Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.

Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment. IMPORTANCE Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism's phenotypic survival to antibiotics; however, this hypothesis remains to be tested. Here, we demonstrate that even before antibiotic treatment, cells that will become persisters have a more acidic intracellular pH than clonal cells that will be either susceptible or VBNC upon antibiotic treatment. Moreover, after antibiotic treatment, persisters become more alkaline than VBNC and susceptible E. coli cells. This newly found phenotypic feature is remarkable because it distinguishes persister and VBNC cells that have often been thought to display the same dormant phenotype. We then show that this differential pH regulation is abolished in the absence of the enzyme tryptophanase via a major remodeling of bacterial metabolism and pH homeostasis. These new whole-genome transcriptome data should be taken into account when modeling bacterial metabolism at the crucial transition from exponential to stationary phase. Overall, our findings indicate that the manipulation of the intracellular pH represents a bacterial strategy for surviving antibiotic treatment. In turn, this suggests a strategy for developing persister-targeting antibiotics by interfering with cellular components, such as tryptophanase, that play a major role in pH homeostasis.

Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner.

Bacterial cells are critically dependent upon pH regulation. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH of Escherichia coli. Indole is an aromatic molecule with diverse signalling roles. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry. We show that under conditions permitting indole production, cells maintain their cytoplasmic pH at 7.2. In contrast, under conditions where no indole is produced, the cytoplasmic pH is near 7.8. We demonstrate that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, we illustrate that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a "memory" to maintain the cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole provides E. coli with a degree of protection against stresses, including some bactericidal antibiotics.

New Approaches and Therapeutic Options for Mycobacterium tuberculosis in a Dormant State.

We are far away from the days when tuberculosis (TB) accounted for 1 in 4 deaths during the 19th century. However, Mycobacterium tuberculosis complex (MTBC) strains are still the leading cause of morbidity and mortality by a single infectious disease, with 9.6 million cases and 1.5 million deaths reported. One-third of the world's population is estimated by the WHO to be infected with latent TB. During the last decade, several studies have aimed to define the characteristics of dormant bacteria in these latent infections. General features of the shift to a dormant state encompass several phenotypic changes that reduce metabolic activity. This low metabolic state is thought to increase the resistance of MTBC strains to host/environmental stresses, including antibiotic action. Once the stress ceases (e.g., interruption of treatment), dormant cells can reactivate and cause symptomatic disease again. Therefore, a proper understanding of dormancy could guide the rational development of new treatment regimens that target dormant cells, reducing later relapse. Here, we briefly summarize the latest data on the genetics involved in the regulation of dormancy and discuss new approaches to TB treatment.

Indole modifies the central carbon flux in the anaerobic metabolism of Escherichia coli: application to the production of hydrogen and other metabolites.

Indole is a bicyclic signaling molecule with effects on both eukaryotic and prokaryotic cells. The majority of studies of indole action have been performed with bacteria cultured under aerobic conditions and little information is available about its effects under anaerobic conditions. Here the effect of the indole on anaerobic metabolism of Escherichia coli WDHL was studied. Indole in the range 0.5-8mM was added to the culture medium and cell growth, hydrogen and metabolite production were compared to cultures lacking indole. Results showed that while 8mM indole abolished growth completely, 4mM indole had a partial bacteriostatic effect and the maximum optical density of the culture decreased by 44% compared to the control cultures. In addition, 4mM indole had an important effect on anaerobic metabolism. Hydrogen production increased from 650±115 to 1137±343mL H2/L, and hydrogen yield increased from 0.45±0.1 to 0.94±0.34mol H2/mol glucose, compared to the control culture. Carbon flux was also affected and the composition of the final by-products changed. Lactate (41mM) was the main metabolite in the control cultures, whereas ethanol (56.2mM) and acetate (41.2mM) were the main metabolites in the cultures with 2mM indole. We conclude that the supplementation of E. coli cultures with exogenous indole is a simple and novel strategy to improve the production of hydrogen as well as other metabolites such as ethanol used as biofuels.