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As antibiotic resistance increases and antibiotic development dwindles, new antimicrobial agents are needed. Recent advances in nanoscale engineering have increased interest in metal oxide nanoparticles, particularly zinc oxide nanoparticles, as antimicrobial agents. Zinc oxide nanoparticles are promising due to their broad-spectrum antibacterial activity and low production cost. Despite many studies demonstrating the effectiveness of zinc oxide nanoparticles, the antibacterial mechanism is still unknown. Previous work has implicated the role of reactive oxygen species such as hydrogen peroxide, physical damage of the cell envelope, and/or release of toxic Zn2+ ions as possible mechanisms of action. To evaluate the role of these proposed methods, we assessed the susceptibility of S. aureus mutant strains, ΔkatA and ΔmprF, to zinc oxide nanoparticles of approximately 50 nm in size. These assays demonstrated that hydrogen peroxide and electrostatic interactions are not crucial for mediating zinc oxide nanoparticle toxicity. Instead, we found that Zn2+ accumulates in Mueller-Hinton Broth over time and that removal of Zn2+ through chelation reverses this toxicity. Furthermore, we found that the physical separation of zinc oxide nanoparticles and bacterial cells using a semi-permeable membrane still allows for growth inhibition. We concluded that soluble Zn2+ is the primary mechanism by which zinc oxide nanoparticles mediate toxicity in Mueller-Hinton Broth. Future work investigating how factors such as particle morphology (e.g., size, polarity, surface defects) and media contribute to Zn2+ dissolution could allow for the synthesis of zinc oxide nanoparticles that possess chemical and morphological properties best suited for antibacterial efficacy.
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Nano- and microscale zinc oxide (ZnO) exhibits significant potential as a novel antibacterial agent in biomedical applications. However, the uncertainty regarding the underlying mechanisms of the observed antimicrobial action inhibits the realization of this potential. Particularly, the nature of interactions at the free crystalline surface and the influence of the local bacterial environment remains unclear. In this investigation, we utilize ZnO particles synthesized via tunable hydrothermal growth method as a platform to elucidate the effects of interactions with phosphate-rich environments and differentiate them from those with bacteria. This is achieved using the time- and energy-dependent surface photovoltage (SPV) to monitor modifications of the surface electronic structure and surface charge dynamics of the ZnO particles due to these interactions. It is found that there exists a dramatic change in the SPV transients after exposure to phosphate-rich environments. It also presents differences in the sub-bandgap surface electronic structure after these exposures. It can be suggested that these phenomena are a consequence of phosphate adsorption at surface traps corresponding to zinc deficiency defects. This effect is shown to be suppressed in the presence of Staphylococcus aureus bacteria. Our results support the previously proposed model of the competitive nature of interactions between S. aureus and aqueous phosphates with the free surface of ZnO and bring greater clarity to the effects of phosphate-rich environments on bacterial growth inhibition of ZnO.
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The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.
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Nano- and microcrystalline ZnO is an inexpensive, easily synthesized material with a multitude of applications. Its usefulness in the present and future stems from its exceptional optoelectronic, structural, and chemical characteristics as well as a broad range of production techniques. One application comes from its ability to inhibit bacterial growth. Despite the well-documented, vigorously studied antimicrobial action of ZnO particles, the most fundamental physical and chemical mechanisms driving growth inhibition are still not well identified. Particularly, the nature of interactions between ZnO surfaces and extracellular material is not totally clear. This is important given the anisotropic lattice of ZnO leading to two characteristically different lattice terminations: polar and nonpolar, polar being electrically charged with many defect sites and nonpolar being electrically neutral while remaining relatively defect-free. In this work, we employ a hydrothermal growth protocol that allows us to produce ZnO microcrystals with dependable control of morphology and, particularly, the relative abundances of polar and nonpolar free surfaces. This functions as a platform for our investigations into surface-surface interactions behind the antibacterial action of ZnO microcrystals. In our studies, we produced ZnO crystals comparable in size or larger than Staphylococcus aureus bacteria. This was done intentionally to ensure that the ZnO particles would not internalize into the bacterial cells. Our experiments were performed in conjunction with surface photovoltage studies of ZnO crystals to characterize electronic structure and charge dynamics that might be contributing to the antibacterial properties of our samples. We report on the interactions between ZnO microcrystalline surfaces and extracellular material of Staphylococcus aureus bacteria.
Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Óxido de Zinco/química , Antibacterianos/química , Cristalização , Testes de Sensibilidade Microbiana , Optogenética , Propriedades de Superfície , Óxido de Zinco/farmacologiaRESUMO
Understanding bacterial virulence provides insight into the molecular basis behind infection and could identify new drug targets. However, assessing potential virulence determinants relies on testing in an animal model. The mouse is a well-validated model but it is constrained by the ethical and logistical challenges of using vertebrate animals. Recently the larva of the greater wax moth Galleria mellonella has been explored as a possible infection model for a number of pathogens. In this study, we developed G. mellonella as an infection model for Bacillus anthracis Sterne. We first validated two different infection assays, a survival assay and a competition assay, using mutants containing disruptions in known B. anthracis virulence genes. We next tested the utility of G. mellonella to assess the virulence of transposon mutants with unknown mutations that had increased susceptibility to hydrogen peroxide in in vitro assays. One of these transposon mutants also displayed significantly decreased virulence in G. mellonella. Further investigation revealed that this mutant had a disruption in the petrobactin biosynthesis operon (asbABCDEF), which has been previously implicated in both virulence and defense against oxidative stress. We conclude that G. mellonella can detect attenuated virulence of B. anthracis Sterne in a manner consistent with that of mammalian infection models. Therefore, G. mellonella could serve as a useful alternative to vertebrate testing, especially for early assessments of potential virulence genes when use of a mammalian model may not be ethical or practical.
Assuntos
Antraz/microbiologia , Bacillus anthracis , Animais , Antraz/metabolismo , Bacillus anthracis/genética , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Genes Bacterianos , Peróxido de Hidrogênio/metabolismo , Camundongos , Viabilidade Microbiana/genética , Mutação , Óperon , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Staphylococcus aureus is a leading cause of infection in the United States, and due to the rapid development of resistance, new antibiotics are constantly needed. trans-Translation is a particularly promising antibiotic target because it is conserved in many bacterial species, is critical for bacterial survival, and is unique among prokaryotes. We have investigated the potential of KKL-40, a small-molecule inhibitor of trans-translation, and find that it inhibits both methicillin-susceptible and methicillin-resistant strains of S. aureus KKL-40 is also effective against Gram-positive pathogens, including a vancomycin-resistant strain of Enterococcus faecalis, Bacillus subtilis, and Streptococcus pyogenes, although its performance with Gram-negative pathogens is mixed. KKL-40 synergistically interacts with the human antimicrobial peptide LL-37, a member of the cathelicidin family, to inhibit S. aureus but not other antibiotics tested, including daptomycin, kanamycin, or erythromycin. KKL-40 is not cytotoxic to HeLa cells at concentrations that are 100-fold higher than the effective MIC. We also find that S. aureus develops minimal resistance to KKL-40 even after multiday passage at sublethal concentrations. Therefore, trans-translation inhibitors could be a particularly promising drug target against S. aureus, not only because of their ability to inhibit bacterial growth but also because of their potential to simultaneously render S. aureus more susceptible to host antimicrobial peptides.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Células HeLa , Humanos , Resistência a Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/microbiologia , CatelicidinasRESUMO
ClpX functions as either an independent chaperone or a component of the ClpXP protease, a conserved intracellular protease that acts as a global regulator in the bacterial cell by degrading regulatory proteins, stress response proteins and rate-limiting enzymes. Previously, we found that loss of clpX in Bacillus anthracis Sterne leads to increased susceptibility to antimicrobial agents that target the cell envelope. The aim of this study was to identify genes within the regulatory network of clpX that contribute to antimicrobial resistance. Using microarray analysis, we found 119 genes that are highly differentially expressed in the ∆clpX mutant, with the majority involved in metabolic, transport or regulatory functions. Several of these differentially expressed genes, including glpF, sigM, mrsA, lrgA and lrgB, are associated with cell wall-active antibiotics in other bacterial species. We focused on lrgA and lrgB, which form the lrgAB operon and are downregulated in ∆clpX, because loss of lrgAB increases autolytic activity and penicillin susceptibility in Staphylococcus aureus. While we observed no changes in autolytic activity in either ∆clpX or ∆lrgAB B. anthracis Sterne, we find that both mutants have increased susceptibility to the antimicrobial peptide LL-37 and daptomycin. However, phenotypes between ∆clpX and ∆lrgAB are not identical as ∆clpX also displays increased susceptibility to penicillin and nisin but ∆lrgAB does not. Therefore, while decreased expression of lrgAB may be partially responsible for the increased antimicrobial susceptibility seen in the ∆clpX mutant, disruption of other pathways must also contribute to this phenotype.
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Bacillus anthracis/genética , Proteínas de Bactérias/genética , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Antibacterianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Deleção de Genes , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
Bacillus anthracis, the causative agent of anthrax, relies on multiple virulence factors to subvert the host immune defense. Using Caenorhabditis elegans as an infection model, we screened approximately 5,000 transposon mutants of B. anthracis Sterne for decreased virulence. One of the attenuated mutants resulted in loss of expression of yceG and yceH, the last two genes in a six-gene cluster of tellurite resistance genes. We generated an analogous insertional mutant to confirm the phenotype and characterize the role of yceGH in resistance to host defenses. Loss of yceGH rendered the mutants more sensitive to tellurite toxicity as well as to host defenses such as reactive oxygen species and the cathelicidin family of antimicrobial peptides. Additionally, we see decreased survival in mammalian models of infection, including human whole blood and in mice. We identify a novel role for the yceGH genes in B. anthracis Sterne virulence and suggest that C. elegans is a useful infection model to study anthrax pathogenesis.
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Antraz/imunologia , Bacillus anthracis/genética , Bacillus anthracis/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Telúrio/imunologia , Animais , Antraz/microbiologia , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Mutação/genética , Mutação/imunologia , Virulência/genética , Virulência/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
The ClpXP protease is a critical bacterial intracellular protease that regulates protein turnover in many bacterial species. Here we identified a pharmacological inhibitor of the ClpXP protease, F2, and evaluated its action in Bacillus anthracis and Staphylococcus aureus. We found that F2 exhibited synergistic antimicrobial activity with cathelicidin antimicrobial peptides and antibiotics that target the cell well and/or cell membrane, such as penicillin and daptomycin, in B. anthracis and drug-resistant strains of S. aureus. ClpXP inhibition represents a novel therapeutic strategy to simultaneously sensitize pathogenic bacteria to host defenses and pharmaceutical antibiotics.
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Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Endopeptidase Clp/antagonistas & inibidores , Proteínas de Escherichia coli/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/genética , Membrana Celular/metabolismo , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Dados de Sequência Molecular , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Tetrazóis/farmacologia , CatelicidinasRESUMO
Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria-Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair-deficient strains (MMRâ») such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.
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Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/genética , Análise Mutacional de DNA/métodos , Mutação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , FenótipoRESUMO
The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.
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Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/fisiologia , Caenorhabditis elegans/microbiologia , Endotoxinas/fisiologia , Proteínas Hemolisinas/fisiologia , Animais , Toxinas de Bacillus thuringiensis , VirulênciaRESUMO
Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes) have been implicated in the initial phase of anthrax, less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.
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Antígenos de Bactérias/farmacologia , Bacillus anthracis , Toxinas Bacterianas/farmacologia , Endocitose/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Animais , Bacillus anthracis/química , Bacillus anthracis/patogenicidade , Caderinas , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Sinergismo Farmacológico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Modelos Animais , Ligação Proteica , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.
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Antibacterianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Soro/imunologia , Soro/microbiologia , Transferrina/farmacologia , Animais , Bacillus anthracis/crescimento & desenvolvimento , Feminino , Humanos , Imunidade Inata , Ferro/metabolismo , CamundongosRESUMO
Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and alpha-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.
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Adenosina Trifosfatases/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Bacillus anthracis/patogenicidade , Farmacorresistência Bacteriana , Endopeptidase Clp/metabolismo , Adenosina Trifosfatases/genética , Animais , Antraz/microbiologia , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Elementos de DNA Transponíveis , Endopeptidase Clp/genética , Cobaias , Hemólise , Humanos , Imunidade Inata , Camundongos , Mutagênese , Fenótipo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Gro alpha) and CXCL2 (Gro beta), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis. CONCLUSIONS/SIGNIFICANCE: These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies.
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Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Meningites Bacterianas/induzido quimicamente , Meningites Bacterianas/patologia , Microcirculação/efeitos dos fármacos , Neutrófilos/fisiologia , Bacillus anthracis , Encéfalo/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Humanos , Modelos Neurológicos , Neutrófilos/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Production of the beta-subunit of FSH is the rate-limiting step in FSH synthesis, and a number of peptide and steroid hormones within the reproductive axis have been found to regulate transcription of the FSH beta-subunit gene. Although both activin and glucocorticoids are notable regulators of FSHbeta by themselves, we find that cotreatment results in a synergistic interaction on the mouse FSHbeta promoter at the level of the gonadotrope using transient transfection of a reporter gene into the LbetaT2 immortalized gonadotrope-derived cell line. This synergistic interaction is specific to FSHbeta, because only additive effects of these two hormones are observed on LH beta-subunit, GnRH receptor, and mouse mammary tumor virus gene expression. Components of both activin and glucocorticoid signaling are found to be necessary for synergy, and there are specific cis elements on the mouse FSHbeta promoter that contribute to the synergistic response as well. We also identify novel activin-responsive regions in the mouse FSHbeta promoter and find that the -120 site can bind Smad2/3 in vitro. In addition, the glucocorticoid receptor and Smad3 are sufficient to confer a striking synergy with glucocorticoids on the mouse FSHbeta promoter. Our studies provide the first evidence of a synergistic interaction between activin and glucocorticoids within the gonadotrope cell and demonstrate that this synergy can occur directly at the level of the mouse FSHbeta promoter.
Assuntos
Ativinas/farmacologia , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Glucocorticoides/farmacologia , Gonadotrofos/efeitos dos fármacos , Gonadotrofos/metabolismo , Ativinas/biossíntese , Ativinas/metabolismo , Animais , Linhagem Celular Transformada , DNA/metabolismo , Sinergismo Farmacológico , Subunidade beta do Hormônio Folículoestimulante/genética , Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3/fisiologiaRESUMO
FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LbetaT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHbeta promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHbeta promoter where six putative hormone response elements exist. The -381 site is critical for FSHbeta induction by all three steroid hormones, whereas the -197 and -139 sites contribute to maximal induction. Interestingly, the -273 and -230 sites are also necessary for androgen and progestin induction of FSHbeta, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHbeta promoter, in vivo, and specifically bind the -381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHbeta by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotrope.
Assuntos
Androgênios/farmacologia , Hormônio Foliculoestimulante/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Progestinas/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Conservada , DNA/genética , DNA/metabolismo , Humanos , Ligantes , Camundongos , Hipófise/citologia , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Elementos de Resposta/genética , Transcrição Gênica/genéticaRESUMO
Appropriate expression of GnRH receptor (GnRHR) is necessary for the correct regulation of the gonadotropins, LH and FSH, by GnRH. GnRHR is primarily expressed in the gonadotrope cells of the anterior pituitary, and a number of regulatory elements important for both basal and hormonal regulation of the gene have been identified. Using the gonadotrope-derived cell line, alpha T3-1, that endogenously expresses GnRHR, we have identified an ATTA element located at -298 relative to the transcriptional start site that is essential for basal expression of the GnRHR gene. LHX3, a member of the LIM homeodomain family, binds the -298 ATTA site in vitro as well as to the endogenous GnRHR promoter in vivo. Additionally, LHX3 specifically activates through this -298 ATTA site in transient transfection assays. LHX3 is essential for pituitary development and has been implicated in the regulation of a number of pituitary specific genes; however, this is the first report identifying its role in the regulation of GnRHR.
Assuntos
Regulação da Expressão Gênica , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores LHRH/genética , Animais , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Camundongos , Mutagênese , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Hipófise , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , TransfecçãoRESUMO
FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.
Assuntos
Ativinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hormônio Foliculoestimulante/metabolismo , Transativadores/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Hormônio Foliculoestimulante/genética , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Homologia de Sequência , Proteínas SmadRESUMO
FSH is a heterodimeric glycoprotein hormone secreted from the gonadotrope cell population of the anterior pituitary. Despite its crucial role in mammalian reproduction, very little is known about regulation of the FSH beta-subunit gene at the molecular level. In this report, we examine the basis for cell-specific expression of FSH beta using the mouse L beta T2 and alpha T3-1 gonadotrope-derived cell lines. Characterization of the hormonal content of L beta T2 and alpha T3-1 cells at the protein level classifies these cells as relatively mature and immature gonadotropes, respectively. We studied L beta T2 cell-specific expression of FSH beta using 398 bp of the mouse FSH beta regulatory region linked to a luciferase reporter gene in transient transfection assays. This mouse FSH beta promoter can direct reporter gene expression specifically to L beta T2 cells when compared with other pituitary- and non-pituitary-derived cell lines, including alpha T3-1 cells. Furthermore, it is induced by activin, and interruption of the autocrine activin loop in L beta T2 cells by the addition of follistatin reduces its expression. Truncation analysis indicates that several regions of the promoter are involved in this specificity and that these can be dissociated from activin regulation. We identify binding sites for the orphan nuclear receptor steroidogenic factor-1 and the heterotrimeric transcription factor nuclear factor Y and show that these elements functionally interact to regulate FSH beta gene expression in an L beta T2 cell-specific manner. Moreover, steroidogenic factor-1 and nuclear factor Y are shown to physically interact with each other. This study is the first to demonstrate the presence of basal FSH beta protein in L beta T2 cells and to identify specific elements within the FSH beta promoter that contribute to basal and cell-specific expression of the gene.