RESUMEN
AIM: To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. MATERIALS AND METHODS: Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. RESULTS: Level of PT production by strains isolated from infected persons varied from 3 +/- 0.5 to 64.8 +/- 12.2 ng/MFU/ml: in 9 strains--from 3 +/- 0.5 to 9.4 +/- 2.1 ng/MFU/ml, in 7--10.5 +/- 1.8 to 18.4 +/- 2.6 ng/MFU/ml, and in 9--23.6 +/- 4.5 to 64.8 +/- 12.2 ng/MFU/ml. CONCLUSION: B. pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.
Asunto(s)
Bordetella pertussis/enzimología , Bordetella pertussis/aislamiento & purificación , Toxina del Pertussis/biosíntesis , Tos Ferina/enzimología , Animales , Bordetella pertussis/patogenicidad , Niño , Preescolar , Femenino , Humanos , Masculino , Conejos , Tos Ferina/microbiologíaRESUMEN
Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.
Asunto(s)
Toxina de Adenilato Ciclasa/inmunología , Bordetella pertussis/inmunología , Antígeno de Macrófago-1/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Tos Ferina/inmunología , Proteínas de Unión al GTP rho/inmunología , Citoesqueleto de Actina/inmunología , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/inmunología , Factores Despolimerizantes de la Actina/metabolismo , Toxina de Adenilato Ciclasa/metabolismo , Animales , Bordetella pertussis/enzimología , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Antígenos CD18/genética , Antígenos CD18/inmunología , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , AMP Cíclico/inmunología , Femenino , GTP Fosfohidrolasas/inmunología , GTP Fosfohidrolasas/metabolismo , Antígeno de Macrófago-1/metabolismo , Macrófagos/metabolismo , Ratones , Neuropéptidos/inmunología , Neuropéptidos/metabolismo , Tos Ferina/enzimología , Proteínas de Unión al GTP rac/inmunología , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1 , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Proteína RCA2 de Unión a GTPRESUMEN
The adenylyl cyclase (AC) toxin CyaA from Bordetella pertussis constitutes an important virulence factor for the pathogenesis of whooping cough. CyaA is activated by calmodulin (CaM) and compromises host defense by excessive cAMP production. Hence, pharmacological modulation of the CyaA/CaM interaction could constitute a promising approach to treat whooping cough, provided that interactions of endogenous effector proteins with CaM are not affected. As a first step toward this ambitious goal we examined the interactions of CyaA with wild-type CaM and four CaM mutants in which most methionine residues were replaced by leucine residues and studied the effects of the CaM antagonists calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). CyaA/CaM interaction was monitored by CaM-dependent fluorescence resonance energy transfer (FRET) between tryptophan residues in CyaA and 2'-(N-methylanthraniloyl)-3'-deoxy-adenosine 5'-triphosphate and catalytic activity. Comparison of the concentration/response curves of CaM and CaM mutants for FRET and catalysis revealed differences, suggesting a two-step activation mechanism of CyaA by CaM. Even in the absence of CaM, calmidazolium inhibited catalysis, and it did so according to a biphasic function. Trifluoperazine and W-7 did not inhibit FRET or catalysis. In contrast to CyaA, some CaM mutants were more efficacious than CaM at activating membranous AC isoform 1. The slope of CyaA activation by CaM was much steeper than of AC1 activation. Collectively, the two-step activation mechanism of CyaA by CaM offers opportunities for pharmacological intervention. The failure of classic CaM inhibitors to interfere with CyaA/CaM interactions and the different interactions of CaM mutants with CyaA and AC1 point to unique CyaA/CaM interactions.
Asunto(s)
Toxina de Adenilato Ciclasa/química , Adenilil Ciclasas/metabolismo , Bordetella pertussis/enzimología , Calmodulina/química , Membrana Celular/efectos de los fármacos , Toxina de Adenilato Ciclasa/genética , Toxina de Adenilato Ciclasa/metabolismo , Unión Competitiva , Bordetella pertussis/patogenicidad , Calmodulina/antagonistas & inhibidores , Calmodulina/genética , Catálisis , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Imidazoles/química , Imidazoles/farmacología , Modelos Moleculares , Estructura Molecular , Mutación , Unión Proteica , Sulfonamidas/química , Sulfonamidas/farmacología , Trifluoperazina/química , Trifluoperazina/farmacología , Virulencia , Tos Ferina/enzimología , Tos Ferina/microbiologíaAsunto(s)
5'-Nucleotidasa/metabolismo , Tos Ferina/inmunología , Animales , Relación Dosis-Respuesta Inmunológica , Inmunidad Celular , Inmunización , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/enzimología , Ratones , Vacuna contra la Tos Ferina/inmunología , Factores de Tiempo , Tos Ferina/enzimologíaAsunto(s)
Toxoide Diftérico , Difteria/enzimología , Péptido Hidrolasas/metabolismo , Vacuna contra la Tos Ferina , Bazo/enzimología , Toxoide Tetánico , Tétanos/enzimología , Tos Ferina/enzimología , Alanina , Animales , Formación de Anticuerpos , Catepsinas/metabolismo , Difteria/inmunología , Histocitoquímica , Leucina , Ratones , Bazo/inmunología , Tétanos/inmunología , Vacunación , Tos Ferina/inmunologíaRESUMEN
Innate immunity plays an important role in protection against respiratory infections in humans and animals. Host defense peptides such as beta-defensins represent major components of innate immunity. We recently developed a novel porcine model of pertussis, an important respiratory disease of young children and infants worldwide. Here, we investigated the role of porcine beta-defensin 1 (pBD-1), a porcine defensin homologue of human beta-defensin 2, in conferring protection against respiratory infection with Bordetella pertussis. In this model, newborn piglets were fully susceptible to infection and developed severe bronchopneumonia. In contrast, piglets older than 4 weeks of age were protected against infection with B. pertussis. Protection was associated with the expression of pBD-1 in the upper respiratory tract. In fact, pBD-1 expression was developmentally regulated, and the absence of pBD-1 was thought to contribute to the increased susceptibility of newborn piglets to infection with B. pertussis. Bronchoalveolar lavage specimens collected from older animals as well as chemically synthesized pBD-1 displayed strong antimicrobial activity against B. pertussis in vitro. Furthermore, in vivo treatment of newborn piglets with only 500 mug pBD-1 at the time of challenge conferred protection against infection with B. pertussis. Interestingly, pBD-1 displayed no bactericidal activity in vitro against Bordetella bronchiseptica, a closely related natural pathogen of pigs. Our results demonstrate that host defense peptides play an important role in protection against pertussis and are essential in modulating innate immune responses against respiratory infections.
Asunto(s)
Bordetella pertussis/inmunología , Tos Ferina/inmunología , Tos Ferina/prevención & control , beta-Defensinas/fisiología , Animales , Animales Recién Nacidos , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Bordetella pertussis/genética , Bordetella pertussis/crecimiento & desarrollo , Bordetella pertussis/ultraestructura , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Femenino , Inmunidad Innata , Lactoferrina/fisiología , Pulmón/microbiología , Pulmón/patología , Muramidasa/fisiología , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/inmunología , Proteínas/genética , Proteínas/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Cloruro de Sodio/metabolismo , Porcinos , Tos Ferina/enzimología , Tos Ferina/microbiología , beta-Defensinas/biosíntesis , beta-Defensinas/genética , beta-Defensinas/uso terapéuticoRESUMEN
We studied the alterations of MDA and three forms of SOD activities such as T-SOD, CuZn-SOD, and Mn-SOD in rat cerebral tissues injected by bordetella pertussis (BP) to elucidate protective mechanism of SOD against the infectious brain injury. The results were that water content(WC), Evans blue content(EB), MDA, and Mn-SOD activities in 4 h and 24 h BP-treated groups increased and T-SOD and CuZn-SOD decreased compared to corresponding normal saline(NS)-treated groups, respectively(P < 0.01); MDA increased and had a positive correlation with WC and EB in 4 h BP treated group (r = 0.9650, r = 0.9441, P < 0.01, P < 0.01, respectively); Mn-SOD activities were elevated and had a negative correlation with WC, EB, and MDA (r = -0.8650, r = -0.9021, r = -0.9346, P < 0.01, P < 0.01, P < 0.01, respectively) in 24 h BP-treated group. The results suggest that the increase of component Mn-SOD activities may play an important role in vivo endogenous protective mechanism against delayed infectious brain injury.
Asunto(s)
Bordetella pertussis , Edema Encefálico/enzimología , Superóxido Dismutasa/fisiología , Tos Ferina , Animales , Edema Encefálico/microbiología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Tos Ferina/complicaciones , Tos Ferina/enzimologíaRESUMEN
(1) Fetal thymuses, organs from patients who died from diseases that are not clinically known to be associated with concomitant lymphoid tissue involvement, as well as thymuses from patients dying from diseases which effect the lymphatic complex of the body, one way or another, have been investigated for their alkaline phosphatase activity, using Gomori technique and applying four different phosphate esters as substrates. (2) Three substrates (beta-glycerophophate, riboflavin 5-phosphate and adenosine triphosphate) showed essentially the same pattern of activity in which the cortex and Hassall's corpuscles were reactive, while the medulla was negative. A reversal of this pattern was demonstrated with 5-monophosphoric acid. (3) Before the age of 32-36 weeks of intra-uterine life there is no alkaline phosphatase activity in the thymus; therafter, the enzyme begins to make its first appearance. (4) There is a definite increase in the intensity of the reaction with advance of intra-uterine life. This increase in phosphatase content is continued postnatally, to reach its maximum at about the age of 10 years: after that, the enzyme activity gradually subsides. (5) There is a tremendous augmentation of phosphatase activity in the case of disease which are known to affect the lymphoid complex. (6) The phosphatase activity of the thymus has been discussed in relation to the prevailing concepts about the function of the thymus, with special emphasis on a possible association with 'lymphocyte-stimulating factor' production and/or secretion.