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Sean Crosson - One of the best experts on this subject based on the ideXlab platform.
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allosteric control of a bacterial Stress Response System by an anti σ factor
Molecular Microbiology, 2018Co-Authors: Justin L Luebke, Daniel S Eaton, Joseph R Sachleben, Sean CrossonAbstract:Summary Bacterial signal transduction Systems commonly use receiver (REC) domains, which regulate adaptive Responses to the environment as a function of their phosphorylation state. REC domains control cell physiology through diverse mechanisms, many of which remain understudied. We have defined structural features that underlie activation of the multi-domain REC protein, PhyR, which functions as an anti-anti-σ factor and regulates transcription of genes required for Stress adaptation and host-microbe interactions in Alphaproteobacteria. Though REC phosphorylation is necessary for PhyR function in vivo, we did not detect expected changes in inter-domain interactions upon phosphorylation by solution X-ray scattering. We sought to understand this result by defining additional molecular requirements for PhyR activation. We uncovered specific interactions between unphosphorylated PhyR and an intrinsically disordered region (IDR) of the anti-σ factor, NepR, by solution NMR spectroscopy. Our data support a model whereby nascent NepR(IDR)-PhyR interactions and REC phosphorylation coordinately impart the free energy to shift PhyR to an open, active conformation that binds and inhibits NepR. This mechanism ensures PhyR is activated only when NepR and an activating phosphoryl signal are present. Our study provides new structural understanding of the molecular regulatory logic underlying a conserved environmental Response System. This article is protected by copyright. All rights reserved.
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wrpa is an atypical flavodoxin family protein under regulatory control of the brucella abortus general Stress Response System
Journal of Bacteriology, 2016Co-Authors: Julien Herrou, Daniel M Czyz, Jonathan W Willett, Hyesook Kim, Gekleng Chhor, Gyorgy Babnigg, Youngchang Kim, Sean CrossonAbstract:The general Stress Response (GSR) System of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in Response to a range of Stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR System under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative Stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. IMPORTANCE Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general Stress Response (GSR) regulatory System of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.
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the brucella abortus general Stress Response System regulates chronic mammalian infection and is controlled by phosphorylation and proteolysis
Journal of Biological Chemistry, 2013Co-Authors: Sean Crosson, Hyesook Kim, Clayton C Caswell, Robert D Foreman, Martin R RoopAbstract:Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general Stress Response (GSR) System, PhyR and σE1, are required for Brucella abortus Stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid Stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR System is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified Stress Response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR System affects both Stress survival and the interface between B. abortus and the host immune System. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR System regulates acute Stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.
Robert M Q Shanks - One of the best experts on this subject based on the ideXlab platform.
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antibiotics used in empiric treatment of ocular infections trigger the bacterial rcs Stress Response System independent of antibiotic susceptibility
Antibiotics, 2021Co-Authors: Nathaniel S Harshaw, Nicholas A Stella, Eric G Romanowski, Kara M Lehner, Regis P Kowalski, Robert M Q ShanksAbstract:The Rcs phosphorelay is a bacterial Stress Response System that responds to envelope Stresses and in turn controls several virulence-associated pathways, including capsule, flagella, and toxin biosynthesis, of numerous bacterial species. The Rcs System also affects antibiotic tolerance, biofilm formation, and horizontal gene transfer. The Rcs System of the ocular bacterial pathogen Serratia marcescens was recently demonstrated to influence ocular pathogenesis in a rabbit model of keratitis, with Rcs-defective mutants causing greater pathology and Rcs-activated strains demonstrating reduced inflammation. The Rcs System is activated by a variety of insults, including β-lactam antibiotics and polymyxin B. In this study, we developed three luminescence-based transcriptional reporters for Rcs System activity and used them to test whether antibiotics used for empiric treatment of ocular infections influence Rcs System activity in a keratitis isolate of S. marcescens. These included antibiotics to which the bacteria were susceptible and resistant. Results indicate that cefazolin, ceftazidime, polymyxin B, and vancomycin activate the Rcs System to varying degrees in an RcsB-dependent manner, whereas ciprofloxacin and tobramycin activated the promoter fusions, but in an Rcs-independent manner. Although minimum inhibitory concentration (MIC) analysis demonstrated resistance of the test bacteria to polymyxin B and vancomycin, the Rcs System was activated by sub-inhibitory concentrations of these antibiotics. Together, these data indicate that a bacterial Stress System that influences numerous pathogenic phenotypes and drug-tolerance is influenced by different classes of antibiotics despite the susceptibility status of the bacterium.
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the rcs Stress Response System regulator gumb modulates serratia marcescens induced inflammation and bacterial proliferation in a rabbit keratitis model and cytotoxicity in vitro
Infection and Immunity, 2021Co-Authors: Eric G Romanowski, John E Romanowski, Deepinder K Dhaliwal, Kathleen A Yates, Nicholas A Stella, Anthony J St Leger, Robert M Q ShanksAbstract:In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs Stress Response System. The role of the Rcs System and bacterial Stress Response Systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs Stress Response System induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs System, and this overall Stress Response System is a key mediator of a bacterium's ability to induce vision-threatening keratitis.
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the rcs Stress Response System modulates serratia marcescens induced inflammation and bacterial proliferation in a rabbit keratitis model
bioRxiv, 2020Co-Authors: Eric G Romanowski, John E Romanowski, Deepinder K Dhaliwal, Kathleen A Yates, Nicholas A Stella, Robert M Q ShanksAbstract:In this study, we tested the hypothesis that the conserved bacterial Rcs Stress Response System mediates corneal pathogenesis associated with Serratia marcescens ocular infections. This was accomplished by modifying Rcs activity using mutant strains. These include a mutant that has a hyper-active Rcs System due to deletion of the IgaA family gene, gumB, and a gumB rcsC double mutant that is defective for Rcs signaling. The role of the Rcs System and bacterial Stress Response Systems for microbial keratitis is not known. Here we observed that the Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h, and demonstrated a notable reduction in inflammation based on inflammatory signs and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid and partially complemented by restoration of shlA cytolysin expression and elimination of capsular polysaccharide production. We observed that inactivation of the Rcs Stress Response System completely restored corneal virulence to the gumB mutant. NanoString transcriptional analysis of bacterial genes expressed during microbial keratitis demonstrated expression of gumB, rcsB, shlA, and three metalloprotease genes. Data suggest that the bacterial capsular polysaccharide is not necessary for infection, but capsule overexpression reduces inflammation. Together, these data indicate that GumB regulates virulence factor production through the Rcs System and this overall Stress Response System is a key mediator of a bacteriums ability to induce vision-threatening keratitis.
Charles A Nelson - One of the best experts on this subject based on the ideXlab platform.
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environmental determinants of physiological reactivity to Stress the interacting effects of early life deprivation caregiving quality and Stressful life events
Development and Psychopathology, 2020Co-Authors: Mark Wade, Margaret A Sheridan, Nathan A Fox, Charles H Zeanah, Charles A Nelson, Katie A MclaughlinAbstract:Children who spend their early lives in institutions experience profound psychosocial deprivation that is associated with altered Stress Response System development. Here, we used data from a longitudinal randomized controlled trial of foster care for institutionally reared children to examine whether caregiving quality and Stressful life events (SLEs) in early adolescence (age 12) influence patterns of hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous System (SNS) reactivity. Controlling for the effect of institutional care, higher caregiving quality at age 12 was associated with heightened cortisol and SNS reactivity. However, moderation analysis revealed that the latter effect was only observed among never-institutionalized children, whereas ever-institutionalized children demonstrated a persistently blunted SNS Response regardless of recent caregiving quality. Among institutionally reared children, SLEs interacted with prior random assignment to foster care, such that those placed in foster care early in development had a SNS Response that approximated never-institutionalized children when SLEs at age 12 were low. In contrast, SNS reactivity was persistently blunted among those with prolonged deprivation, regardless of recent SLEs. Early-life deprivation is associated with persistent blunting of Stress Response Systems, but normalization may be achievable if SLEs are limited following placement into enriched family-based care.
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causal effects of the early caregiving environment on development of Stress Response Systems in children
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Katie A Mclaughlin, Margaret A Sheridan, Florin Tibu, Nathan A Fox, Charles H Zeanah, Charles A NelsonAbstract:Disruptions in Stress Response System functioning are thought to be a central mechanism by which exposure to adverse early-life environments influences human development. Although early-life adversity results in hyperreactivity of the sympathetic nervous System (SNS) and hypothalamic–pituitary–adrenal (HPA) axis in rodents, evidence from human studies is inconsistent. We present results from the Bucharest Early Intervention Project examining whether randomized placement into a family caregiving environment alters development of the autonomic nervous System and HPA axis in children exposed to early-life deprivation associated with institutional rearing. Electrocardiogram, impedance cardiograph, and neuroendocrine data were collected during laboratory-based challenge tasks from children (mean age = 12.9 y) raised in deprived institutional settings in Romania randomized to a high-quality foster care intervention (n = 48) or to remain in care as usual (n = 43) and a sample of typically developing Romanian children (n = 47). Children who remained in institutional care exhibited significantly blunted SNS and HPA axis Responses to psychosocial Stress compared with children randomized to foster care, whose Stress Responses approximated those of typically developing children. Intervention effects were evident for cortisol and parasympathetic nervous System reactivity only among children placed in foster care before age 24 and 18 months, respectively, providing experimental evidence of a sensitive period in humans during which the environment is particularly likely to alter Stress Response System development. We provide evidence for a causal link between the early caregiving environment and Stress Response System reactivity in humans with effects that differ markedly from those observed in rodent models.
Hyesook Kim - One of the best experts on this subject based on the ideXlab platform.
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wrpa is an atypical flavodoxin family protein under regulatory control of the brucella abortus general Stress Response System
Journal of Bacteriology, 2016Co-Authors: Julien Herrou, Daniel M Czyz, Jonathan W Willett, Hyesook Kim, Gekleng Chhor, Gyorgy Babnigg, Youngchang Kim, Sean CrossonAbstract:The general Stress Response (GSR) System of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in Response to a range of Stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR System under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative Stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. IMPORTANCE Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general Stress Response (GSR) regulatory System of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.
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the brucella abortus general Stress Response System regulates chronic mammalian infection and is controlled by phosphorylation and proteolysis
Journal of Biological Chemistry, 2013Co-Authors: Sean Crosson, Hyesook Kim, Clayton C Caswell, Robert D Foreman, Martin R RoopAbstract:Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general Stress Response (GSR) System, PhyR and σE1, are required for Brucella abortus Stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid Stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR System is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified Stress Response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR System affects both Stress survival and the interface between B. abortus and the host immune System. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR System regulates acute Stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.
Carsten T Wotjak - One of the best experts on this subject based on the ideXlab platform.
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role of the endocannabinoid System in regulation of the hypothalamic pituitary adrenocortical axis
Progress in Brain Research, 2008Co-Authors: M A Steiner, Carsten T WotjakAbstract:The endocannabinoid System has been recognized as a major neuromodulatory System, which functions to maintain brain homoeostasis. Endocannabinoids are synthesized and released from the postsynapse and act as retrograde neuronal messengers, which bind to cannabinoid type 1 receptors at the presynapse. Here, they inhibit the release of neurotransmitters, including glutamate and GABA. By these means, endocannabinoids control the activation of various neuronal circuits including those involved in neuroendocrine Stress processing. Accordingly, exogenous cannabinoids such as the major active component of marijuana, Δ9-tetrahydrocannabinol, have long been known to activate the major neuroendocrine Stress Response System of mammals, the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the function of the endocannabinoid System in the regulation of Stress hormone secretion has only recently begun to be understood. It is the focus of the present review to provide the reader with an overview of our current knowledge of the role of endocannabinoid signalling in HPA axis regulation under basal as well as under Stressful conditions. This includes the specific sites of action, potential underlying neuronal pathways and interactions between behavioural and neuroendocrine Stress coping. Furthermore, the potential role of HPA axis activity dysregulations, caused by deficits in the endocannabinoid System, for the pathophysiology of psychiatric diseases is discussed.
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role of the endocannabinoid System in regulation of the hypothalamic pituitary adrenocortical axis
Progress in Brain Research, 2008Co-Authors: M A Steiner, Carsten T WotjakAbstract:The endocannabinoid System has been recognized as a major neuromodulatory System, which functions to maintain brain homoeostasis. Endocannabinoids are synthesized and released from the postsynapse and act as retrograde neuronal messengers, which bind to cannabinoid type 1 receptors at the presynapse. Here, they inhibit the release of neurotransmitters, including glutamate and GABA. By these means, endocannabinoids control the activation of various neuronal circuits including those involved in neuroendocrine Stress processing. Accordingly, exogenous cannabinoids such as the major active component of marijuana, Delta(9)-tetrahydrocannabinol, have long been known to activate the major neuroendocrine Stress Response System of mammals, the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the function of the endocannabinoid System in the regulation of Stress hormone secretion has only recently begun to be understood. It is the focus of the present review to provide the reader with an overview of our current knowledge of the role of endocannabinoid signalling in HPA axis regulation under basal as well as under Stressful conditions. This includes the specific sites of action, potential underlying neuronal pathways and interactions between behavioural and neuroendocrine Stress coping. Furthermore, the potential role of HPA axis activity dysregulations, caused by deficits in the endocannabinoid System, for the pathophysiology of psychiatric diseases is discussed.