RESUMEN
Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals) catalyzes deglycosylation of N-glycans on glycoproteins. A genetic disorder caused by mutations in the NGLY1 gene leads to NGLY1 deficiency with symptoms including motor deficits and neurological problems. Effective therapies have not been established, though, a recent study used the administration of an adeno-associated viral vector expressing human NGLY1 to dramatically rescue motor functions in young Ngly1-/- rats. Thus, early therapeutic intervention may improve symptoms arising from central nervous system dysfunction, and assay methods for measuring NGLY1 activity in biological samples are critical for early diagnostics. In this study, we established an assay system for plate-based detection of endogenous NGLY1 activity using a FRET-based probe. Using this method, we revealed significant changes in NGLY1 activity in rat brains during aging. This novel assay offers reliable disease diagnostics and provides valuable insights into the regulation of PNGase/NGLY1 activity in diverse organisms under different stress conditions.
Asunto(s)
Trastornos Congénitos de Glicosilación , Transferencia Resonante de Energía de Fluorescencia , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Animales , Humanos , Masculino , Ratas , Envejecimiento/metabolismo , Encéfalo/metabolismo , Trastornos Congénitos de Glicosilación/diagnóstico , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficienciaRESUMEN
Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.
Asunto(s)
Trastornos Congénitos de Glicosilación , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Humanos , Citosol/metabolismo , Glicosilación , Glicoproteínas/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genéticaRESUMEN
Exenatide was the first marketed GLP-1 receptor agonist for the treatment of type 2 diabetes. Modification to the chemical structure or the formulation has the potential to increase the stability of exenatide. We introduced human complex-type sialyloligosaccharide to exenatide at the native Asn28 position. The synthesis was achieved using both solid phase peptide synthesis (SPPS) and Omniligase-1-mediated chemoenzymatic ligation. The results demonstrate that glycosylation increases the proteolytic stability of exenatide while retaining its full biological activity.
Asunto(s)
Diabetes Mellitus Tipo 2 , Humanos , Exenatida , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes , Glicosilación , Péptido Hidrolasas , Receptor del Péptido 1 Similar al Glucagón/agonistas , PonzoñasRESUMEN
Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.
Asunto(s)
Receptores de Somatostatina , Somatostatina , Humanos , Receptores de Somatostatina/metabolismo , Glicosilación , Somatostatina/metabolismo , PolisacáridosRESUMEN
The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.
Asunto(s)
Glucagón , Hipoglucemia , Cisteína , HumanosRESUMEN
Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.
Asunto(s)
Péptidos/química , Cromatografía Líquida de Alta Presión , Glicosilación , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Oligosacáridos/química , Oligosacáridos/metabolismo , Péptidos/análisis , Péptidos/metabolismo , SolubilidadRESUMEN
N(α) -Trifluoroacetyl-Cys-Leu-NH2 (TfaC-Leu-NH2 ) was incorporated into thioesters through its side-chain thiol group to develop a more reactive peptide-thioester than the commonly used peptide-3-mercaptopropionic acid (MPA)-thioester. The TfaC-thioester could be readily synthesized by solid-phase peptide synthesis (SPPS) with Boc chemistry using in situ neutralization protocols in sufficient yield without any side reaction associated with the use of TfaC. This thioester proved to display a much higher reactivity in the thiol-free native chemical ligation (NCL) reaction than the MPA-thioester and to be comparable to the thioarylester, such as the 4-mercaptophenylacetic acid (MPAA)-thioester, in terms of the ligation rate. We were able to demonstrate the usefulness of the TfaC-thioester by using it to synthesize neuromedin S via a one-pot sequential NCL approach followed by desulfurization. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 503-511, 2016.
Asunto(s)
Neuropéptidos/química , Neuropéptidos/síntesis química , Compuestos de Sulfhidrilo/químicaRESUMEN
Cancer-related γ-glutamyl cyclotransferase (GGCT) specifically converts γ-glutamyl amino acids (γ-Glu-Xaa) into pyroglutamate and the corresponding amino acids (Xaa). Here we report a novel GGCT fluorogenic probe "LISA-101" containing a masked O-acylated fluorophore "resorufin" on the side chain of the P amino acid (Xaa). Upon GGCT treatment, the P amino acid was liberated and spontaneously released the intact fluorophore. Thus, the fluorescence was regained. LISA-101 will expand the strategies for cancer studies.
Asunto(s)
Anticuerpos Monoclonales , Colorantes Fluorescentes , Neoplasias/diagnóstico , gamma-Glutamilciclotransferasa , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Humanos , Células MCF-7 , Estructura Molecular , gamma-Glutamilciclotransferasa/síntesis química , gamma-Glutamilciclotransferasa/metabolismoRESUMEN
An isopeptide of amyloid ß peptide 1-42 (isoAß42) was considered as a non-aggregative precursor molecule for the highly aggregative Aß42. It has been applied to biological studies after several pretreatments. Here we report that isoAß42 is monomeric with a random coil structure at 40 µM without any pretreatment. But we also found that isoAß42 retains a slight aggregative nature, which is significantly weaker than that of the native Aß42.
Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/química , Conformación ProteicaRESUMEN
Phosphonium and uronium salt-based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine-assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6-trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4'-dimethoxydiphenylmethyl and 4-methoxybenzyloxymethyl groups were demonstrated to be acid-labile S-protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave-assisted SPPS performed at a high temperature.
Asunto(s)
Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida , Secuencia de Aminoácidos , Aminoácidos/química , Dimetilformamida/química , Fluorenos/química , Microondas , Compuestos Organofosforados/química , Piperidinas/química , Solventes/química , Estereoisomerismo , Triazoles/químicaRESUMEN
The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid ß peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/aislamiento & purificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/aislamiento & purificación , Técnicas de Síntesis en Fase Sólida/métodos , Péptidos beta-Amiloides/química , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/síntesis química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/aislamiento & purificación , Fragmentos de Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, inâ vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.
Asunto(s)
Polisacáridos , Polisacáridos/química , Polisacáridos/síntesis química , Humanos , Glicosilación , Péptidos/química , Péptidos/síntesis química , Proteínas/química , Proteínas/síntesis química , Proteínas/metabolismo , Glicopéptidos/síntesis química , Glicopéptidos/químicaRESUMEN
Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an â¼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.
Asunto(s)
Péptido 1 Similar al Glucagón , Glicosilación , Humanos , Animales , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/química , Péptidos Similares al Glucagón/farmacología , Péptidos Similares al Glucagón/química , Péptidos Similares al Glucagón/análogos & derivados , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Hipoglucemiantes/farmacología , Hipoglucemiantes/química , Hipoglucemiantes/síntesis química , Masculino , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Ingeniería de Proteínas , RatonesRESUMEN
The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.
Asunto(s)
Angiotensina II/metabolismo , Angiotensina II/orina , Secuencia de Aminoácidos , Glicosilación , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/orina , Distribución TisularRESUMEN
Light it up: human chromosome 7 ORF 24, a tumor-related protein, has been identified as a γ-glutamyl cyclotransferase (GGCT) in the glutathione homeostasis cycle. The singular substrate preference of the enzyme has hampered chemical probe development, and no fluorogenic probe has been reported. Here we report the first fluorogenic dipeptide probe, LISA-4, which should contribute toward further understanding of GGCT.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Nitrógeno/metabolismo , Oxígeno/metabolismo , gamma-Glutamilciclotransferasa/metabolismo , Sitios de Unión , Biocatálisis , Cromosomas Humanos Par 7 , Dipéptidos/química , Dipéptidos/metabolismo , Colorantes Fluorescentes/química , Humanos , Simulación del Acoplamiento Molecular , Nitrógeno/química , Sistemas de Lectura Abierta , Oxígeno/química , Estructura Terciaria de Proteína , gamma-Glutamilciclotransferasa/genéticaRESUMEN
Rational design of inhibitors: The cis-amide backbone at position 7 in the serine protease inhibitor marinostatin was replaced with an E or Z olefin. The E olefin analogue was not active, but the Z analogue was. The cis conformation might play a critical role in organizing a canonical structure for binding to proteases.
Asunto(s)
Bacillus subtilis/enzimología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Pseudoalteromonas/química , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Subtilisinas/antagonistas & inhibidores , Alquenos/química , Alquenos/farmacología , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad , Subtilisinas/metabolismoRESUMEN
N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.
Asunto(s)
Aminoácidos/química , Anilidas/síntesis química , Fluorenos/química , Péptidos/síntesis química , Compuestos de Azufre/síntesis química , Acilación , Ésteres , Cinética , Fosfatos/química , Técnicas de Síntesis en Fase Sólida , Compuestos de Sulfhidrilo/químicaRESUMEN
The 4-methoxybenzyloxymethyl (MBom) group was introduced at the Nπ-position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The Nπ-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His(π-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired τ-isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale production without any obstacles.
Asunto(s)
Anisoles/síntesis química , Histidina/síntesis química , Neuropéptidos/síntesis química , Anisoles/aislamiento & purificación , Cristalización , Histidina/aislamiento & purificación , Microondas , Técnicas de Síntesis en Fase SólidaRESUMEN
Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.
Asunto(s)
Alcoholes/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Ciclotidas/química , Ciclotidas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hedyotis/metabolismo , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Cromatografía Liquida , Ciclización , Hedyotis/crecimiento & desarrollo , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Pliegue de Proteína , Espectrometría de Masas en TándemRESUMEN
CD4(+)CD25(+) regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4(+)CD25(+) Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25(+) cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25(+) cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4(+)CD25(-) T cells alone or a combination of CD4(+)CD25(+) and CD4(+)CD25(-) T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4(+)CD25(+) Treg cells to CD4(+)CD25(-) effector T cells, as demonstrated by the lack of response of CD4(+)CD25(+) T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis. Our data show that CD4(+)CD25(+) Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection.