The Experts below are selected from a list of 1596 Experts worldwide ranked by ideXlab platform
Thomas K Wood - One of the best experts on this subject based on the ideXlab platform.
-
escherichia coli cryptic prophages sense nutrients to control Persister Cell resuscitation
bioRxiv, 2021Co-Authors: Michael J Benedik, Sooyeon Song, Junseob Kim, Ryota Yamasaki, Thomas K WoodAbstract:ABSTRACT We determined previously that some cryptic prophages are not genomic junk but instead enable Cells to combat myriad stresses as part of an active stress response. However, how these phage fossils affect the extreme stress response of dormancy; i.e., how cryptic prophages affect Persister Cell formation and resuscitation, has not been fully explored. Persister Cells form as a result of stresses such as starvation, antibiotics, and oxidative conditions, and resuscitation of these Persister Cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. Unlike for the active stress response, here we find that deletion of each of the nine Escherichia coli cryptic prophages has no effect on Persister Cell formation. Strikingly, elimination of each cryptic prophage results in an increase in Persister Cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system as a result of inactivating transcriptional regulator AlpA of the CP4-57 cryptic prophage, since we found ΔalpA increases Persister resuscitation, and AlpA represses phosphate regulator PhoR. Therefore, we report a novel Cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the Cell from dormancy and prevents premature resuscitation in the absence of nutrients.
-
interkingdom signal indole inhibits pseudomonas aeruginosa Persister Cell waking
Journal of Applied Microbiology, 2019Co-Authors: Weiwei Zhang, Ryota Yamasaki, Sooyeon Song, Thomas K WoodAbstract:Aims Persister Cells are stressed Cells that have transient tolerance to antibiotics; these Cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how Persisters resuscitate, so we explored the waking of Cells in the presence of the interkingdom signal indole. Methods and results To generate a large population of Persister Cells, we induced the Persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pretreating Cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single Cell observations, that proline is sufficient to wake the Persister Cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa Persister Cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa Persister Cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary-phase cultures, the indole produced by E. coli completely inhibits Persister Cell resuscitation of P. aeruginosa. Conclusions Therefore, E. coli has devised a method to outcompete its competitors by preventing their resuscitation with indole. Significance and impact of the study This work provides insight into why indole is produced by commensal bacteria.
-
interkingdom signal indole inhibits pseudomonas aeruginosa Persister Cell waking
bioRxiv, 2019Co-Authors: Weiwei Zhang, Ryota Yamasaki, Thomas K WoodAbstract:ABSTRACT Aims Persister Cells are stressed Cells that have transient tolerance to antibiotics; these Cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how Persisters resuscitate, so we explored the waking of a Cells in the presence of the interkingdom signal indole. Methods and Results To generate a large population of Persister Cells, we induced the Persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pre-treating Cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single Cell observations, that proline is sufficient to wake the Persister Cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa Persister Cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa Persister Cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary phase cultures, the indole produced by E. coli completely inhibits Persister Cell resuscitation of P. aeruginosa. Conclusions Therefore, E. col has devised a method to outcompete its competitors by preventing their resuscitation with indole. Significance This work provides insight into why indole is produced by commensal bacteria.
-
Persister Cells resuscitate using membrane sensors that activate chemotaxis lower camp levels and revive ribosomes
Social Science Research Network, 2019Co-Authors: Ryota Yamasaki, Michael J Benedik, Sooyeon Song, Thomas K WoodAbstract:Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all Cells experience nutrient stress, which causes a sub-population to become dormant. However, how Persister Cells wake to reconstitute infections is not understood well. Here, using single-Cell observations, we determined that Escherichia coli Persister Cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of Persister Cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating Cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, Persister Cells wake by perceiving nutrients via membrane receptors which relay the signal to ribosomes via the secondary messenger cAMP, and Persisters wake and utilize chemotaxis to acquire nutrients.
-
ribosome dependence of Persister Cell formation and resuscitation
Journal of Microbiology, 2019Co-Authors: Thomas K Wood, Sooyeon Song, Ryota YamasakiAbstract:Since most bacterial Cells are starving, they must enter a resting stage. Persister is the term used for metabolically-dormant Cells that are not spores, and these Cells arise from stress such as that from antibiotics as well as that from starvation. Because of their lack of metabolism, Persister Cells survive exposure to multiple stresses without undergoing genetic change; i.e., they have no inherited phenotype and behave as wild-type Cells once the stress is removed and nutrients are presented. In contrast, mutations allow resistant bacteria to grow in the presence of antibiotics and slow growth allows tolerant Cells to withstand higher concentrations of antibiotics; hence, there are three closely-related phenotypes: persistent, resistant, and tolerant. In addition, since dormancy is so prevalent, Persister Cells must have a means for resuscitating (since so many Cells should obtain this resting state). In this review, we focus on what is known about the formation and resuscitation of Persister Cells.
Hanne Ingmer - One of the best experts on this subject based on the ideXlab platform.
-
Phenol-Soluble Modulins Modulate Persister Cell Formation in Staphylococcus aureus.
Frontiers in microbiology, 2020Co-Authors: Mara Baldry, Martin S. Bojer, Martin Vestergaard, Zahra Najarzadeh, Rikke Louise Meyer, Daniel E. Otzen, Hanne IngmerAbstract:Staphylococcus aureus is a human pathogen that can cause chronic and recurrent infections and is recalcitrant to antibiotic chemotherapy. This trait is partly attributed to its ability to form Persister Cells, which are subpopulations of Cells that are tolerant to lethal concentrations of antibiotics. Recently, we showed that the phenol-soluble modulins (PSMs) expressed by S. aureus reduce Persister Cell formation. PSMs are a versatile group of toxins that, in addition to toxicity, form amyloid-like fibrils thought to support biofilm structures. Here, we examined individual or combined synthetic PSMα peptides and their equivalent amyloid-like fibrils on ciprofloxacin-selected S. aureus Persister Cells. We found that PSMα2 and the mixture of all four alpha peptides consistently were able to reduce Persister frequency in all growth phases, and this activity was specifically linked to the presence of the soluble peptide as no effect was seen with fibrillated peptides. Persister reduction was particularly striking in a mutant that, due to mutations in the Krebs cycle, has enhanced ability to form Persisters with PSMα4 and the combination of peptides being most effective. In biofilms, only the combination of peptides displayed Persister reducing activity. Collectively, we report the individual contributions of PSMα peptides to Persister Cell reduction and that the combination of peptides generally was most effective. Strikingly, the fibrillated peptides lost activity and thus, if formed in bacterial cultures, they will be inactive against Persister Cells. Further studies will be needed to address the biological role of phenol-soluble modulins in reducing Persister Cells.
-
high Persister Cell formation by clinical staphylococcus aureus strains belonging to clonal complex 30
Microbiology, 2020Co-Authors: Liping Liu, Martin S. Bojer, Ying Wang, Paal Skytt Andersen, Hanne IngmerAbstract:Bacterial Persisters form a subpopulation of Cells that survive lethal concentrations of antibiotics without being genetically different from the susceptible population. They are generally considered to be phenotypic variants that spontaneously have entered a dormant state with low ATP levels or reduced membrane potential. In Staphylococcus aureus, a serious opportunistic human pathogen, Persisters are believed to contribute to chronic infections that are a major global healthcare problem. While S. aureus Persisters have mostly been studied in laboratory strains, we have here investigated the ability of clinical strains to form Persisters. For 44 clinical strains belonging to the major clonal complexes CC5, CC8, CC30 or CC45, we examined Persister Cell formation in stationary phase when exposed to 100 times the MIC of ciprofloxacin, an antibiotic that targets DNA replication. We find that while all strains are able to form Persisters, those belonging to CC30 displayed on average 100-fold higher Persister Cell frequencies when compared to strains of other CCs. Importantly, there was no correlation between Persister formation and the Cellular ATP content of the individual strains, but the group of CC30 strains displayed slightly lower membrane potential compared to the non-CC30 group. CC30 strains have previously been associated with chronic and reoccuring infections and we hypothesize that there could be a correlation between lineage-specific characteristics displayed via in vitro Persister assays and the observed clinical spectrum of disease.
-
Inactivation of TCA cycle enhances Staphylococcus aureus Persister Cell formation in stationary phase
Scientific Reports, 2018Co-Authors: Ying Wang, Martin S. Bojer, Shilpa Elizabeth George, Zhihao Wang, Peter Ruhdal Jensen, Christiane Wolz, Hanne IngmerAbstract:Persister Cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of Cells in stationary phase. By enrichment from a transposon mutant library in S . aureus we identified mutants that in this growth phase displayed enhanced Persister Cell formation. We found that inactivation of either sucA or sucB , encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10–100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F_1F_0 ATPase. In S . aureus , TCA cycle activity and gene expression are de-repressed in stationary phase but single Cells with low expression may be prone to form Persisters. While ATP levels were not consistently affected in high Persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that Persister Cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.
-
Quorum Sensing-Regulated Phenol-Soluble Modulins Limit Persister Cell Populations in Staphylococcus aureus
Frontiers Media S.A., 2018Co-Authors: Martin S. Bojer, Søren Lindemose, Martin Vestergaard, Hanne IngmerAbstract:Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are Persisters being individual Cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen Staphylococcus aureus and the influence of growth phase on Persister formation, we noted that spent supernatants of stationary phase cultures of S. aureus or S. epidermidis, but not of distantly related bacteria, significantly reduced the Persister Cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase S. aureus Cells. Curiously, the Persister reducing activity of S. aureus supernatants was also effective against Persisters formed by either S. carnosus or Listeria monocytogenes. The Persister reducing component, which resisted heat but not proteases and was produced in the late growth phase in an agr quorum-sensing dependent manner, was identified to be the phenol-soluble modulin (PSM) toxins. S. aureus express several PSMs, each with distinct cytolytic and antimicrobial properties; however, the Persister reducing activity was specifically linked to synthesis of the PSMα family. Correspondingly, a high-Persister phenotype of a PSMα mutant was observed upon fluoroquinolone or aminoglycoside challenge, demonstrating that the Persister reducing activity of PSMs can be endogenously synthesized or extrinsically added. Given that PSMs have been associated with lytic activity against bacterial membranes we propose that PSM toxins increase the susceptibility of Persister Cells to killing by intraCellularly acting antibiotics and that chronic and re-occurring infections with quorum sensing, agr negative mutants may be difficult to treat with antibiotics because of Persister Cells formed in the absence of PSM toxins
-
Table_1.pdf
2018Co-Authors: Martin S. Bojer, Søren Lindemose, Martin Vestergaard, Hanne IngmerAbstract:Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are Persisters being individual Cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen Staphylococcus aureus and the influence of growth phase on Persister formation, we noted that spent supernatants of stationary phase cultures of S. aureus or S. epidermidis, but not of distantly related bacteria, significantly reduced the Persister Cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase S. aureus Cells. Curiously, the Persister reducing activity of S. aureus supernatants was also effective against Persisters formed by either S. carnosus or Listeria monocytogenes. The Persister reducing component, which resisted heat but not proteases and was produced in the late growth phase in an agr quorum-sensing dependent manner, was identified to be the phenol-soluble modulin (PSM) toxins. S. aureus express several PSMs, each with distinct cytolytic and antimicrobial properties; however, the Persister reducing activity was specifically linked to synthesis of the PSMα family. Correspondingly, a high-Persister phenotype of a PSMα mutant was observed upon fluoroquinolone or aminoglycoside challenge, demonstrating that the Persister reducing activity of PSMs can be endogenously synthesized or extrinsically added. Given that PSMs have been associated with lytic activity against bacterial membranes we propose that PSM toxins increase the susceptibility of Persister Cells to killing by intraCellularly acting antibiotics and that chronic and re-occurring infections with quorum sensing, agr negative mutants may be difficult to treat with antibiotics because of Persister Cells formed in the absence of PSM toxins.
Ryota Yamasaki - One of the best experts on this subject based on the ideXlab platform.
-
escherichia coli cryptic prophages sense nutrients to control Persister Cell resuscitation
bioRxiv, 2021Co-Authors: Michael J Benedik, Sooyeon Song, Junseob Kim, Ryota Yamasaki, Thomas K WoodAbstract:ABSTRACT We determined previously that some cryptic prophages are not genomic junk but instead enable Cells to combat myriad stresses as part of an active stress response. However, how these phage fossils affect the extreme stress response of dormancy; i.e., how cryptic prophages affect Persister Cell formation and resuscitation, has not been fully explored. Persister Cells form as a result of stresses such as starvation, antibiotics, and oxidative conditions, and resuscitation of these Persister Cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. Unlike for the active stress response, here we find that deletion of each of the nine Escherichia coli cryptic prophages has no effect on Persister Cell formation. Strikingly, elimination of each cryptic prophage results in an increase in Persister Cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system as a result of inactivating transcriptional regulator AlpA of the CP4-57 cryptic prophage, since we found ΔalpA increases Persister resuscitation, and AlpA represses phosphate regulator PhoR. Therefore, we report a novel Cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the Cell from dormancy and prevents premature resuscitation in the absence of nutrients.
-
reactive oxygen species penetrate Persister Cell membranes of escherichia coli for effective Cell killing
Frontiers in Cellular and Infection Microbiology, 2020Co-Authors: Aki Kawano, Ryota Yamasaki, Tatsuya Sakakura, Yoshiyuki Takatsuji, Tetsuya Haruyama, Yoshie Yoshioka, Wataru AriyoshiAbstract:Persister Cells are difficult to eliminate because they are tolerant to antibiotic stress. In the present study, using artificially induced Escherichia coli Persister Cells, we found that reactive oxygen species (ROS) have greater effects on Persister Cells than on exponential Cells. Thus, we examined which types of ROS could effectively eliminate Persister Cells and determined the mechanisms underlying the effects of these ROS. Ultraviolet (UV) light irradiation can kill Persister Cells, and bacterial viability is markedly increased under UV shielding. UV induces the production of ROS, which kill bacteria by moving toward the shielded area. Electron spin resonance-based analysis confirmed that hydroxyl radicals are produced by UV irradiation, although singlet oxygen is not produced. These results clearly revealed that ROS sterilizes Persister Cells more effectively compared to the sterilization of exponential Cells (** p < 0.01). These ROS do not injure the bacterial Cell wall but rather invade the Cell, followed by Cell killing. Additionally, the sterilization effect on Persister Cells was increased by exposure to oxygen plasma during UV irradiation. However, vapor conditions decreased Persister Cell sterilization by reducing the levels of hydroxyl radicals. We also verified the effect of ROS against bacteria in biofilms that are more resistant than planktonic Cells. Although UV alone could not completely sterilize the biofilm bacteria, UV with ROS achieved complete sterilization. Our results demonstrate that Persister Cells strongly resist the effects of antibiotics and starvation stress but are less able to withstand exposure to ROS. It was shown that ROS does not affect the Cell membrane but penetrates it and acts internally to kill Persister Cells. In particular, it was clarified that the hydroxy radical is an effective sterilizer to kill Persister Cells.
-
interkingdom signal indole inhibits pseudomonas aeruginosa Persister Cell waking
Journal of Applied Microbiology, 2019Co-Authors: Weiwei Zhang, Ryota Yamasaki, Sooyeon Song, Thomas K WoodAbstract:Aims Persister Cells are stressed Cells that have transient tolerance to antibiotics; these Cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how Persisters resuscitate, so we explored the waking of Cells in the presence of the interkingdom signal indole. Methods and results To generate a large population of Persister Cells, we induced the Persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pretreating Cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single Cell observations, that proline is sufficient to wake the Persister Cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa Persister Cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa Persister Cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary-phase cultures, the indole produced by E. coli completely inhibits Persister Cell resuscitation of P. aeruginosa. Conclusions Therefore, E. coli has devised a method to outcompete its competitors by preventing their resuscitation with indole. Significance and impact of the study This work provides insight into why indole is produced by commensal bacteria.
-
interkingdom signal indole inhibits pseudomonas aeruginosa Persister Cell waking
bioRxiv, 2019Co-Authors: Weiwei Zhang, Ryota Yamasaki, Thomas K WoodAbstract:ABSTRACT Aims Persister Cells are stressed Cells that have transient tolerance to antibiotics; these Cells undergo no genetic change, but instead, their tolerance is due to reduced metabolism. Unfortunately, little is known about how Persisters resuscitate, so we explored the waking of a Cells in the presence of the interkingdom signal indole. Methods and Results To generate a large population of Persister Cells, we induced the Persister phenotype in the opportunistic pathogen Pseudomonas aeruginosa by pre-treating Cells with carbonyl cyanide m-chlorophenylhydrazone to reduce translation by depleting ATP levels, and found, via single Cell observations, that proline is sufficient to wake the Persister Cells. P. aeruginosa is often present in the gastrointestinal tract, and indole from commensal bacteria such as Escherichia coli has been shown to inhibit P. aeruginosa quorum sensing and pathogenicity without influencing growth. Furthermore, indole is not toxic to P. aeruginosa Persister Cells. However, we find here that physiological concentrations of indole inhibit P. aeruginosa Persister Cell resuscitation with an efficiency of higher than 95%. Critically, when contacted with E. coli stationary phase cultures, the indole produced by E. coli completely inhibits Persister Cell resuscitation of P. aeruginosa. Conclusions Therefore, E. col has devised a method to outcompete its competitors by preventing their resuscitation with indole. Significance This work provides insight into why indole is produced by commensal bacteria.
-
Persister Cells resuscitate using membrane sensors that activate chemotaxis lower camp levels and revive ribosomes
Social Science Research Network, 2019Co-Authors: Ryota Yamasaki, Michael J Benedik, Sooyeon Song, Thomas K WoodAbstract:Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all Cells experience nutrient stress, which causes a sub-population to become dormant. However, how Persister Cells wake to reconstitute infections is not understood well. Here, using single-Cell observations, we determined that Escherichia coli Persister Cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of Persister Cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating Cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, Persister Cells wake by perceiving nutrients via membrane receptors which relay the signal to ribosomes via the secondary messenger cAMP, and Persisters wake and utilize chemotaxis to acquire nutrients.
Martin S. Bojer - One of the best experts on this subject based on the ideXlab platform.
-
Phenol-Soluble Modulins Modulate Persister Cell Formation in Staphylococcus aureus.
Frontiers in microbiology, 2020Co-Authors: Mara Baldry, Martin S. Bojer, Martin Vestergaard, Zahra Najarzadeh, Rikke Louise Meyer, Daniel E. Otzen, Hanne IngmerAbstract:Staphylococcus aureus is a human pathogen that can cause chronic and recurrent infections and is recalcitrant to antibiotic chemotherapy. This trait is partly attributed to its ability to form Persister Cells, which are subpopulations of Cells that are tolerant to lethal concentrations of antibiotics. Recently, we showed that the phenol-soluble modulins (PSMs) expressed by S. aureus reduce Persister Cell formation. PSMs are a versatile group of toxins that, in addition to toxicity, form amyloid-like fibrils thought to support biofilm structures. Here, we examined individual or combined synthetic PSMα peptides and their equivalent amyloid-like fibrils on ciprofloxacin-selected S. aureus Persister Cells. We found that PSMα2 and the mixture of all four alpha peptides consistently were able to reduce Persister frequency in all growth phases, and this activity was specifically linked to the presence of the soluble peptide as no effect was seen with fibrillated peptides. Persister reduction was particularly striking in a mutant that, due to mutations in the Krebs cycle, has enhanced ability to form Persisters with PSMα4 and the combination of peptides being most effective. In biofilms, only the combination of peptides displayed Persister reducing activity. Collectively, we report the individual contributions of PSMα peptides to Persister Cell reduction and that the combination of peptides generally was most effective. Strikingly, the fibrillated peptides lost activity and thus, if formed in bacterial cultures, they will be inactive against Persister Cells. Further studies will be needed to address the biological role of phenol-soluble modulins in reducing Persister Cells.
-
high Persister Cell formation by clinical staphylococcus aureus strains belonging to clonal complex 30
Microbiology, 2020Co-Authors: Liping Liu, Martin S. Bojer, Ying Wang, Paal Skytt Andersen, Hanne IngmerAbstract:Bacterial Persisters form a subpopulation of Cells that survive lethal concentrations of antibiotics without being genetically different from the susceptible population. They are generally considered to be phenotypic variants that spontaneously have entered a dormant state with low ATP levels or reduced membrane potential. In Staphylococcus aureus, a serious opportunistic human pathogen, Persisters are believed to contribute to chronic infections that are a major global healthcare problem. While S. aureus Persisters have mostly been studied in laboratory strains, we have here investigated the ability of clinical strains to form Persisters. For 44 clinical strains belonging to the major clonal complexes CC5, CC8, CC30 or CC45, we examined Persister Cell formation in stationary phase when exposed to 100 times the MIC of ciprofloxacin, an antibiotic that targets DNA replication. We find that while all strains are able to form Persisters, those belonging to CC30 displayed on average 100-fold higher Persister Cell frequencies when compared to strains of other CCs. Importantly, there was no correlation between Persister formation and the Cellular ATP content of the individual strains, but the group of CC30 strains displayed slightly lower membrane potential compared to the non-CC30 group. CC30 strains have previously been associated with chronic and reoccuring infections and we hypothesize that there could be a correlation between lineage-specific characteristics displayed via in vitro Persister assays and the observed clinical spectrum of disease.
-
Inactivation of TCA cycle enhances Staphylococcus aureus Persister Cell formation in stationary phase
Scientific Reports, 2018Co-Authors: Ying Wang, Martin S. Bojer, Shilpa Elizabeth George, Zhihao Wang, Peter Ruhdal Jensen, Christiane Wolz, Hanne IngmerAbstract:Persister Cells constitute a small subpopulation of bacteria that display remarkably high antibiotic tolerance and for pathogens such as Staphylococcus aureus are suspected as culprits of chronic and recurrent infections. Persisters formed during exponential growth are characterized by low ATP levels but less is known of Cells in stationary phase. By enrichment from a transposon mutant library in S . aureus we identified mutants that in this growth phase displayed enhanced Persister Cell formation. We found that inactivation of either sucA or sucB , encoding the subunits of the α-ketoglutarate dehydrogenase of the tricarboxylic acid cycle (TCA cycle), increased survival to lethal concentrations of ciprofloxacin by 10–100 fold as did inactivation of other TCA cycle genes or atpA encoding a subunit of the F_1F_0 ATPase. In S . aureus , TCA cycle activity and gene expression are de-repressed in stationary phase but single Cells with low expression may be prone to form Persisters. While ATP levels were not consistently affected in high Persister mutants they commonly displayed reduced membrane potential, and persistence was enhanced by a protein motive force inhibitor. Our results show that Persister Cell formation in stationary phase does not correlate with ATP levels but is associated with low membrane potential.
-
Quorum Sensing-Regulated Phenol-Soluble Modulins Limit Persister Cell Populations in Staphylococcus aureus
Frontiers Media S.A., 2018Co-Authors: Martin S. Bojer, Søren Lindemose, Martin Vestergaard, Hanne IngmerAbstract:Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are Persisters being individual Cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen Staphylococcus aureus and the influence of growth phase on Persister formation, we noted that spent supernatants of stationary phase cultures of S. aureus or S. epidermidis, but not of distantly related bacteria, significantly reduced the Persister Cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase S. aureus Cells. Curiously, the Persister reducing activity of S. aureus supernatants was also effective against Persisters formed by either S. carnosus or Listeria monocytogenes. The Persister reducing component, which resisted heat but not proteases and was produced in the late growth phase in an agr quorum-sensing dependent manner, was identified to be the phenol-soluble modulin (PSM) toxins. S. aureus express several PSMs, each with distinct cytolytic and antimicrobial properties; however, the Persister reducing activity was specifically linked to synthesis of the PSMα family. Correspondingly, a high-Persister phenotype of a PSMα mutant was observed upon fluoroquinolone or aminoglycoside challenge, demonstrating that the Persister reducing activity of PSMs can be endogenously synthesized or extrinsically added. Given that PSMs have been associated with lytic activity against bacterial membranes we propose that PSM toxins increase the susceptibility of Persister Cells to killing by intraCellularly acting antibiotics and that chronic and re-occurring infections with quorum sensing, agr negative mutants may be difficult to treat with antibiotics because of Persister Cells formed in the absence of PSM toxins
-
Table_1.pdf
2018Co-Authors: Martin S. Bojer, Søren Lindemose, Martin Vestergaard, Hanne IngmerAbstract:Incomplete killing of bacterial pathogens by antibiotics is an underlying cause of treatment failure and accompanying complications. Among those avoiding chemotherapy are Persisters being individual Cells in a population that for extended periods of time survive high antibiotic concentrations proposedly by being in a quiescent state refractory to antibiotic killing. While investigating the human pathogen Staphylococcus aureus and the influence of growth phase on Persister formation, we noted that spent supernatants of stationary phase cultures of S. aureus or S. epidermidis, but not of distantly related bacteria, significantly reduced the Persister Cell frequency upon ciprofloxacin challenge when added to exponentially growing and stationary phase S. aureus Cells. Curiously, the Persister reducing activity of S. aureus supernatants was also effective against Persisters formed by either S. carnosus or Listeria monocytogenes. The Persister reducing component, which resisted heat but not proteases and was produced in the late growth phase in an agr quorum-sensing dependent manner, was identified to be the phenol-soluble modulin (PSM) toxins. S. aureus express several PSMs, each with distinct cytolytic and antimicrobial properties; however, the Persister reducing activity was specifically linked to synthesis of the PSMα family. Correspondingly, a high-Persister phenotype of a PSMα mutant was observed upon fluoroquinolone or aminoglycoside challenge, demonstrating that the Persister reducing activity of PSMs can be endogenously synthesized or extrinsically added. Given that PSMs have been associated with lytic activity against bacterial membranes we propose that PSM toxins increase the susceptibility of Persister Cells to killing by intraCellularly acting antibiotics and that chronic and re-occurring infections with quorum sensing, agr negative mutants may be difficult to treat with antibiotics because of Persister Cells formed in the absence of PSM toxins.
Alessandra Alves De Souza - One of the best experts on this subject based on the ideXlab platform.
-
The MqsRA toxin-antitoxin system from xylella fastidiosa plays a key role in bacterial fitness, pathogenicity, and Persister Cell formation
'Frontiers Media SA', 2020Co-Authors: Merfa Marcus, Niza Bárbara, Takita, Marco A., Alessandra Alves De SouzaAbstract:Through the formation of Persister Cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of Persister Cells because they are able to induce Cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in Persister Cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coil as related to the formation of Persister Cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively Cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of Persister Cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the Cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X fastidiosa and reveal new insights into the physiology of phytopathogen host interactions7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2010/50712-9; 2013/17485-7; 2013/02014-
-
the mqsra toxin antitoxin system from xylella fastidiosa plays a key role in bacterial fitness pathogenicity and Persister Cell formation
Frontiers in Microbiology, 2016Co-Authors: Marcus V Merfa, Barbara Niza, Marco Aurelio Takita, Alessandra Alves De SouzaAbstract:Through the formation of Persister Cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of Persister Cells because they are able to induce Cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in Persister Cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of Persister Cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively Cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of Persister Cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the Cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions.