The Persistence-Inducing Toxin HokB Forms Dynamic Pores That Cause ATP Leakage

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coil via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB. IMPORTANCE There is increasing awareness of the clinical importance of persistence. Indeed, persistence is linked to the recalcitrance of chronic infections, and evidence is accumulating that persister cells constitute a pool of viable cells from which resistant mutants can emerge. Unfortunately, persistence is a poorly understood process at the mechanistic level. In this study, we unraveled the pore-forming activity of HokB in E. coil and discovered that these pores lead to leakage of intracellular ATP, which is correlated with the induction of persistence. Moreover, we established a link between persistence and pore activity, as the number of HokBinduced persister cells was strongly reduced using a channel blocker. The latter opens opportunities to reduce the number of persister cells in a clinical setting

A novel bacterial cell death pathway targeted by a poisoned cell cycle sentinel

The relentless increase in bacterial resistance is a major global health threat. The expanding number of people that succumb to bacterial infections due to a lack of effective treatment is extremely alarming. Mankind is therefore in urgent need of novel and innovative antibacterial compounds. The development of such antibacterial therapies could be aided by a deepened understanding of bacterial physiology. Besides an obvious fundamental interest, increased insight into how bacteria proliferate and sustain viability could reveal new points of interference that can be targeted by future antibiotics. While it has been firmly established that the GTPase Obg is vital for bacterial proliferation and plays an important role in the bacterial cell cycle, its precise cellular function is currently unknown. Nonetheless, efforts are being made to identify antibacterial compounds that target the Obg protein. Investigation of Obg functionality is hampered by the inability to create obg deletion mutants, the fact that depletion and overexpression of Obg often have similar effects and the occurrence of very pleiotropic phenotypes. We here use a dominant-negative isoform of the ObgE protein (Obg of Escherichia coli), ObgE*, as a tool to study ObgE function. Insights gleaned from this approach could support the development of Obg-targeting therapies. The mutation present in ObgE* is intriguing because it changes ObgE from a protein that is essential for bacterial survival to a highly lethal one. Upon expression of ObgE* in E. coli, cells rapidly lose viability because ObgE* irreversibly blocks progression through the cell cycle. It most likely does so by disturbing the assembly of FtsZ at midcell, thereby inhibiting cell division. Since the mutant ObgE protein described here strongly interferes with normal cell cycle progression, and because ObgE is known to be involved in cell cycle control, we hypothesize that the normal role of wild-type ObgE in the bacterial cell cycle is disturbed by the mutation present in ObgE*. Studying how ObgE* affects cell cycle progression and cell division can thus provide insight into the enigmatic role of ObgE in these processes. ObgE*-mediated toxicity can be almost completely neutralized by mutations in lpxA. The LpxA gene product catalyzes a reversible reaction that, in the forward direction, uses peptidoglycan and phospholipid precursors to produce an intermediate in lipopolysaccharide synthesis. LpxA therefore functions as a hub in cell envelope metabolism and changes in LpxA function could affect not only lipopolysaccharide synthesis but also the production of peptidoglycan and/or membrane phospholipids. Since changes in cell envelope metabolism restore the cell cycle progression defect imposed by ObgE*, our results strongly implicate cell envelope homeostasis in regulation of the bacterial cell cycle. This possible connection has been severely understudied but is potentially a very significant factor in bacterial proliferation. In conclusion, our results reveal that ObgE* inhibits cell cycle progression at the stage of cell division most likely by interfering with cell envelope metabolism. A connection between ObgE and the bacterial cell envelope has never been reported before. Moreover, although it is known that ObgE influences the cell cycle, this is the first time that Obg is suggested to affect cell division directly. These newly discovered connections are therefore of high interest for further research on the Obg protein and will likewise increase our understanding of the bacterial cell cycle and, more specifically, how it is influenced by the cell envelope. Importantly, Obg, the bacterial cell cycle, cell division and the cell envelope are all considered highly attractive targets for antibiotic development. Uncovering previously unknown connections between them could therefore lead to the discovery of new points of interference that can be targeted by novel antibacterial compounds.nrpages: 236status: publishe

Bacterial Heterogeneity and Antibiotic Survival: Understanding and Combatting Persistence and Heteroresistance

For decades, mankind has dominated the battle against bacteria, yet the tide is slowly turning. Our antibacterial strategies are becoming less effective, allowing bacteria to get the upper hand. The alarming rise in antibiotic resistance is an important cause of anti-infective therapy failure. However, other factors are at play as well. It is widely recognized that bacterial populations display high levels of heterogeneity. Population heterogeneity generates phenotypes specialized in surviving antibiotic attacks. Nonetheless, the presence of antibiotic-insensitive subpopulations is not considered when initiating treatment. It is therefore time to reevaluate how we combat bacterial infections. We here focus on antibiotic persistence and heteroresistance, phenomena in which small fractions of the population are tolerant (persisters) and resistant to antibiotics, respectively. We discuss molecular mechanisms involved, their clinical importance, and possible therapeutic strategies. Moving forward, we argue that these heterogeneous phenotypes should no longer be ignored in clinical practice and that better diagnostic and therapeutic approaches are urgently needed.status: publishe

An integrative view of cell cycle control in Escherichia coli

Bacterial proliferation depends on the cells' capability to proceed through consecutive rounds of the cell cycle. The cell cycle consists of a series of events during which cells grow, copy their genome, partition the duplicated DNA into different cell halves and, ultimately, divide to produce two newly formed daughter cells. Cell cycle control is of the utmost importance to maintain the correct order of events and safeguard the integrity of the cell and its genomic information. This review covers insights into the regulation of individual key cell cycle events in Escherichia coli. The control of initiation of DNA replication, chromosome segregation and cell division is discussed. Furthermore, we highlight connections between these processes. Although detailed mechanistic insight into these connections is largely still emerging, it is clear that the different processes of the bacterial cell cycle are coordinated to one another. This careful coordination of events ensures that every daughter cell ends up with one complete and intact copy of the genome, which is vital for bacterial survival.status: publishe

Reactive oxygen species do not contribute to ObgE*-mediated programmed cell death

Programmed cell death (PCD) in bacteria is considered an important target for developing novel antimicrobials. Development of PCD-specific therapies requires a deeper understanding of what drives this process. We recently discovered a new mode of PCD in Escherichia coli that is triggered by expression of a mutant isoform of the essential ObgE protein, ObgE*. Our previous findings demonstrate that ObgE*-mediated cell death shares key characteristics with apoptosis in eukaryotic cells. It is well-known that reactive oxygen species (ROS) are formed during PCD in eukaryotes and play a pivotal role as signaling molecules in the progression of apoptosis. Therefore, we explored a possible role for ROS in bacterial killing by ObgE*. Using fluorescent probes and genetic reporters, we found that expression of ObgE* induces formation of ROS. Neutralizing ROS by chemical scavenging or by overproduction of ROS-neutralizing enzymes did not influence toxicity of ObgE*. Moreover, expression of ObgE* under anaerobic conditions proved to be as detrimental to bacterial viability as expression under aerobic conditions. In conclusion, ROS are byproducts of ObgE* expression that do not play a role in the execution or progression of ObgE*-mediated PCD. Targeted therapies should therefore look to exploit other aspects of ObgE*-mediated PCD.status: publishe

Slapende bacteriën ontsnappen aan antibiotica

status: publishe

A single-amino-acid substitution in Obg activates a new programmed cell death pathway in Escherichia coli

Programmed cell death (PCD) is an important hallmark of multicellular organisms. Cells self-destruct through a regulated series of events for the benefit of the organism as a whole. The existence of PCD in bacteria has long been controversial due to the widely held belief that only multicellular organisms would profit from this kind of altruistic behavior at the cellular level. However, over the past decade, compelling experimental evidence has established the existence of such pathways in bacteria. Here, we report that expression of a mutant isoform of the essential GTPase ObgE causes rapid loss of viability in Escherichia coli. The physiological changes that occur upon expression of this mutant protein—including loss of membrane potential, chromosome condensation and fragmentation, exposure of phosphatidylserine on the cell surface, and membrane blebbing—point to a PCD mechanism. Importantly, key regulators and executioners of known bacterial PCD pathways were shown not to influence this cell death program. Collectively, our results suggest that the cell death pathway described in this work constitutes a new mode of bacterial PCD.status: publishe

GTP Binding Is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.status: publishe

GTP Binding Is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*–GTP, but not ObgE*–GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function

A Single-Amino-Acid Substitution in Obg Activates a New Programmed Cell Death Pathway in Escherichia coli.

Programmed cell death (PCD) is an important hallmark of multicellular organisms. Cells self-destruct through a regulated series of events for the benefit of the organism as a whole. The existence of PCD in bacteria has long been controversial due to the widely held belief that only multicellular organisms would profit from this kind of altruistic behavior at the cellular level. However, over the past decade, compelling experimental evidence has established the existence of such pathways in bacteria. Here, we report that expression of a mutant isoform of the essential GTPase ObgE causes rapid loss of viability in Escherichia coli. The physiological changes that occur upon expression of this mutant protein-including loss of membrane potential, chromosome condensation and fragmentation, exposure of phosphatidylserine on the cell surface, and membrane blebbing-point to a PCD mechanism. Importantly, key regulators and executioners of known bacterial PCD pathways were shown not to influence this cell death program. Collectively, our results suggest that the cell death pathway described in this work constitutes a new mode of bacterial PCD.SCOPUS: ar.jinfo:eu-repo/semantics/publishe