RESUMEN
NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.
Asunto(s)
Anticuerpos Biespecíficos , Humanos , Degranulación de la Célula , Ligandos , Receptores KIR , Citotoxicidad Inmunológica , Línea Celular Tumoral , Receptores KIR2DL1RESUMEN
Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.
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Bacteriófagos , Listeria monocytogenes , Listeria , Listeria/genética , Bacteriófagos/genética , Listeria monocytogenes/genética , Industria de Alimentos , FenotipoRESUMEN
Gene regulation in cis by riboswitches is prevalent in bacteria. The yybP-ykoY riboswitch family is quite widespread, yet its ligand and function remained unknown. Here, we characterize the Lactococcus lactis yybP-ykoY orphan riboswitch as a Mn(2+)-dependent transcription-ON riboswitch, with a â¼30-40 µM affinity for Mn(2+). We further determined its crystal structure at 2.7 Å to elucidate the metal sensing mechanism. The riboswitch resembles a hairpin, with two coaxially stacked helices tethered by a four-way junction and a tertiary docking interface. The Mn(2+)-sensing region, strategically located at the highly conserved docking interface, has two metal binding sites. Whereas one site tolerates the binding of either Mg(2+) or Mn(2+), the other site strongly prefers Mn(2+) due to a direct contact from the N7 of an invariable adenosine. Mutagenesis and a Mn(2+)-free E. coli yybP-ykoY structure further reveal that Mn(2+) binding is coupled with stabilization of the Mn(2+)-sensing region and the aptamer domain.
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Escherichia coli/genética , Lactococcus lactis/genética , Magnesio/metabolismo , ARN Bacteriano/química , Secuencias Reguladoras de Ácido Ribonucleico , Riboswitch/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Secuencia de Bases , Cristalografía por Rayos X , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/metabolismo , Magnesio/toxicidad , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido Nucleico , Percepción de Quorum , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
Rheumatoid arthritis (RA) patients present higher risk of SARS-CoV-2 infection (COVID-19), and proper management of the disease in this population requires a better understanding of how the immune system controls the virus. We analyzed the T cell and B cell phenotypes, and their repertoire in a pair of monozygotic twins with RA mismatched for COVID-19 infection. Twin- was not infected, while Twin+ was infected and effectively controlled the infection. We found no significant changes on the αß T cell composition, while γδ T cells and B cells presented considerable expansion of memory population in Twin+ and robust T/B cell responses to several SARS-CoV-2 peptides. T cell receptor ß/γ-chain and immunoglobulin heavy chain next-generation sequencing depicted a remarkable higher diversity in Twin+ compared with Twin-, despite no significant changes being found in variable/joining family usage. Repertoire overlap analyses showed that, although being identical twins, very few clones were shared between them, indicating that COVID-19 may lead to deep changes on the immune cell repertoire in RA patients. Altogether, our results indicate that RA patients may develop robust and persistent COVID-19-specific T/B cell responses; γδ T cells and B cells may play a key role in the management of COVID-19 in RA, and the infection may lead to a profound reshaping of immune cell receptor specificities.
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Artritis Reumatoide , COVID-19 , Enfermedades en Gemelos/genética , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta , SARS-CoV-2 , Linfocitos T , Gemelos Monocigóticos/genéticaRESUMEN
OBJECTIVE: We describe for the first time clinical characteristics in a series of 20 pre-surgically investigated patients with mild malformation of cortical development with oligodendroglial hyperplasia (MOGHE) who were operated on in our epilepsy center. We aimed to better diagnose this entity and help surgical planning. METHODS: Data on 20 patients with histologically confirmed MOGHE were retrospectively evaluated as to age at epilepsy onset and operation, seizure semiology, magnetic resonance imaging (MRI) localization, electroencephalography (EEG) patterns, extent of the operative resection, and postoperative seizure outcome. RESULTS: Epilepsy began mainly in early childhood; however, symptoms did not manifest until adolescence or adulthood in 30% of patients. All patients had pathologic MRI findings. In 45% of patients the lesion was initially overlooked. Most commonly, the lesion was seen in the frontal lobe. Seizure semiology was characterized as follows: (1) epileptic spasms at epilepsy onset were common and (2) nocturnal hyperkinetic seizures during the course of the disease were rare. EEG always showed frequent interictal epileptic discharges. Two peculiar patterns were observed: (1) during sleep stage I-II, sub-continuous repetitive (0.5-1.5/s) unilateral plump spike/polyspike slow waves were seen and (2) during wakefulness, unilateral paroxysms of 2-2.5/s spike-wave complexes occurred. In total, 60% of patients were seizure-free 1 year postoperatively. Postoperative seizure outcome was positively correlated with the extent of resection, age at epilepsy onset, and age at operation. Postoperative long-term outcomes remained stable in patients undergoing larger operations. SIGNIFICANCE: MRI, EEG, and semiology already contribute to the diagnosis of probable MOGHE preoperatively. Because postoperative seizure outcomes depend on the extent of the resection, prior knowledge of a probable MOGHE helps to plan the resection and balance the risks and benefits of such an intervention. In patients undergoing larger operations, epilepsy surgery achieved good postoperative results; the first long-term outcome data were stable in these patients.
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Epilepsia , Convulsiones , Adolescente , Adulto , Preescolar , Electroencefalografía/métodos , Humanos , Hiperplasia/cirugía , Imagen por Resonancia Magnética/métodos , Estudios Retrospectivos , Convulsiones/etiología , Convulsiones/cirugía , Resultado del TratamientoRESUMEN
Although the impact of donor graft composition on clinical outcomes after hematopoietic stem cell transplantation (HSCT) has been studied, little is known about the role of intragraft γδ TCR repertoire on clinical outcomes following HSCT. Using a high-throughput sequencing platform, we sought to analyze the TCR γ-chain (TRG) repertoire of γδ T cells within donor stem cell grafts and address its potential impact on clinical response in the corresponding patients. A total of 20 peripheral blood stem cell grafts were analyzed, and donors were classified as CMV+/- The respective acute myeloid leukemia recipients were followed for disease relapse and acute graft-versus-host disease (aGvHD) development post-HSCT. In all samples, TRG repertoire showed a reduced diversity and displayed overrepresented clones. This was more prominent in grafts from CMV+ donors, which presented a more private repertoire, lower diversity, skewed distribution, and reduced usage of the V9-JP pairing. Grafts given to nonrelapse patients presented a more public repertoire and increased presence of long sequence clonotypes. Variable-joining gene segment usage was not associated with aGvHD development, but a higher usage of V2-JP1 pairing and lower usage of V4-J2/V5-J2/V8-JP2 were observed in grafts given to nonrelapse patients. Our work identified five private overrepresented and one public CDR3 sequence (CATWDGPYYKKLF) associated with CMV infection, in addition to 12 highly frequent public sequences present exclusively in grafts given to nonrelapse patients. Our findings show that, despite CMV infection reshaping the TRG repertoire, TRG composition is not associated with aGvHD development, and several public sequences are associated with clinical remission.
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Infecciones por Citomegalovirus/inmunología , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Trasplantes/virología , Resultado del Tratamiento , Adulto , Anciano , Células Clonales , Femenino , Enfermedad Injerto contra Huésped/epidemiología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/virología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/virología , Adulto JovenRESUMEN
Bacteria tightly regulate intracellular zinc levels to ensure sufficient zinc to support essential functions, while preventing toxicity. The bacterial response to zinc limitation includes the expression of putative zinc metallochaperones belonging to subfamily 1 of the COG0523 family of G3E GTPases. However, the client proteins and the metabolic processes served by these chaperones are unclear. Here, we demonstrate that the Bacillus subtilis YciC zinc metallochaperone (here renamed ZagA for ZTP activated GTPase A) supports de novo folate biosynthesis under conditions of zinc limitation, and interacts directly with the zinc-dependent GTP cyclohydrolase IA, FolE (GCYH-IA). Furthermore, we identify a role for the alarmone ZTP, a modified purine biosynthesis intermediate, in the response to zinc limitation. ZTP, a signal of 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria, transiently accumulates as FolE begins to fail, stimulates the interaction between ZagA and FolE, and thereby helps to sustain folate synthesis despite declining zinc availability.
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Aminoimidazol Carboxamida/análogos & derivados , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Metalochaperonas/metabolismo , Ribonucleótidos/metabolismo , Zinc/metabolismo , Aminoimidazol Carboxamida/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Metalochaperonas/genética , Unión ProteicaRESUMEN
The ability of bacteria to adapt to stress depends on the conditional expression of specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σM, σW, σX and σV. These four σ factors recognize overlapping sets of promoters, although the sequences that determine this overlapping recognition are incompletely understood. A major role in promoter selectivity has been ascribed to the core -10 and -35 promoter elements. Here, we demonstrate that a homopolymeric T-tract motif, proximal to the -35 element, functions in combination with the core promoter sequences to determine selectivity for ECF sigma factors. This motif is most critical for promoter activation by σV, and contributes variably to activation by σM, σX and σW. We propose that this motif, which is a feature of the deduced promoter consensus for a subset of ECF σ factors from many species, imparts intrinsic DNA curvature to influence promoter activity. The differential effect of this region among ECF σ factors thereby provides a mechanism to modulate the nature and extent of regulon overlap.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica/métodos , Regiones Promotoras Genéticas/genética , Factor sigma/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Pared Celular/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Regulón/genética , Factor sigma/metabolismoRESUMEN
Lymphocyte reconstitution is pivotal for successful long-term outcome after allogeneic hematopoietic stem cell transplantation (HSCT), and conditioning regimen and post-transplantation immunosuppression are risk factors for prolonged immunodeficiency. Nevertheless, the effects of different immunosuppressive protocols on lymphocyte output and replicative capacity have not been investigated. Here we assessed T cell receptor excision circles (TREC), kappa-deleting recombination excision circles (KREC), and T cell telomere length (TL) as proxy markers for immune reconstitution in patients in a prospective randomized trial comparing graft-versus-host disease (GVHD) prophylaxis after transplantation (cyclosporine/methotrexate versus tacrolimus/sirolimus; nâ¯=â¯200). Results showed that medians of TREC, KREC, and TL were not significantly different between the prophylaxis groups at any assessment time point during follow-up (24 months), but the kinetics of TREC, KREC, and TL were significantly influenced by other transplantation-related factors. Older recipient age, the use of antithymocyte globulin before graft infusion, and use of peripheral blood stem cell grafts were associated with lower TREC levels, whereas acute GVHD transiently affected KREC levels. Patients with lymphocyte excision circle levels above the median at ≤6 months post-transplantation had reduced transplantation-related mortality and superior 5-year overall survival (P < .05). We noticed significant T cell telomere shortening in the patient population as a whole during follow-up. Our results suggest that lymphocyte reconstitution after transplantation is not altered by different immunosuppressive protocols. This study has been registered at ClinicalTrials.gov (identifier: NCT00993343).
Asunto(s)
Linfocitos B/inmunología , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/métodos , Linfocitos T/inmunología , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo/métodos , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Acute graft-versus-host disease (aGVHD) is 1 of the main major complications of post-hematopoietic stem cell transplantation (HSCT). Identifying patients at risk of severe aGVHD may lead to earlier intervention and treatment, resulting in increased survival and a better quality of life. We aimed to identify biomarkers in donor grafts and patient plasma around the time of transplantation that might be predictive of aGVHD development. We build on our previously published methods by using multiplex assays and multicolor flow cytometry. We identified 5 easily assessable cellular markers in donor grafts that combined could potentially be used to calculate risk for severe aGVHD development. Most noteworthy are the T cell subsets expressing IL-7 receptor-α (CD127) and PD-1. Additionally, we identified a potential role for elevated tumor necrosis factor-α levels in both graft and patient before HSCT in development of aGVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/sangre , Trasplante de Células Madre Hematopoyéticas , Subunidad alfa del Receptor de Interleucina-7/sangre , Receptor de Muerte Celular Programada 1/sangre , Calidad de Vida , Donantes de Tejidos , Enfermedad Aguda , Adolescente , Adulto , Anciano , Aloinjertos , Biomarcadores/sangre , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/etiología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/terapia , Factores de RiesgoRESUMEN
BACKGROUND: The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum. METHODS: T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity. RESULTS: CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+. CONCLUSION: Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product.
Asunto(s)
Antígenos CD19/metabolismo , Medios de Cultivo/farmacología , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/citología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Humanos , Fenotipo , Linfocitos T/efectos de los fármacosRESUMEN
Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Algoritmos , Secuencia de Bases , Carbocianinas/química , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , ATPasas Transportadoras de Cobre , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Cinética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Factor sigma/química , Factor sigma/genética , Factor sigma/metabolismo , Transactivadores/química , Transactivadores/genéticaRESUMEN
Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.
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Amidohidrolasas/genética , Bacillus subtilis/crecimiento & desarrollo , N-Formilmetionina/metabolismo , Biosíntesis de Proteínas/genética , ARN de Transferencia de Metionina/genética , Amidohidrolasas/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Biopelículas/crecimiento & desarrollo , Peróxido de Hidrógeno/farmacología , Ácidos Hidroxámicos/farmacología , Estrés Oxidativo/fisiología , Paraquat/farmacología , Biosíntesis de Proteínas/fisiología , Piruvaldehído/farmacologíaRESUMEN
Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilisâ PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis.
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Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Hierro/toxicidad , Adenosina Trifosfatasas/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Manganeso/metabolismo , Mutación , Regulón , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS), a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.
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Bacillus subtilis/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Metales/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Bacillus subtilis/genética , Proteínas Portadoras/genética , Escherichia coli/genética , HumanosRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up.
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Receptores de Antígenos de Linfocitos T , Trasplante de Células Madre , Aloinjertos , Biomarcadores/sangre , Resultados de Cuidados Críticos , Enfermedad Injerto contra Huésped/diagnóstico , Humanos , Receptores de Antígenos de Linfocitos T/sangreRESUMEN
Intracellular zinc levels are tightly regulated since zinc is an essential cofactor for numerous enzymes, yet can be toxic when present in excess. The majority of intracellular zinc is tightly associated with proteins and is incorporated during synthesis from a poorly defined pool of kinetically labile zinc. In Bacillus subtilis, this labile pool is sensed by equilibration with the metalloregulator Zur, as an indication of zinc sufficiency, and by CzrA, as an indication of zinc excess. Here, we demonstrate that the low-molecular-weight thiol bacillithiol (BSH) serves as a major buffer of the labile zinc pool. Upon shift to conditions of zinc excess, cells transiently accumulate zinc in a low-molecular-weight pool, and this accumulation is largely dependent on BSH. Cells lacking BSH are more sensitive to zinc stress, and they induce zinc efflux at lower external zinc concentrations. Thiol reactive agents such as diamide and cadmium induce zinc efflux by interfering with the Zn-buffering function of BSH. Our data provide new insights into intracellular zinc buffering and may have broad relevance given the presence of BSH in pathogens and the proposed role of zinc sequestration in innate immunity.
Asunto(s)
Bacillus subtilis/metabolismo , Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Zinc/metabolismo , Tampones (Química) , Cisteína/metabolismo , Glucosamina/metabolismo , HomeostasisRESUMEN
Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Iones/química , Fenómenos Fisiológicos Bacterianos , ADN/química , Proteínas de Unión al ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Metales/química , Modelos Biológicos , Unión Proteica , Compuestos de Sulfhidrilo/química , Transcripción GenéticaRESUMEN
Bacterial transcription factors often function as DNA-binding proteins that selectively activate or repress promoters, although the biochemical mechanisms vary. In most well-understood examples, activators function by either increasing the affinity of RNA polymerase (RNAP) for the target promoter, or by increasing the isomerization of the initial closed complex to the open complex. We report that Bacillus subtilis Btr, a member of the AraC family of activators, functions principally as a ligand-dependent activator of promoter clearance. In the presence of its co-activator, the siderophore bacillibactin (BB), the Btr:BB complex enhances productive transcription, while having only modest effects on either RNAP promoter association or the production of abortive transcripts. Btr binds to two direct repeat sequences adjacent to the -35 region; recognition of the downstream motif is most important for establishing a productive interaction between the Btr:BB complex and RNAP. The resulting Btr:BB dependent increase in transcription enables the production of the ferric-BB importer to be activated by the presence of its cognate substrate.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Sideróforos/metabolismo , Activación Transcripcional , Factor de Transcripción de AraC/metabolismo , Bacillus subtilis/genética , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/metabolismo , Ligandos , Modelos Genéticos , Mutación , ARN/biosíntesis , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
A single nucleotide mismatch within intron 1 differentiates HLA-A*02:01:01:251 from the HLA-A*02:01:01:01 allele.