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Bacterial persister cells constitute a subpopulation of genetically identical, metabolically slow-growing cells that are highly tolerant of antibiotics and other environmental stress conditions. Studies have demonstrated that gene loci known as toxin-antitoxin (TA) system play a central role in the persister state. Under normal growth conditions, antitoxins potently inhibit the activities of the toxins. In contrast, under conditions of stress, the antitoxins are selectively degraded, freeing the toxins to inhibit essential cellular processes, such as DNA replication and protein translation. This inhibition results in rapid growth arrest. In this Review, we highlight recent discoveries of these TA modules with a focus on the newly uncovered mechanisms, especially conditional cooperativity, that are used Escherichia coli and many other bacterial species can enter into a viable but nonculturable (VBNC) state, which is a survival strategy adopted by cells exposed to adverse environmental conditions to regulate cell growth and persistence. We will also discuss the resuscitation of VBNC state of Escherichia coli by pyruvate sensing and transport.
Toxin-antitoxin systems (TA systems) are widely present in many bacteria and archea. These Toxin-antitoxin systems are made up of a protein toxin and its related antitoxin. Toxin-antitoxin systems plays a key role in persister cell formation. In persister cell formation the toxins are activated randomly or by introducing some environmental stress, due to activation of toxins other physiological processes gets stopped and the cell enters dormancy or we can say formation of persister cells takes place. In normal growing cells these toxins are inactivated by antitoxins, in some cases when these antitoxins are not present or are inactive the toxins are inactivated by promoter mutations. Bacteria have different ways to survive stress conditions, Gram positive bacteria like Bacillus perform sporulation and make spores for survival to these types of stress conditions. Other bacteria have two distinct mechanisms in which they enter a dormant stage they are 1- VBNC (Viable but Non Culturable) cells 2- Persister cell formation. VBNC is a state in which bacteria are in deep dormancy, they experience a loss of culturability but they remain viable means they maintain viability. The reason for going into VBNC state may be stress due to less nutrient availability or antibiotic stress. When the stress conditions are eliminated from the environment the cells resuscitate from VBNC state, the process of re-establishing culturability is termed as resuscitation, some mechanisms of it is still unknown. There are many mechanisms of resuscitation of which one of them is Pyruvate Sensing and transport.
Toxin Antitoxin Systems And Its Types
Toxin-antitoxin systems consists of a protein toxin and its related antitoxin. Both toxin and antitoxin are transcribed at the same time from a single operon. It is found that in most cases antitoxin is found upstream to the toxin. Their expression is also coupled so that amount of both remains same. However recent studies have demonstrated that antitoxin is produced at a much higher level compared to toxins. The role of TA system is clear, when the antitoxin inhibits the toxin, the organisms grow normally. However during conditions of stress or lower nutrition availability, the antitoxin is inactivated allowing the toxins to function and inhibit growth. Therefore due to such type of mechanism of action of toxins, dormancy is induced and bacteria enters a non-replicating dormant stage.
There are in total seven types of Toxin antitoxin systems, which are classified based on antitoxin’s mechanism of action. The Type I systems characterize an RNA antitoxin, which binds to the toxin messenger RNA (mRNA) and prevents its translation. Examples of these systems include hok/sok, fst/RNAII, and tisB/IstR. Type II systems are the most abundant and best studied family of Toxin Antitoxin systems. The type II systems primarily contribute to formation of persister. These systems utilize a protein antitoxin that directly binds to its cognate toxin, neutralizing its toxic activity. The most studied of these systems is the MazEF system, and the RelBE and HipBA systems have been used widely for research. The Type III systems use an RNA antitoxin that binds to the protein toxin to inactivate it. These systems include the ToxIN and CptIN systems, which contribute to phage resistance.
The Type IV systems shows a protein antitoxin. These antitoxins protect the cellular targets of their related toxins. YeeU/YeeV is an example of a Type IV system, where the YeeV toxin prevents polymerization of the cytoskeletal proteins MreB and FtsZ. YeeU, the related antitoxin of YeeV, directly binds these proteins and enhances their bundling, preventing YeeV from acting on them. The Type V systems encode a protein antitoxin, which degrades the mRNA of its related toxin, preventing its translation. The example of this is the GhoT toxin damages the bacterial membrane leading to lysis. However, the GhoS antitoxin specifically degrades GhoT mRNA, preventing the expression of its toxic activity. The Type VI systems utilize a protein antitoxin, which promotes degradation of the toxin by ClpXP. The SocAB system consists of a SocB toxin that inhibits replication by binding the β sliding clamp. However, the SocA antitoxin controls SocB activity by acting as an adaptor between ClpXP and SocB, resulting in ClpXP-mediated degradation of SocB.
The Type VII systems have been recently discovered, in this the antitoxin is an enzyme that inactivates the toxin by oxidizing a cysteine residue. Examples of it is Hha, TomB. Figure 1 Toxin-antitoxin systems. Toxins are shown in orange and antitoxins in blue; activities that are nontoxic are in black font, whereas those that are toxic are in grey. Type I: the sRNA antitoxin base pairs with toxin sRNA to inhibit translation; the membrane lytic toxins function to depolarize the cell membrane and disrupt ATP synthesis. Type II: the antitoxin and toxin are proteins; under growth conditions, the toxin is bound to the antitoxin, which inhibits its activity. Both the antitoxin and, in most cases, the TA complex bind the TA promoter to repress transcription. Under stress conditions, cellular proteases such as lon and ClpXp are activated that preferentially cleave the antitoxins, freeing the toxins to inhibit growth by inhibiting translation or replication. Type III: the antitoxin sRNA is processed by the endoribonuclease (RNase) toxin, resulting in the formation of RNA pseudo knot–toxin complexes, which inhibit toxin activity. Type IV: the protein antitoxin stabilizes bacterial filaments, while the protein toxin destabilizes them; in the absence of the antitoxin, this toxin-mediated destabilization inhibits cell division. Type V: the antitoxin GhoS is an RNase specific for the toxin GhoT mA; under conditions of stress, the mRNA of the antitoxin is degraded by the Mqsr toxin, resulting in GhoT translation and membrane lysis. Type VI: the SocA antitoxin is an adaptor protein that binds the SocB toxin to promote its degradation by ClpXp. When not degraded, the toxin binds the sliding clamp to inhibit DNA replication.
Toxins Of Toxin-Antitoxin Systems Are Primarily Inactivated Through Promoter Mutation
Toxin Antitoxin (TA) systems have many functions such as in stress conditions, in persister cell formation and many more. TA systems have two components one is the protein toxin and its related antitoxin. The function of antitoxin is to supress the action of toxin in normally growing cell. In some cases the antitoxins are not functional or are inactivated, or in some cases also not present as in horizontal gene transfer. So in such conditions the toxins must be inactivated, so that is done primarily mutating the promoter of the toxin or mutating the chromosomal copies of gene. For the studies of these the toxins Ral R (type 1), MqsR (type 2), GhoT (type 5) and Hha (type 7) were used and the toxin inactivation and mutation analysis was done. Overall by using four toxins from four different TA systems, it was found that toxins were primarily inactivated by changes in their promoter rather than changes in the structural genes.
Bacterial Persister And Vbnc (Viable But Non Culturable) Cells
Bacteria have many ways to tackle the stress conditions they face, the stress conditions may be nutrition depletion or antibiotic exposure to the bacterial cells. Bacterial cells enter a dormant stage to tackle such situations. Bacteria often have two dormant phenotype:
- The VBNC (Viable but Non Culturable) state
- The Persister state
Bacterial persistence is a phenomenon where bacteria enter a dormant state, allowing non-replicating organisms to survive during stressful conditions. It was first observed in 1942 in staphylococcus population VBNC is a state where bacteria are in deep dormancy, they experience a loss of culturability but they remain viable i.e. they sustain their viability.
Both persisters and VBNC have been linked to chronic infections, both occur in biofilms. Antibiotic tolerance is also a distinguishing feature of such type of cells as cellular targets of most antibiotics are inactive during dormancy. Using certain techniques and some starvation conditions VBNC were created and it was found that majority of the remaining bacterial population was spherical with empty cystol and they can’t resuscitate but some of the bacteria resuscitated and behaved like persister cells( similar in antibiotic tolerance, morphology, resuscitation rates and metabolic activity). Hence it was concluded that VBNC and persisters describe the same stress induced phenotype but it is seen that persisters are more prevalent in the environment.
Figure 2 Experimental dormancy dynamics of antibiotic persistence and the VBNC state. Persister cells are isolated by exposing a growing culture of cells to a lethal dose of antibiotics. The cells that remain Culturable after treatment are called persisters. This typically produces the classic biphasic killing curve, with the slope of the initial phase (green dotted line) generated by the death of sensitive cells and the slope of the second phase (yellow dotted line) generated by the surviving persisters. Similarly, VBNC cells are isolated by applying a stress (e.g., cold temperature) or an antibiotic to a growing culture. Upon exposure to this stress, cells lose culturability in a variable amount of time (depending on the stress used) (blue line), however a large portion of the population remains viable but nonculturable (red dotted line) as is determined by a variety of assays that test for gene expression, membrane stability, and metabolic activity. When the inducing stress is removed and adequate conditions are met (vertical dotted grey line) cells begin to alter their physiology towards resuscitation. After a lag period (dependent on the stress and bacterial species) cells regain the ability to grow on nutrient media.
Role Of Toxin Antitoxin (Ta) System In Persister Cell Formation
Most of the bacterial cell die when they encounter stress conditions or antibiotics, only a few of the bacteria are able to resist such conditions and these multidrug tolerant phenotypes are called bacterial persisters. One of the way by which the bacteria do this is with the help of toxin antitoxin systems. Toxin-antitoxin systems plays a key role in persister cell formation. In persister cell formation the toxins are activated randomly or by introducing some environmental stress, due to activation of toxins other physiological processes gets stopped and the cell enters dormancy or we can say formation of persister cells takes place. In normal growing cells these toxins are inactivated by antitoxins which are present in very large amount compared to toxins.
The initial studies about the TA systems were started by Balaban, he used a strain carrying one of the gain of function alleles, known as hipA7, to tract bacterial persistence in a micro fluid device. In his studies he showed that hipA7 persisters were growth arrested bacteria in whole bacterial population, shortly after then HipA was shown to be a toxin that causes self-inhibition of growth in the absence its cognate HipB antitoxin, making HipBA as a toxin antitoxin system. These discoveries linked TA system with bacterial persistence for the first time. This HipBA was type 2 TA system, other type 2 systems involved in persistence are MazE, relE, ChpS, YefM, higB, mazF, yafQ, and yoeB in E. coli. Similar to type II TA system, the role of type I TA in persister formation has also been shown. An example is TisAB/IstR-1 system of E. coli where, TisB is a toxin and IstR-1is an anti-sense RNA acting as anti-toxin by binding to tisA (an untranslated open reading frame). The role of this locus in inducing persistence was extensively examined in bacteria due to SOS response and DNA repair. Additionally, the hokB/sokB type I TA modules in E.coli also play a role in persister formation.
Figure 3 TA systems and persistence. In growing cells (blue), the concentration of antitoxin exceeds that of the toxin. In persister cells (orange), the situation is reversed, with toxin concentration exceeding antitoxin concentration. Switching from one phenotypic state to the other requires the ratio of toxin to antitoxin to change. In a growing bacterial population, a small fraction of cells are in the persister state. Exposure to stress, such as nutrient deprivation or antibiotics, leads to the activation of bacterial proteases, especially Lon and ClpXP, which preferentially cleave antitoxins, resulting in an excess of toxin. This results is the rapid killing of the majority of actively growing cells; the exposure to stress may also activate a very small fraction of cells to switch into the persister state. Less is known about how cells emerge from the persister state, but one potential mechanism observed in multiple type IITA systems is conditional cooperativity.
Resuscitation of Viable But Nonculturable (Vbnc) Cells
Escherichia coli and many other bacteria can enter VBNC state, which is a survival strategy adapted by many cells in adverse conditions. VBNC cells can however resume cell division, the process of re-establishing culturability is known as resuscitation. There are many factors that can promote the restoration of culturability e.g. YeaZ promoting factor, catalase, α-ketoglutamate and pyruvate. E. coli and many other organisms use two-component systems (TCSs) for environmental sensing and cellular responses. A membrane-integrated histidine kinase perceives the stimulus, and a cytosolic response regulator mediates an appropriate output. There are two TCSs in E. coli, i.e., the BtsS/BtsR and YpdA/YpdB systems. Both TCSs respond to extracellular pyruvate, although with different affinities, and thus constitute a pyruvate-sensing network, BtsSR/YpdAB. YpdA/YpdB activation results in yhjX expression, whereas BtsS/BtsR activation promotes btsT expression.
A comprehensive study was carried out using two strains of Escherichia coli, one wild type and other mutant. The mutant lacked the pyruvate signalling network, which prevents expression of the transporters BtsT and YhjX. It was found that VBNC cells requires an amino-acid rich complex medium, and supplementation with 2 Mm pyruvate and also isotonic dilution that increases resuscitating cells. When external pyruvate is absent only small number of wild type cells were Culturable but none of the mutants grew out as colonies. When external pyruvate was present to promote resuscitation, the number of wild type cells increased and mutant also showed a basal level of resuscitation. These results showed that resuscitation in presence of pyruvate requires BtsSR/YpdAB signalling network. These studies showed the importance of pyruvate sensing and transport in resuscitation of Viable but non Culturable cells. There are many other methods of resuscitation of which one method is discussed here.
Toxin Antitoxin system are widely involved in persister cell formation. In normal growing cells toxins are inhibited by antitoxin in some cases when antitoxin is not functional or not present the toxins are primarily inactive by promoter mutations. Persister cells and VBNC are very closely related, they only have a certain differences. Both of them are formed in high stress conditions. Both of them are antibiotic resistant and hence involved in a large level of infections, so practices can be done to make drugs which can act on TA systems so that the persistence could be stopped. The methods of resuscitation were studied but further research in them is needed as very little is known about the mechanism of resuscitation.
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