RESUMEN
Brucellosis, caused by a facultative intracellular parasite Brucella species, is the most common bacterial zoonotic infection worldwide. Brucella can survive and proliferate in several phagocytic and nonphagocytic cell types. Human brucellosis has similar clinical symptoms with systemic diseases, which may lead to delay of diagnosis and increasing of complications. Therefore, investigating the proliferation of Brucella in host cells is important to understand the pathogenesis of the disease. Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has been recommended by World Health Organization as an antimalarial drug. However, there have been few studies regarding its effectiveness against bacteria. In the present study, it was revealed that B. suis vaccine strain 2 (S2) grew in BV2 cells without significant cytotoxicity, and less than 20 µM DHA had no inhibitory effects on BV2 cells. Furthermore, DHA reduced B. suis S2 growth in BV2 cells, and increased the percentage of apoptosis and the expression of cleaved caspase3 in B. suis S2infected cells. Collectively, the present data indicated that DHA induced the caspasedependent apoptotic pathway to inhibit the intracellular B. suis S2 growth.
Asunto(s)
Apoptosis/efectos de los fármacos , Artemisininas/farmacología , Brucella suis/efectos de los fármacos , Brucella suis/fisiología , Viabilidad Microbiana/efectos de los fármacos , Microglía/microbiología , Animales , Brucelosis/metabolismo , Brucelosis/microbiología , Humanos , RatonesRESUMEN
BACKGROUND: Brucellosis, also known as undulant, Mediterranean or Malta fever, is a systemic infection that causes fever, sweats, arthralgias and myalgias. A globally important disease, brucellosis is re-emerging in Australia in association with feral pig hunting activities. OBJECTIVE: This article aims to provide clinicians with an overview of brucellosis, covering epidemiology, clinical features, diagnosis, management and prevention. DISCUSSION: Brucellosis should be suspected in all patients with non-specific, flu-like illness who fall into one of the major risk groups (feral pig hunters, overseas travellers and migrants). Depression is common and often severe, relative to other symptoms. Early diagnosis and treatment are important for preventing complications, which include osteoarticular, genitourinary or, more rarely, neurological or cardiovascular diseases. Diagnosing acute infections is based on serology and blood cultures; imaging and biopsy may be required for diagnosis of focal infections. Dual therapy with doxycycline and gentamicin is the recommended treatment. Relapse occurs in up to 10% of patients. Prevention is achieved through the use of protective gear during hunting and avoidance of unpasteurised dairy products in countries where occur in animals.
Asunto(s)
Brucelosis/diagnóstico , Brucelosis/terapia , Animales , Anorexia/etiología , Antibacterianos/uso terapéutico , Artralgia/etiología , Australia/epidemiología , Brucella abortus/efectos de los fármacos , Brucella abortus/patogenicidad , Brucella canis/efectos de los fármacos , Brucella canis/patogenicidad , Brucella melitensis/efectos de los fármacos , Brucella melitensis/patogenicidad , Brucella suis/efectos de los fármacos , Brucella suis/patogenicidad , Brucelosis/epidemiología , Bovinos , Productos Lácteos/efectos adversos , Productos Lácteos/virología , Reservorios de Enfermedades/veterinaria , Reservorios de Enfermedades/virología , Perros , Doxiciclina/uso terapéutico , Fatiga/etiología , Fiebre/etiología , Gentamicinas/uso terapéutico , Cabras , Cefalea/etiología , Humanos , Factores de Riesgo , Ovinos , Porcinos , Viaje/estadística & datos numéricos , Zoonosis/diagnóstico , Zoonosis/fisiopatologíaRESUMEN
The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209+ MZ macrophages (MZMs) and the CD169+ marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection.
Asunto(s)
Brucelosis/inmunología , Interacciones Huésped-Patógeno , Interferón gamma/inmunología , Macrófagos/inmunología , Receptores de Interferón/inmunología , Bazo/inmunología , Animales , Antibacterianos/farmacología , Linfocitos B/inmunología , Linfocitos B/microbiología , Brucella abortus/efectos de los fármacos , Brucella abortus/inmunología , Brucella abortus/patogenicidad , Brucella melitensis/efectos de los fármacos , Brucella melitensis/inmunología , Brucella melitensis/patogenicidad , Brucella suis/efectos de los fármacos , Brucella suis/inmunología , Brucella suis/patogenicidad , Brucelosis/tratamiento farmacológico , Brucelosis/genética , Brucelosis/microbiología , Quimiocina CCL19/genética , Quimiocina CCL19/inmunología , Quimiocina CCL21/genética , Quimiocina CCL21/inmunología , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Enfermedad Crónica , Regulación de la Expresión Génica , Interferón gamma/genética , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/inmunología , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Rifampin/farmacología , Transducción de Señal , Bazo/microbiología , Estreptomicina/farmacología , Linfocitos T/inmunología , Linfocitos T/microbiología , Receptor de Interferón gammaRESUMEN
Carbonic anhydrases have started to emerge as new potential antibacterial targets for several pathogens. Two ß-carbonic anhydrases, denominated bsCA I and bsCA II, have been isolated and characterized from the bacterial pathogen Brucella suis, the causative agent of brucellosis or Malta fever. These enzymes have been investigated in detail and a wide range of classical aromatic and heteroaromatic sulfonamides as well as carbohydrate-based compounds have been found to inhibit selectively and efficiently Brucella suis carbonic anhydrases. Inhibition of these metalloenzymes constitutes a novel approach for the potential development of new anti-Brucella agents. This review aims at discussing the recent literature on this topic.
Asunto(s)
Antibacterianos/farmacología , Brucella suis/efectos de los fármacos , Brucella suis/enzimología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Sulfonamidas/farmacología , Antibacterianos/química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.
Asunto(s)
Brucella suis/patogenicidad , Brucelosis/veterinaria , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/patología , Placenta/patología , Trofoblastos/patología , Factor de Transcripción Activador 6/metabolismo , Animales , Apoptosis/fisiología , Vacuna contra la Brucelosis , Brucella suis/clasificación , Brucella suis/efectos de los fármacos , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Proliferación Celular , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Estrógenos/metabolismo , Femenino , Enfermedades de las Cabras/microbiología , Cabras , Proteínas de Choque Térmico/biosíntesis , Placenta/microbiología , Embarazo , Progesterona/metabolismo , Prolactina/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción CHOP/biosíntesis , Tunicamicina/farmacología , eIF-2 Quinasa/metabolismoRESUMEN
A series of new Schiff bases derived from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide containing either a hydrophobic or a hydrophilic tail, have been investigated as inhibitors of three ß-carbonic anhydrases (CA, EC 4.2.1.1) from three different microorganisms. Their antifungal, antibacterial and antiprotozoan activities have been determined against the pathogenic fungus Cryptococcus neoformans, the bacterial pathogen Brucella suis and the protozoan parasite Leishmania donovani chagasi, responsible for Leishmaniasis. The results of these inhibition studies show that all three enzymes were efficiently inhibited by the Schiff base sulfonamides with KI values in the nanomolar or submicromolar range, depending on the nature of the tail, coming from the aryl/heteroaryl moiety present in the starting aldehyde employed in the synthesis. Furthermore, the compounds hereby investigated revealed high ß-CAs selectivity over the ubiquitous, physiologically relevant and off-target human isoforms (CA I and II) and to be more potent as antifungal and antibacterial than as antiprotozoan potential drugs.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antiprotozoarios/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Antibacterianos/química , Antifúngicos/química , Antiprotozoarios/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Brucella suis/efectos de los fármacos , Anhidrasa Carbónica I/metabolismo , Anhidrasa Carbónica II/metabolismo , Anhidrasas Carbónicas/metabolismo , Cryptococcus neoformans/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Humanos , Leishmania infantum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Bases de Schiff , Sulfanilamida , Sulfanilamidas/químicaRESUMEN
Plazomicin is a next-generation aminoglycoside with a potentially unique set of clinical characteristics compared with other aminoglycosides. This study assessed the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of plazomicin against 15 clinical isolates as well as three reference strains representing Brucella abortus, Brucella melitensis and Brucella suis. These data were compared with those obtained for six other aminoglycosides and two aminocyclitols. Plazomicin and gentamicin were the only drugs demonstrating bactericidal activity towards two of the three Brucella spp., whilst plazomicin was the only drug exhibiting bactericidal activity against B. suis. This is the first study to assess the bactericidal nature of plazomicin against Brucella spp. in vitro.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Brucella abortus/efectos de los fármacos , Brucella melitensis/efectos de los fármacos , Brucella suis/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Sisomicina/análogos & derivados , Brucella abortus/aislamiento & purificación , Brucella melitensis/aislamiento & purificación , Brucella suis/aislamiento & purificación , Brucelosis/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Sisomicina/farmacologíaRESUMEN
Histidinol dehydrogenase (HDH) has been established as a virulence factor for the human pathogen bacterium Brucella suis. Targeting such a virulence factor is a relevant anti-infectious approach as it could decrease the frequency of antibiotic resistance appearance. In this paper, we describe the synthesis of a family of oxo- and thioxo-imidazo[1,5-c]pyrimidines, potential enzyme inhibitors. Beyond their anti-HDH activity, the synthesis approach of these molecules, never described before, is highly original and these oxo- and thioxo- derivatives can improve dramatically the efficiency of the histidine protection pathway for the synthesis of histidine analogues.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/farmacología , Brucella suis/enzimología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Histidina/química , Bibliotecas de Moléculas Pequeñas/farmacología , Brucella suis/efectos de los fármacos , Brucelosis/microbiología , Histidina/metabolismo , Humanos , Imidazoles/química , Estructura Molecular , Pirimidinas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-Actividad , Factores de Virulencia/síntesis química , Factores de Virulencia/farmacologíaRESUMEN
Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 µM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 µM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.
Asunto(s)
Brucella suis/efectos de los fármacos , Brucella suis/crecimiento & desarrollo , Brucelosis/microbiología , Macrófagos/microbiología , Ácidos Nucleicos de Péptidos/farmacología , Animales , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brucella suis/genética , Brucella suis/metabolismo , Línea Celular , Péptidos de Penetración Celular , Medios de Cultivo , Farmacorresistencia Bacteriana , Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Brucella suis is a dangerous biological warfare agent already used for military purposes. This bacteria cause brucellosis, a zoonosis highly infective and difficult to fight. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. We present here the first three-dimensional structure of B. suis NH (BsNH) and propose this enzyme as a molecular target to the drug design in the fight against brucellosis. In addition, we performed molecular docking studies, aiming to analyze the three-dimensional positioning of nine known inhibitors of Chritidia fasciculata NH (CfNH) in the active sites of BsNH and CfNH. We also analyzed the main interactions of some of these compounds inside the active site of BsNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compound as lead for the design of potential inhibitors of BsNH. Most of the docking and MD results corroborated to each other and the docking results also suggested a good correlation with experimental data.
Asunto(s)
Proteínas Bacterianas/química , Brucella suis/enzimología , Simulación de Dinámica Molecular , N-Glicosil Hidrolasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Armas Biológicas , Brucella suis/química , Brucella suis/efectos de los fármacos , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Enlace de Hidrógeno , Cinética , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/antagonistas & inhibidores , N-Glicosil Hidrolasas/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Alineación de SecuenciaRESUMEN
AIMS: The purpose of this study was to evaluate the effects of environmental conditions and material type on persistence and inactivation of Brucella suis. METHODS AND RESULTS: Brucella suis (approx. 1 × 10(8) CFU) was spiked onto surfaces (glass, aluminium and wood) by liquid inoculation. Persistence was evaluated over 56 days at 22 ± 2°C, 40 ± 15% r.h. and 5 ± 3°C, 30 ± 15% r.h. In addition, three readily available decontaminants (pH-adjusted bleach, 70% ethanol and 1% citric acid) were evaluated for their effectiveness at inactivating Br. suis on these materials. Decontaminations were conducted following 0 and 28 days exposure to the two conditions. Results indicated that Br. suis can persist on environmental surfaces for at least 56 days. Persistence was highest at low temperature. Decontamination was most challenging on wood with all three decontaminants. CONCLUSIONS: Following a Br. suis contamination incident, passive decontamination (through attenuation) may not be feasible, as this organism can persist for months. In addition, the results suggest that some sporicidal decontaminants may be ineffective on materials such as wood, even for vegetative biological agents such as Br. suis. SIGNIFICANCE AND IMPACT OF STUDY: This study aids incident commanders and remediation experts to make informed decisions regarding decontamination after a biological contamination incident.
Asunto(s)
Brucella suis/crecimiento & desarrollo , Materiales de Construcción/microbiología , Descontaminación/métodos , Aluminio , Brucella suis/efectos de los fármacos , Ácido Cítrico/farmacología , Etanol/farmacología , Vidrio , Humedad , Ácido Hipocloroso/farmacología , Temperatura , Factores de Tiempo , Madera/microbiologíaRESUMEN
Positive cultures from hepatic abscess drainage are extremely rare, and in this case the infection would have remained undiagnosed if Brucella suis had not been isolated. Failure to correctly diagnose this zoonosis delays patient treatment and is dangerous, as in this case that could have been a laboratory-acquired disease. This type of infection is preventable if proper safety protocols are established and followed.
Asunto(s)
Brucella suis/aislamiento & purificación , Brucelosis/diagnóstico , Doxiciclina/uso terapéutico , Absceso Hepático/microbiología , Rifampin/uso terapéutico , Anciano , Brucella suis/efectos de los fármacos , Brucelosis/tratamiento farmacológico , Brucelosis/microbiología , Drenaje , Humanos , Absceso Hepático/cirugía , Masculino , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
A new selective medium, named LNIV-M, has been developed for isolation of Brucella suis. In this work, we evaluated the growth of B. suis reference and field strains from domestic pigs in different basal media and the susceptibility to different antibiotics contained in the currently used Farrell's and modified Thayer-Martin media. We also determined the efficacy of LNIV-M and its diagnostic performance for isolating B. suis from wild boar tissue samples. A total of 1649 samples from 918 hunter-harvested wild boars were cultured in LNIV-M, Farrell's and modified Thayer-Martin media. One hundred and thirty-nine (8.4%) samples from 63 (6.9%) animals resulted in a positive culture. LNIV-M detected 93.6% and 62.6% of positive animals and samples, respectively, while Farrell's and modified Thayer-Martin media detected, respectively, 92.1% and 79.4% of positive animals and 58.3% and 59.7% of samples. These results confirm the adequate diagnostic performance of LNIV-M in the isolation of B. suis.
Asunto(s)
Técnicas Bacteriológicas/veterinaria , Brucella suis/fisiología , Brucelosis/veterinaria , Medios de Cultivo/química , Enfermedades de los Porcinos/diagnóstico , Animales , Antibacterianos/farmacología , Técnicas Bacteriológicas/normas , Brucella suis/efectos de los fármacos , Brucella suis/aislamiento & purificación , Brucelosis/diagnóstico , Brucelosis/microbiología , Medios de Cultivo/normas , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Sus scrofa , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
Brucella, a facultative intracellular pathogen, is one of the most common zoonotic diseases worldwide. Considering the alarming health problem caused by the emergence of resistance and multi-resistance of intracellular pathogen, the challenge is currently to identify and to validate novel pharmaceutical targets in this bacteria species. Brucella's genome encodes metalloproteins involved in various biosynthetic processes, some of them being essential during intracellular growth phase and virulence. The potential of prokaryotic zinc metalloproteins such as carbonic anhydrase (CA) and histidinol dehydrogenase (HDH) as anti-Brucella targets has only recently been taken into consideration in the search of novel anti-infective agents that lack of cross-resistance to existing drugs. These enzymes have a growing significance in modern medicine as they are required for growth and/or virulence in several intracellular pathogen species. This review illustrates and describes the progress which has been made in the design and the discovery of selective inhibitors of these bacterial enzymes as new potential anti-Brucella agents.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/metabolismo , Brucella suis/enzimología , Anhidrasas Carbónicas/metabolismo , Metaloproteínas/metabolismo , Zinc/metabolismo , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Brucella suis/efectos de los fármacos , Brucella suis/patogenicidad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Metaloproteínas/antagonistas & inhibidores , Estructura Molecular , Virulencia/efectos de los fármacosRESUMEN
In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination of classical approaches and docking studies. The IC(50)-values of these inhibitors on HDH activity were in the nanomolar range, and several of them showed a 70-100% inhibition of Brucella growth in minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication of B. suis in human macrophages at concentrations as low as 5 µM, with an overall survival of intramacrophagic bacteria reduced by a factor 10(3). Docking studies with two inhibitors showed a good fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor NAD(+). Experimental data confirmed competition between inhibitors and NAD(+) at this site. Hence, these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/farmacología , Brucella suis/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Unión Competitiva , Brucella suis/enzimología , Brucella suis/patogenicidad , Brucella suis/fisiología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Cetonas/química , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Datos de Secuencia Molecular , NAD/metabolismo , Conformación Proteica , Especificidad por Sustrato , Replicación Viral/efectos de los fármacosRESUMEN
A ß-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO(2) to bicarbonate, with a k(cat) of 1.1×10(6), and k(cat)/K(m) of 8.9×10(7)M(-1)s(-1). A panel of sulfonamides and sulfamates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3nM-8.56µM. The best bsCA II inhibitors were some glycosylated sulfanilamides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3-87nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial ß-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics.
Asunto(s)
Antibacterianos/farmacología , Brucella suis/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Sulfonamidas/farmacología , Ácidos Sulfónicos/farmacología , Antibacterianos/química , Brucella suis/enzimología , Brucella suis/crecimiento & desarrollo , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/aislamiento & purificación , Clonación Molecular , Diseño de Fármacos , Descubrimiento de Drogas , Concentración 50 Inhibidora , Cinética , Sulfonamidas/química , Ácidos Sulfónicos/químicaRESUMEN
The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis DeltabepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the DeltabepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the DeltabepE DeltabepG double mutant showed a more severe phenotype than the DeltabepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella suis/efectos de los fármacos , Farmacorresistencia Bacteriana , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Brucella suis/patogenicidad , Brucella suis/fisiología , Ácido Desoxicólico/farmacología , Células Epiteliales/microbiología , Etidio/farmacología , Eliminación de Gen , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Violeta de Genciana/farmacología , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , VirulenciaRESUMEN
BACKGROUND: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. RESULTS: Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. CONCLUSION: We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.
Asunto(s)
Brucella suis/clasificación , Brucella suis/enzimología , Ureasa/inmunología , Ureasa/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Brucella suis/efectos de los fármacos , Brucella suis/inmunología , Brucelosis/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Filogenia , Tiourea/farmacología , Ureasa/clasificaciónRESUMEN
Brucella suis histidinol dehydrogenase (HDH) can be efficiently targeted by substrate analogues. The growth of this pathogen in minimal medium was inhibited and the multiplication in human macrophages was totally abolished in the presence of the drugs. These effects have been shown to be correlated with the previously described inhibition of Brucella HDH activity.
Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Brucella suis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Histidinol/análogos & derivados , Histidinol/farmacología , Macrófagos/microbiología , Factores de Virulencia/antagonistas & inhibidores , Brucella suis/crecimiento & desarrollo , Brucella suis/patogenicidad , Brucelosis/microbiología , Línea Celular , Medios de Cultivo , Histidina/farmacología , Humanos , Cetonas/farmacología , Macrófagos/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis, purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and constitute attractive candidates for the development of novel anti-Brucella agents.