RESUMEN
RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.
Asunto(s)
Quitridiomicetos , Técnicas de Silenciamiento del Gen , Glutamato-Cisteína Ligasa , Interferencia de ARN , ARN Mensajero , ARN Interferente Pequeño , ARN Interferente Pequeño/genética , ARN Mensajero/genética , Animales , Quitridiomicetos/genética , Quitridiomicetos/patogenicidad , Glutamato-Cisteína Ligasa/genética , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Anfibios/microbiología , Glutatión/metabolismoRESUMEN
Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralise oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, suggesting that glutathione is essential for life cycle completion in Bd. Supplementation with <2 mM exogenous glutathione accelerated zoospore development, but concentrations >2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutics.
Asunto(s)
Batrachochytrium , Cadmio , Glutatión , Peróxido de Hidrógeno , Glutatión/metabolismo , Cadmio/toxicidad , Animales , Batrachochytrium/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Micosis/microbiología , Micosis/veterinaria , Micosis/metabolismo , Anfibios/microbiologíaRESUMEN
Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.
Asunto(s)
Quitridiomicetos , Virus Fúngicos , Anfibios , Animales , Australia , Batrachochytrium , Virus Fúngicos/genéticaRESUMEN
Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.
Asunto(s)
Anuros/microbiología , Quitridiomicetos , Susceptibilidad a Enfermedades/veterinaria , Micosis/veterinaria , Serotonina/metabolismo , Enfermedades de la Piel/veterinaria , Piel/metabolismo , Animales , Anuros/inmunología , Anuros/metabolismo , Australia , Proliferación Celular/efectos de los fármacos , Quitridiomicetos/efectos de los fármacos , Susceptibilidad a Enfermedades/metabolismo , Susceptibilidad a Enfermedades/microbiología , Cromatografía de Gases y Espectrometría de Masas , Micosis/inmunología , Micosis/metabolismo , Serotonina/farmacología , Piel/química , Piel/microbiología , Enfermedades de la Piel/metabolismo , Esporangios/efectos de los fármacos , Esporangios/crecimiento & desarrollo , Linfocitos T/efectos de los fármacosRESUMEN
The fungus Batrachochytrium dendrobatidis is causing global amphibian declines. Here we describe a simple, rapid and inexpensive methylene blue staining protocol to determine B. dendrobatidis viability, regardless of life-stage. The viability of cells in suspension or adherent monolayers can be determined using either manual microscopy counting or colorimetric assay.
Asunto(s)
Anfibios/microbiología , Quitridiomicetos/fisiología , Esporangios , Esporas Fúngicas/aislamiento & purificación , Animales , Azul de Metileno/química , Viabilidad Microbiana , Técnicas Microbiológicas , Coloración y EtiquetadoRESUMEN
Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBF's efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 µg/ml for 2 h, while the minimal inhibitory concentration was 2 µg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.
Asunto(s)
Antifúngicos/administración & dosificación , Antifúngicos/farmacocinética , Anuros/microbiología , Quitridiomicetos/efectos de los fármacos , Micosis/veterinaria , Terbinafina/administración & dosificación , Terbinafina/farmacocinética , Animales , Antifúngicos/farmacología , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Micosis/tratamiento farmacológico , Piel/efectos de los fármacos , Piel/metabolismo , Piel/microbiología , Esporas Fúngicas/efectos de los fármacos , Terbinafina/farmacología , Resultado del TratamientoRESUMEN
Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.
Asunto(s)
Caspasa 3/metabolismo , Caspasa 7/metabolismo , Muerte Celular/genética , Células Epiteliales/metabolismo , Etiquetado Corte-Fin in Situ/métodos , Transferasas/genética , Animales , Anuros , Apoptosis , EpidermisRESUMEN
BACKGROUND: Amphibians are declining at an alarming rate, and one of the major causes of decline is the infectious disease chytridiomycosis. Parasitic fungal sporangia occur within epidermal cells causing epidermal disruption, but these changes have not been well characterised. Apoptosis (planned cell death) can be a damaging response to the host but may alternatively be a mechanism of pathogen removal for some intracellular infections. METHODS: In this study we experimentally infected two endangered amphibian species Pseudophryne corroboree and Litoria verreauxii alpina with the causal agent of chytridiomycosis. We quantified cell death in the epidermis through two assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL) and caspase 3/7. RESULTS: Cell death was positively associated with infection load and morbidity of clinically infected animals. In infected amphibians, TUNEL positive cells were concentrated in epidermal layers, correlating to the localisation of infection within the skin. Caspase activity was stable and low in early infection, where pathogen loads were light but increasing. In animals that recovered from infection, caspase activity gradually returned to normal as the infection cleared. Whereas, in amphibians that did not recover, caspase activity increased dramatically when infection loads peaked. DISCUSSION: Increased cell death may be a pathology of the fungal parasite, likely contributing to loss of skin homeostatic functions, but it is also possible that apoptosis suppression may be used initially by the pathogen to help establish infection. Further research should explore the specific mechanisms of cell death and more specifically apoptosis regulation during fungal infection.
RESUMEN
The potential cytotoxicity of cadmium selenide (CdSe) quantum dots (QDs) presents a barrier to their use in biomedical imaging or as diagnostic and therapeutic agents. Sulforaphane (SFN) is a chemoprotective compound derived from cruciferous vegetables which can up-regulate antioxidant enzymes and induce apoptosis and autophagy. This study reports the effects of SFN on CdSe QD-induced cytotoxicity in immortalised human hepatocytes and in the livers of mice. CdSe QDs induced dose-dependent cell death in hepatocytes with an IC50 = 20.4 µM. Pre-treatment with SFN (5 µM) increased cell viability in response to CdSe QDs (20 µM) from 49.5 to 89.3%. SFN induced a pro-oxidant effect characterized by depletion of intracellular reduced glutathione during short term exposure (3-6 h), followed by up-regulation of antioxidant enzymes and glutathione levels at 24 h. SFN also caused Nrf2 translocation into the nucleus, up-regulation of antioxidant enzymes and autophagy. siRNA knockdown of Nrf2 suggests that the Nrf2 pathway plays a role in the protection against CdSe QD-induced cell death. Wortmannin inhibition of SFN-induced autophagy significantly suppressed the protective effect of SFN on CdSe QD-induced cell death. Moreover, the role of autophagy in SFN protection against CdSe QD-induced cell death was confirmed using mouse embryonic fibroblasts lacking ATG5. CdSe QDs caused significant liver damage in mice, and this was decreased by SFN treatment. In conclusion, SFN attenuated the cytotoxicity of CdSe QDs in both human hepatocytes and in the mouse liver, and this protection was associated with the induction of Nrf2 pathway and autophagy.
Asunto(s)
Compuestos de Cadmio/toxicidad , Isotiocianatos/farmacología , Hígado/patología , Puntos Cuánticos/toxicidad , Compuestos de Selenio/toxicidad , Adenina/análogos & derivados , Adenina/farmacología , Androstadienos/farmacología , Animales , Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Técnicas de Silenciamiento del Gen , Glutatión/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espacio Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Hígado/efectos de los fármacos , Hígado/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Ratones Endogámicos ICR , Factor 2 Relacionado con NF-E2/metabolismo , Sustancias Protectoras/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfóxidos , Transcripción Genética/efectos de los fármacos , WortmaninaRESUMEN
Adaptation of D. radiodurans cells to extreme irradiation environments requires dynamic interactions between gene expression and metabolic regulatory networks, but studies typically address only a single layer of regulation during the recovery period after irradiation. Dynamic transcriptome analysis of D. radiodurans cells using strand-specific RNA sequencing (ssRNA-seq), combined with LC-MS based metabolite analysis, allowed an estimate of the immediate expression pattern of genes and antioxidants in response to irradiation. Transcriptome dynamics were examined in cells by ssRNA-seq covering its predicted genes. Of the 144 non-coding RNAs that were annotated, 49 of these were transfer RNAs and 95 were putative novel antisense RNAs. Genes differentially expressed during irradiation and recovery included those involved in DNA repair, degradation of damaged proteins and tricarboxylic acid (TCA) cycle metabolism. The knockout mutant crtB (phytoene synthase gene) was unable to produce carotenoids, and exhibited a decreased survival rate after irradiation, suggesting a role for these pigments in radiation resistance. Network components identified in this study, including repair and metabolic genes and antioxidants, provided new insights into the complex mechanism of radiation resistance in D. radiodurans.
Asunto(s)
Antioxidantes/metabolismo , Deinococcus/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Transcriptoma , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reparación del ADN , Deinococcus/genética , Deinococcus/efectos de la radiación , Redes y Vías Metabólicas/genética , Metabolómica , Análisis de Componente Principal , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Tolerancia a Radiación , Análisis de Secuencia de ARNRESUMEN
AIMS: In bacillithiol (BSH)-utilizing organisms, protein S-bacillithiolation functions as a redox switch in response to oxidative stress and protects critical Cys residues against overoxidation. In Bacillus subtilis, both the redox-sensing repressor OhrR and the methionine synthase MetE are redox controlled by S-bacillithiolation in vivo. Here, we identify pathways of protein de-bacillithiolation and test the hypothesis that YphP(BrxA) and YqiW(BrxB) act as bacilliredoxins (Brx) to remove BSH from OhrR and MetE mixed disulfides. RESULTS: We present evidence that the BrxA and BrxB paralogs have de-bacillithiolation activity. This Brx activity results from attack of the amino-terminal Cys residue in a CGC motif on protein BSH-mixed disulfides. B. subtilis OhrR DNA-binding activity is eliminated by S-thiolation on its sole Cys residue. Both the BrxA and BrxB bacilliredoxins mediate de-bacillithiolation of OhrR accompanied by the transfer of BSH to the amino-terminal cysteine of their CGC active site motif. In vitro studies demonstrate that BrxB can restore DNA-binding activity to OhrR which is S-bacillithiolated, but not to OhrR that is S-cysteinylated. MetE is most strongly S-bacillithiolated at Cys719 in vitro and can be efficiently de-bacillithiolated by both BrxA and BrxB. INNOVATION AND CONCLUSION: We demonstrate that BrxA and BrxB function in the reduction of BSH mixed protein disulfides with two natural substrates (MetE, OhrR). These results provide biochemical evidence for a new class of bacterial redox-regulatory proteins, the bacilliredoxins, which function analogously to glutaredoxins. Bacilliredoxins function in concert with other thiol-disulfide oxidoreductases to maintain redox homeostasis in response to disulfide stress conditions.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Metiltransferasas/metabolismo , Oxidación-Reducción , Proteínas Represoras/metabolismo , Bacillus subtilis/genética , Cisteína/metabolismo , Disulfuros/metabolismo , Glucosamina/metabolismo , Estrés Oxidativo , Azufre/metabolismoRESUMEN
In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB led to a decrease in BSH levels. The fosB and bshA mutants of strain COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels compared with those in the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to that in COL under thiol stress conditions. However, the bshA mutant of COL demonstrated decreased survival compared to that of the parent in human whole-blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independently of its capability to produce staphyloxanthin.
Asunto(s)
Proteínas Bacterianas/fisiología , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Estrés Oxidativo/fisiología , Staphylococcus aureus/metabolismo , Amidohidrolasas/deficiencia , Análisis de Varianza , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Cisteína/genética , Cisteína/fisiología , Glucosamina/genética , Glucosamina/fisiología , Glicosiltransferasas/genética , Peróxido de Hidrógeno/farmacología , Análisis por Micromatrices , Pruebas de Sensibilidad Microbiana , Mutación , NADP/metabolismo , Peroxidasa/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Xantófilas/biosíntesisRESUMEN
Bacillithiol (BSH) is the major low-molecular-weight (LMW) thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa , redox potential and thiol-disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol-disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM, which is significantly greater than previously observed from single measurements taken during mid-exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.
Asunto(s)
Bacillus subtilis/química , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Aminas/química , Bacillus subtilis/metabolismo , Ácidos Carboxílicos/química , Cisteína/química , Glucosamina/química , Glutatión/química , Cinética , Oxidación-Reducción , Compuestos de Sulfhidrilo/químicaRESUMEN
BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a high content of bacillithiol (~70%) was still observed in the B. anthracis ∆BA1557 strain. Genomic analysis led to the proposal that another deacetylase could exhibit cross-functionality in bacillithiol biosynthesis. In the present study, BA1557, its paralogue BA3888 and orthologous Bacillus cereus enzymes BC1534 and BC3461 have been characterized for their deacetylase activity towards N-acetylglucosamine malate, thus providing biochemical evidence for this proposal. In addition, the involvement of deacetylase enzymes is also expected in bacillithiol-detoxifying pathways through formation of S-mercapturic adducts. The kinetic analysis of bacillithiol-S-bimane conjugate favours the involvement of BA3888 as the B. anthracis bacillithiol-S-conjugate amidase (Bca). The high degree of specificity of this group of enzymes for its physiological substrate, along with their similar pH-activity profile and Zn²âº-dependent catalytic acid-base reaction provides further evidence for their cross-functionalities.
Asunto(s)
Amidohidrolasas/metabolismo , Bacillus anthracis/metabolismo , Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Acetilación , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Amidohidrolasas/química , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacillus anthracis/enzimología , Bacillus cereus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biocatálisis , Dominio Catalítico , Secuencia Conservada , Cisteína/metabolismo , Glucosamina/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Malatos/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Zinc/metabolismoRESUMEN
Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low molecular weight thiol found in low GC Gram-positive bacteria, such as Staphylococcus aureus. Like other low molecular weight thiols, BSH is likely involved in protection against a number of stresses. We examined S. aureus transposon mutants disrupted in each of the three genes associated with BSH biosynthesis. These mutants are sensitive to alkylating stress, oxidative stress, and metal stress indicating that BSH and BSH-dependent enzymes are involved in protection of S. aureus. We further demonstrate that BshB, a deacetylase involved in the second step of BSH biosynthesis, also acts as a BSH conjugate amidase and identify S. aureus USA 300 LAC 2626 as a BSH-S-transferase, which is able to conjugate chlorodinitrobenzene, cerulenin, and rifamycin to BSH.
Asunto(s)
Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Mutación , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/genética , Cromatografía Líquida de Alta Presión , Cisteína/metabolismo , Glucosamina/metabolismo , Yodoacetamida/farmacología , Metales/farmacología , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Oxidantes/farmacología , Piruvaldehído/farmacología , Staphylococcus aureus/enzimología , Compuestos de Sulfhidrilo/metabolismo , Factores de TiempoRESUMEN
AIMS: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the S-bacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress. RESULTS: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin-NADP(+) oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5'-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. S-bacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis. INNOVATION: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses. CONCLUSION: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects conserved metabolic enzymes and essential proteins against overoxidation.
Asunto(s)
Bacillus/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Ácido Hipocloroso/metabolismo , Estrés Fisiológico , Bacillus/efectos de los fármacos , Vías Biosintéticas , Cisteína/metabolismo , Glucosamina/metabolismo , Metabolómica , Metiltransferasas/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteoma/metabolismo , Proteómica , Hipoclorito de Sodio/metabolismo , Hipoclorito de Sodio/farmacologíaRESUMEN
FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Farmacorresistencia Bacteriana , Fosfomicina/química , Staphylococcus aureus/enzimología , Transferasas/química , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cisteína/análogos & derivados , Cisteína/genética , Cisteína/metabolismo , Fosfomicina/farmacología , Glucosamina/análogos & derivados , Glucosamina/genética , Glucosamina/metabolismo , Cinética , Magnesio/química , Magnesio/metabolismo , Manganeso/química , Manganeso/metabolismo , Staphylococcus aureus/genética , Transferasas/genética , Transferasas/metabolismoRESUMEN
Many bacteria produce siderophores for sequestration of growth-essential iron. Analysis of the Salinispora genomes suggests that these marine actinomycetes support multiple hydroxamate- and phenolate-type siderophore pathways. We isolated and characterized desferrioxamines (DFOs) B and E from all three recognized Salinispora species and linked their biosyntheses in S. tropica CNB-440 and S. arenicola CNS-205 to the des locus through PCR-directed mutagenesis. Gene inactivation of the predicted iron-chelator biosynthetic loci sid2-4 did not abolish siderophore chemistry. Additionally, these pathways could not restore the native growth characteristics of the des mutants in iron-limited media, although differential iron-dependent regulation was observed for the yersiniabactin-like sid2 pathway. Consequently, this study indicates that DFOs are the primary siderophores in laboratory cultures of Salinispora.
Asunto(s)
Deferoxamina/metabolismo , Hierro/metabolismo , Micromonosporaceae/metabolismo , Sideróforos/metabolismo , Secuencia de Bases , Orden Génico , Genes Bacterianos , Micromonosporaceae/genética , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Sideróforos/genéticaRESUMEN
The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng µg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.