required for formation and germination of the akinetes in the filamentous cyanobacterium <i>Anabaena variabilis</i> ATCC 29413.

Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS

The bactericidal activity of several commonly used antibiotics have been shown to partially rely on the production of reactive oxygen species (ROS). Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. However, even dormant cells must maintain a membrane potential. Here we used Bacillus subtilis as model system to study the effect of membrane depolarization on dormant cells. Surprisingly, we found that membrane depolarization also leads to ROS production. In contrast to previous studies, this does not require the Fenton reaction and results primarily in superoxide radicals. Genetic analysis revealed that Rieske factor QcrA, the iron-sulfur subunit of complex III, is a primary source of superoxide radicals. Interestingly, the membrane distribution of QcrA changed upon membrane depolarization, suggesting a dissociation of complex III. Our data reveal an alternative mechanism by which antibiotics can cause lethal ROS levels, and may partially explain why membrane-targeting antibiotics are effective in eliminating persisters.

A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy

Cancer cells disseminate from primary tumors and seed in distant organs, where they can remain dormant for many years before forming clinically detectable metastases. Little is known about how extracellular matrix (ECM) sensing and remodeling can induce and sustain dormancy of disseminated tumor cells (DTCs). Another unanswered question is whether dormant cells themselves are able to assemble ECM niches to sustain their phenotype. From an analysis of the ECM proteome, we found that dormant cancer cells assemble an ECM niche enriched in type III collagen. Tumor-derived, but not stroma-derived type III collagen is required to sustain tumor dormancy as its disruption restores proliferation of dormant cancer cells. Mechanistically, we show that type III collagen interacts with DDR1 to activate STAT1 signaling to induce and maintain dormancy. Second harmonic generation two-photon microscopy further reveals that the dormancy-to-reactivation transition is accompanied by changes in collagen three-dimensional architecture and type III collagen abundance. In vivo, exogenous type III collagen stops tumor growth by inducing dormancy and prevents reawakening of residual dormant cells by prolonging their quiescence. Analysis of clinical samples reveals that type III collagen levels are increased in tumors from head and neck squamous cell carcinoma (HNSCC) lymph node negative patients when compared lymph node positive patients. Our data reveal a novel mechanism by which dormant DTCs depend on the assembly of a type III collagen-rich ECM niche to maintain quiescence. Manipulation of these mechanisms could serve as a self-sustained barrier to metastasis through DTCs dormancy induction.

2-Heptylcyclopropane-1-Carboxylic Acid Disperses and Inhibits Bacterial Biofilms

Fatty-acid signaling molecules can inhibit biofilm formation, signal dispersal events, and revert dormant cells within biofilms to a metabolically active state. We synthesized 2-heptylcyclopropane-1-carboxylic acid (2CP), an analog of cis-2-decenoic acid (C2DA), which contains a cyclopropanated bond that may lock the signaling factor in an active state and prevent isomerization to its least active trans-configuration (T2DA). 2CP was compared to C2DA and T2DA for ability to disperse biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. 2CP at 125 μg/ml dispersed approximately 100% of S. aureus cells compared to 25% for C2DA; both 2CP and C2DA had significantly less S. aureus biofilm remaining compared to T2DA, which achieved no significant dispersal. 2CP at 125 μg/ml dispersed approximately 60% of P. aeruginosa biofilms, whereas C2DA and T2DA at the same concentration dispersed 40%. When combined with antibiotics tobramycin, tetracycline, or levofloxacin, 2CP decreased the minimum concentration required for biofilm inhibition and eradication, demonstrating synergistic and additive responses for certain combinations. Furthermore, 2CP supported fibroblast viability above 80% for concentrations below 1 mg/ml. This study demonstrates that 2CP shows similar or improved efficacy in biofilm dispersion, inhibition, and eradication compared to C2DA and T2DA and thus may be promising for use in preventing infection for healthcare applications.

Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.

Breast cancer dormancy: need for clinically relevant models to address current gaps in knowledge

Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient’s lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance.