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1.
Mol Microbiol ; 117(2): 493-507, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34931374

RESUMEN

TcdB is a potent cytotoxin produced by pathogenic Clostridioides difficile that inhibits Rho GTPases by mono-glucosylation. TcdB enters cells via receptor-mediated endocytosis. The pathogenic glucosyltransferase domain (GTD) egresses endosomes by pH-mediated conformational changes, and is subsequently released in an autoproteolytic manner. We here investigated the uptake, localization and degradation of TcdB. TcdB colocalized with lysosomal marker protein LAMP1, verifying the endosomal-lysosomal route of the toxin. In pulse assays endocytosed TcdB declined to a limit of detection within 2 hr, whereas the released GTD accumulated for up to 8 hr. We observed that autoproteolytic deficient TcdB NXN C698S was degraded significantly faster than wildtype TcdB, suggesting interference of TcdB with lysosomal degradation process. In fact, TcdB reduced lysosomal degradation of endosome cargo as tested with DQ-Green BSA. Lysosomal dysfunction was accompanied by perinuclear accumulation of LAMP1 and a weaker detection in immunoblots. Galectin-8 or galectin-3 was not recruited to lysosomes speaking against lysosome membrane damage. Changes in the autophagosomal marker LC3B suggested additional indirect effect of lysosomal dysfunction on the autophagic flux. In contrast to necrotic signaling induced in by TcdB, lysosomal dysfunction was not abolished by calcium channel blocker nifedipin, indicating separate cytopathogenic effects induced by TcdB during endo-lysosomal trafficking.


Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides , Lisosomas/metabolismo
2.
Toxins (Basel) ; 12(12)2020 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-33255261

RESUMEN

Toxin B (TcdB) produced by Clostridioides difficile is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4-/-) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain VPI10463 (TcdBVPI) and the endemic strain R20291 (TcdBR20) which does not interact with frizzled-1,2,7. TcdBVPI devoid of CSPG4 binding (TcdBVPI ΔCROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4-/- cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdBVPI variant (TcdBVPI F1597S) that in accordance with TcdBR20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdBR20 on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.


Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Clostridioides difficile/metabolismo , Receptores Frizzled/metabolismo , Proteínas de la Membrana/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/genética , Línea Celular , Clostridioides difficile/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Virulencia/metabolismo
3.
Front Microbiol ; 9: 2314, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30416488

RESUMEN

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101-1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin-receptor interaction.

4.
Cell Microbiol ; 20(10): e12865, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29904993

RESUMEN

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.


Asunto(s)
Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidad , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Línea Celular , Glicosilación , Células HEK293 , Humanos , ATPasas de Translocación de Protón/metabolismo , Especies Reactivas de Oxígeno/metabolismo
5.
Toxins (Basel) ; 10(6)2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29865182

RESUMEN

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.


Asunto(s)
ADP Ribosa Transferasas/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Glucosiltransferasas/química , ADP Ribosa Transferasas/genética , Proteínas Bacterianas/genética , Transporte Biológico , Línea Celular Tumoral , Citosol/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Dominios Proteicos , Proteínas Recombinantes de Fusión/administración & dosificación
6.
Toxins (Basel) ; 6(7): 2162-76, 2014 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-25054784

RESUMEN

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1-1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1-542) or intermediate (TcdA1102-1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.


Asunto(s)
Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Oligopéptidos/metabolismo , Células 3T3 , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Enterotoxinas/toxicidad , Glucosiltransferasas/metabolismo , Ratones , Oligopéptidos/toxicidad , Estructura Terciaria de Proteína
7.
Cell Microbiol ; 16(11): 1678-92, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24898616

RESUMEN

TcdA and TcdB are the main pathogenicity factors of Clostridium difficile-associated diseases. Both toxins inhibit Rho GTPases, and consequently, apoptosis is induced in the affected cells. We found that TcdB at higher concentrations exhibits cytotoxic effects that are independent on Rho glucosylation. TcdB and the glucosyltransferase-deficient mutant TcdB D286/288N induced pyknotic cell death which was associated with chromatin condensation and reduced H3 phosphorylation. Affected cells showed ballooning of the nuclear envelope and loss of the integrity of the plasma membrane. Furthermore, pyknotic cells were positively stained with dihydroethidium indicating production of reactive oxygen species. In line with this, pyknosis was reduced by apocynin, an inhibitor of the NADPH oxidase. Bafilomycin A1 prevented cytotoxic effects showing that the newly observed pyknosis depends on intracellular action of TcdB rather than on a receptor-mediated effect. Blister formation and chromatin condensation was specifically induced by the glucosyltransferase domain of TcdB from strain VPI10473 since neither TcdBF from cdi1470 nor the chimera of TcdB harbouring the glucosyltransferase domain of TcdBF was able to induce these effects. In summary, TcdB induces two different and independent phenotypes: (i) cell rounding due to glucosylation of Rho GTPases and (ii) shrinkage of cells and nuclear blister induced by the high concentrations of TcdB independent of Rho glucosylation.


Asunto(s)
Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Muerte Celular , Núcleo Celular/patología , Cromatina/metabolismo , Glucosiltransferasas/toxicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Línea Celular , Membrana Celular/patología , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glicosilación , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Histonas/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Membrana Nuclear/patología , Fosforilación , Procesamiento Proteico-Postraduccional , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al GTP rho/metabolismo
8.
PLoS One ; 7(9): e44358, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22970203

RESUMEN

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.


Asunto(s)
Neuropéptidos/metabolismo , Fosfoserina/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismo , Animales , Activación Enzimática , Células HEK293 , Humanos , Ratones , Proteínas Mutantes/metabolismo , FN-kappa B/metabolismo , Neuropéptidos/deficiencia , Fenotipo , Fosforilación , Unión Proteica , Seudópodos/metabolismo , Relación Estructura-Actividad , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteína de Unión al GTP rac1
9.
Microb Pathog ; 52(1): 92-100, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22107906

RESUMEN

The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Silenciador del Gen , Toxinas Bacterianas/genética , Línea Celular , Clostridioides difficile/genética , Enterotoxinas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Transporte de Proteínas
10.
PLoS One ; 6(3): e17623, 2011 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-21445253

RESUMEN

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA(1-1874)) and found that this mutant was still cytopathic. However, TcdA(1-1874) possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP-mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA(1-1874) to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA(1-1874) exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.


Asunto(s)
Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Oligopéptidos/metabolismo , Células 3T3 , Animales , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cartilla de ADN , Endocitosis/efectos de los fármacos , Enterotoxinas/toxicidad , Células HT29 , Humanos , Ratones , Microscopía Fluorescente , Oligopéptidos/química
11.
Naunyn Schmiedebergs Arch Pharmacol ; 383(3): 285-95, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21267712

RESUMEN

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile and are the causative agents for clinical symptoms, such as secretory diarrhoea and pseudomembranous colitis. Mast cells are essentially involved in the toxin-induced colonic inflammatory processes. To study the direct effects of these toxins on the expression of inflammatory genes, a DNA microarray containing evaluated probes of 90 selected inflammatory genes was applied to the immature mast cell line HMC-1. TcdA and TcdB induced up-regulation of only a limited number of genes within the early phase of cell treatment. Interleukin-8 (IL-8), transcription factor c-jun and heme oxygenase-1 messenger RNA (mRNA) increased more than 2-fold. In contrast, IL-16, known as a CD4(+) T-cell chemoattractant factor and the chemokine receptor cKit were down-regulated. Stimulation of HMC-1 cells with IL-8 had no effect on IL-16 mRNA level, indicating that both cytokines were independently affected by the toxins. Regulation of both cytokines, however, depended on glucosylation of Rho GTPases as tested by application of enzyme-deficient TcdA or TcdB. Down-regulation of total and secreted IL-16 protein was checked by enzyme-linked immunosorbent assay. The data implicate that TcdA and TcdB affect lymphocyte migration by modulating release of the chemoattractant factor IL-16 from mast cells. In addition, this is the first report showing that Rho GTPases are involved in the regulation of IL-16 expression.


Asunto(s)
Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Regulación hacia Abajo/genética , Enterotoxinas/farmacología , Interleucina-16/metabolismo , Mastocitos/metabolismo , Sustitución de Aminoácidos/fisiología , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Línea Celular , Enterotoxinas/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Perfilación de la Expresión Génica , Glicosilación/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Humanos , Imidazoles/farmacología , Interleucina-16/genética , Interleucina-8/genética , Interleucina-8/farmacología , Mastocitos/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-kit/genética , Piridinas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/metabolismo
12.
Naunyn Schmiedebergs Arch Pharmacol ; 383(3): 253-62, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21046073

RESUMEN

Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC(50) regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.


Asunto(s)
Toxinas Bacterianas/metabolismo , Proteasas de Cisteína/metabolismo , Citotoxinas/metabolismo , Enterotoxinas/metabolismo , Sustitución de Aminoácidos/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Membrana Celular/metabolismo , Forma de la Célula/efectos de los fármacos , Citosol/metabolismo , Citotoxinas/química , Citotoxinas/farmacología , Ditiotreitol/química , Relación Dosis-Respuesta a Droga , Enterotoxinas/química , Enterotoxinas/farmacología , Glucosiltransferasas/metabolismo , Glicosilación , Células HT29 , Humanos , Ratones , Células 3T3 NIH , Fragmentos de Péptidos/metabolismo , Ácido Fítico/química , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
13.
Cell Microbiol ; 11(12): 1816-26, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19709124

RESUMEN

Clostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdA-catalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71.


Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/metabolismo , Enterotoxinas/metabolismo , Serina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Células CACO-2 , Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/microbiología , Factor de Crecimiento Epidérmico/metabolismo , Interacciones Huésped-Patógeno , Humanos , Intestinos/microbiología , Intestinos/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especificidad por Sustrato , Virulencia , Proteína de Unión al GTP rhoA/metabolismo
14.
J Med Microbiol ; 57(Pt 6): 765-770, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18480335

RESUMEN

The intestinal epithelial cell line HT-29 was used to study the apoptotic effect of Clostridium difficile toxin A (TcdA). TcdA is a 300 kDa single-chain protein, which glucosylates and thereby inactivates small GTPases of the Rho family (Rho, Rac and Cdc42). The effect of TcdA-catalysed glucosylation of the Rho GTPases is well known: reorganization of the actin cytoskeleton with accompanying morphological changes in cells, leading to complete rounding of cells and destruction of the intestinal barrier function. Less is known about the mechanism by which apoptosis is induced in TcdA-treated cells. In this study, TcdA induced the activation of caspase-3, -8 and -9. Apoptosis, as estimated by the DNA content of cells, started as early as 24 h after the addition of TcdA. The impact of Rho glucosylation was obvious when mutant TcdA with reduced or deficient glucosyltransferase activity was applied. TcdA mutant W101A, with 50-fold reduced glucosyltransferase activity, induced apoptosis only at an equipotent concentration compared with wild-type TcdA at a 50% effective concentration of 0.2 nM. The enzyme-deficient mutant TcdA D285/287N was not able to induce apoptosis. Apoptosis induced by TcdA strictly depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Destruction of the actin cytoskeleton by latrunculin B was not sufficient to induce apoptosis, indicating that apoptosis induced by TcdA must be due to another mechanism. In summary, TcdA-induced apoptosis (cytotoxic effect) depends on the glucosylation of Rho GTPases, but is not triggered by destruction of the actin cytoskeleton (cytopathic effect).


Asunto(s)
Apoptosis/efectos de los fármacos , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Células Epiteliales/efectos de los fármacos , Mucosa Intestinal/citología , Proteínas de Unión al GTP rho/metabolismo , Caspasas/metabolismo , Activación Enzimática , Glicosilación , Células HT29 , Humanos , Mucosa Intestinal/efectos de los fármacos
15.
Infect Immun ; 74(10): 6006-10, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16988280

RESUMEN

Mutation of tryptophan-101 in Clostridium difficile toxin A, a 308-kDa glucosyltransferase, resulted in a 50-fold-reduced cytopathic activity in cell culture experiments. The mutant toxin A was characterized and applied to distinguish between glucosyltransferase-dependent and -independent effects with respect to RhoB up-regulation as a cellular stress response.


Asunto(s)
Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Glucosiltransferasas/farmacología , Proteína de Unión al GTP rhoB/agonistas , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células CACO-2 , Enterotoxinas/química , Enterotoxinas/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/genética , Humanos , Ratones , Mutación , Triptófano/química , Triptófano/genética , Regulación hacia Arriba , Proteína de Unión al GTP rhoB/metabolismo
16.
Microb Pathog ; 38(2-3): 77-83, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15748809

RESUMEN

Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest. We, therefore, expressed recombinant His-tagged toxin A using the Bacillus megaterium expression system. Specific antisera raised against the native toxin A from C. difficile and the recombinant toxin, respectively, showed identical sensitivity and specificity in Western blot and ELISA analyses towards both toxins. By comparison of both toxins in functional studies we showed that the recombinant toxin was about two times more cytotoxic than the native toxin, and the glucosyltransferase-activity of the recombinant toxin was even 10-fold increased. However, recombinant toxin A showed one essential difference to the classically purified one. The reported transferase-independent effect of toxin A to release cytochrome c from isolated mitochondria was not exhibited by the recombinant toxin A. This putative mitochondrial effect decreased with increased purity of toxin A, and was absent with recombinant toxin, strongly suggesting an clostridial contamination responsible. In summary, we tested the recombinant toxin A to be at least an adequate substitute for the native toxin, bearing the advantage of a rapid single-step purification and the absence of biological active contaminations.


Asunto(s)
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Clostridioides difficile/enzimología , Enterotoxinas/metabolismo , Enterotoxinas/toxicidad , Animales , Anticuerpos Antibacterianos/inmunología , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Western Blotting , Células Cultivadas , Clonación Molecular , Citocromos c/análisis , Enterotoxinas/genética , Enterotoxinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Glucosiltransferasas/genética , Glucosiltransferasas/inmunología , Glucosiltransferasas/aislamiento & purificación , Glucosiltransferasas/metabolismo , Glucosiltransferasas/toxicidad , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Células 3T3 NIH , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad
17.
J Biol Chem ; 280(2): 1499-505, 2005 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-15531589

RESUMEN

Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h, a steady state was reached, with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.


Asunto(s)
Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Estrés Fisiológico/genética , Proteína de Unión al GTP rhoB/genética , Células CACO-2 , Humanos , Imidazoles/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Piridinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rhoB/biosíntesis , Proteína de Unión al GTP rhoB/metabolismo
18.
Biochem Biophys Res Commun ; 307(3): 584-8, 2003 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12893263

RESUMEN

Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B. We used the Bacillus megaterium expression system for expression of recombinant toxin A. The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector. The three parts were sequentially fused to the complete gene. The holotoxin gene was ligated into the expression vector pWH1520. This vector was modified to generate a toxin with a C-terminally located His-tag. Gene expression in the B. megaterium system resulted in an approximate 300 kDa protein, which was identified by specific antibody as toxin A. Recombinant, His-tagged toxin A was purified by Ni(2+) as well as thyroglobulin affinity chromatography. Characterization of the recombinant toxin A showed identical cytotoxicity and in vitro-glucosyltransferase activity as the native toxin A from C. difficile.


Asunto(s)
Bacillus megaterium/genética , Toxinas Bacterianas/genética , Enterotoxinas/genética , Animales , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Línea Celular , Enterotoxinas/metabolismo , Enterotoxinas/toxicidad , Expresión Génica , Vectores Genéticos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad
19.
Circulation ; 107(10): 1383-9, 2003 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-12642358

RESUMEN

BACKGROUND: Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. METHODS AND RESULTS: Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). CONCLUSIONS: The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.


Asunto(s)
Enfermedad de la Arteria Coronaria/enzimología , Enfermedad de la Arteria Coronaria/fisiopatología , Endotelio Vascular/fisiopatología , NADPH Oxidasas/metabolismo , Xantina Oxidasa/metabolismo , Adulto , Vasos Coronarios/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Endotelio Vascular/enzimología , Humanos , Hipercolesterolemia/enzimología , Hipercolesterolemia/fisiopatología , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Superóxidos/metabolismo , Vasodilatación
20.
Hypertension ; 41(5): 1092-5, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12654707

RESUMEN

Impaired flow-dependent, endothelium-mediated vasodilation is an early finding in patients with coronary artery disease (CAD). Experimental and some clinical studies observed that angiotensin type-1 receptor antagonists (AT1A) enhance endothelium-dependent relaxation in CAD. The present study was designed to determine whether AT1A improves flow-dependent dilation (FDD) in patients with CAD and, if so, whether bradykinin and NO are involved. High-resolution ultrasound was used to measure radial artery diameter at rest and during reactive hyperemia, causing endothelium-mediated vasodilation. Twenty patients with CAD were randomly assigned to receive intrabrachial infusion of candesartan (800 microg/min) with and without icatibant, a bradykinin B2-receptor antagonist (90 microg/min; group A) or N-monomethyl-l-arginine (L-NMMA), an NO-synthase inhibitor (7 micromol/min; group B). The AT1A candesartan improved FDD by >40%, an effect that was inhibited by icatibant (group A: control, 7.3+/-0.9; candesartan, 10.3+/-1.1; candesartan+icatibant, 5.0+/-0.5%). Similarly, L-NMMA blunted the beneficial effect of candesartan (group B: control, 6.3+/-0.6; candesartan, 8.9+/-0.6; candesartan+L-NMMA: 4.7+/-0.5%; each P<0.01). The angiotensin type-1 receptor antagonist candesartan improves flow-dependent, endothelium-mediated vasodilation in patients with CAD. This effect is inhibited by either icatibant and or L-NMMA, suggesting that both bradykinin and NO contribute to the vascular effects of AT1-receptor antagonists in this patient population.


Asunto(s)
Antagonistas de Receptores de Angiotensina , Antihipertensivos/farmacología , Bencimidazoles/farmacología , Bradiquinina/análogos & derivados , Enfermedad de la Arteria Coronaria/fisiopatología , Endotelio Vascular/efectos de los fármacos , Receptores de Bradiquinina/fisiología , Tetrazoles/farmacología , Antagonistas Adrenérgicos beta/farmacología , Compuestos de Bifenilo , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/farmacología , Humanos , Infusiones Intraarteriales , Persona de Mediana Edad , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroprusiato/farmacología , Arteria Radial/efectos de los fármacos , Arteria Radial/fisiopatología , Receptor de Angiotensina Tipo 1 , Receptor de Bradiquinina B2 , omega-N-Metilarginina/farmacología
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