RESUMEN
While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.
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Trastorno del Espectro Autista , Trastornos del Neurodesarrollo , Síndrome de Prader-Willi , Adolescente , Humanos , Trastorno del Espectro Autista/genética , Hiperfagia/genética , Hiperfagia/complicaciones , Trastornos del Neurodesarrollo/genética , Obesidad/complicaciones , Síndrome de Prader-Willi/complicaciones , Síndrome de Prader-Willi/genética , ProteínasRESUMEN
Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.
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Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Discapacidad Intelectual/genética , Empalme Alternativo/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Haploinsuficiencia/genética , Trastornos del Neurodesarrollo/genética , Ribonucleoproteínas Nucleares Heterogéneas/genéticaRESUMEN
Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.
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Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Ratones , Humanos , Infecciones Estafilocócicas/microbiología , Biopelículas , Modelos Animales de Enfermedad , Serina , AntibacterianosRESUMEN
ASXL3-related disorder, sometimes referred to as Bainbridge-Ropers syndrome, was first identified as a distinct neurodevelopmental disorder by Bainbridge et al. in 2013. Since then, there have been a number of case series and single case reports published worldwide. A comprehensive review of the literature was carried out. Abstracts were screened, relevant literature was analysed, and descriptions of common phenotypic features were quantified. ASXL3 variants were collated and categorised. Common phenotypic features comprised global developmental delay or intellectual disability (97%), feeding problems (76%), hypotonia (88%) and characteristic facial features (93%). The majority of genetic variants were de novo truncating variants in exon 11 or 12 of the ASXL3 gene. Several gaps in our knowledge of this disorder were identified, namely, underlying pathophysiology and disease mechanism, disease contribution of missense variants, relevance of variant location, prevalence and penetrance data. Clinical information is currently limited by patient numbers and lack of longitudinal data, which this review aims to address.
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Anomalías Múltiples , Discapacidades del Desarrollo , Facies , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Niño , Humanos , Discapacidades del Desarrollo/genética , Factores de Transcripción/genética , Fenotipo , Síndrome , Discapacidad Intelectual/genética , Proteínas Represoras/genéticaRESUMEN
Objectives. To determine facility-level factors associated with COVID-19 outbreaks in US Immigration and Customs Enforcement (ICE) detention centers. Methods. We obtained COVID-19 case counts at 88 ICE detention facilities from May 6, 2020, through June 21, 2021, from the COVID Prison Project. We obtained information about facility population size, facility type (dedicated to immigrants or mixed with other incarcerated populations), and facility operator (public vs private contractor) from third-party sources. We defined the threshold for a COVID-19 outbreak as a cumulative 3-week incidence of 10% or more of the detained population. Results. Sixty-three facilities (72%) had at least 1 outbreak. Facilities with any outbreak were significantly more likely to be privately operated (P < .001), to have larger populations (113 vs 37; P = .002), and to have greater changes in their population size over the study period (â56% vs -26%; P < .001). Conclusions. Several facility-level factors were associated with the occurrence of COVID-19 outbreaks in ICE facilities. Public Health Implications. Structural and organizational factors that promote respiratory infection spread in ICE facilities must be addressed to protect detainee health. (Am J Public Health. 2024;114(9):909-912. https://doi.org/10.2105/AJPH.2024.307704).
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COVID-19 , Humanos , COVID-19/epidemiología , Estados Unidos/epidemiología , Brotes de Enfermedades , Emigración e Inmigración/legislación & jurisprudencia , Emigración e Inmigración/estadística & datos numéricos , SARS-CoV-2 , Prisiones/estadística & datos numéricos , Cárceles Locales/estadística & datos numéricosRESUMEN
De novo truncating and splicing pathogenic variants in the Additional Sex Combs-Like 3 (ASXL3) gene are known to cause neurodevelopmental delay, intellectual disability, behavioral difficulties, hypotonia, feeding problems and characteristic facial features. We previously reported 45 patients with ASXL3-related disorder including three individuals with a familial variant. Here we report the detailed clinical and molecular characteristics of these three families with inherited ASXL3-related disorder. First, a father and son with c.2791_2792del p.Gln931fs pathogenic variant. The second, a mother, daughter and son with c.4534C > T, p.Gln1512Ter pathogenic variant. The third, a mother and her daughter with c.4441dup, p.Leu1481fs maternally inherited pathogenic variant. This report demonstrates intrafamilial phenotypic heterogeneity and confirms heritability of ASXL3-related disorder.
Asunto(s)
Anomalías Múltiples , Discapacidades del Desarrollo , Discapacidad Intelectual , Niño , Femenino , Humanos , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Fenotipo , Síndrome , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Acute pain accounts for over 70% of Emergency Department (ED) visits. Sub-dissociative dose ketamine (0.1-0.6 mg/kg) is safe and effective for the management of acute pain in the ED. However, the optimal dose of intravenous ketamine that provides effective analgesia and minimizes the risk of adverse effects has yet to be identified. The objective of this study was to describe an effective analgesia dose range of IV ketamine for acute pain in the ED. METHODS: This multi-center, retrospective cohort study evaluated adult patients who received analgesic and sub-dissociative dose ketamine for the management of acute pain between May 5, 2018, and August 30, 2021, in 21 emergency departments at academic, community, and critical access hospitals across four states. Patients were excluded if they received ketamine for an indication other than pain, such as procedural sedation or intubation, or for whom there was incomplete documentation for the primary outcome. Patients who received a ketamine dose <0.3 mg/kg were stratified into the low-dose group, and those who received a dose of 0.3 mg/kg or higher to the high-dose group. The primary outcome was change in pain scores within 60 min using a standard 11-point numeric rating scale (NRS). Secondary outcomes included incidence of adverse effects and use of rescue analgesics. Continuous variables were compared between dose groups using student t-test or Wilcoxon Rank-Sum test. Linear regression was used to assess the association between the change in NRS pain scores within 60 min and dose after adjusting for baseline pain, requiring an additional dose of ketamine, and receiving an opioid. RESULTS: From 3796 patient encounters screened for receipt of ketamine, 384 patients met inclusion criteria including 258 in the low-dose group, and 126 in the high-dose group. The primary reason for exclusion was incomplete documentation of pain scores, or ketamine used for sedation. Median baseline pain scores were 8.2 in the low-dose group and 7.8 in the high-dose group (difference 0.5; 95% CI 0 to 1, p = 0.04). Both groups demonstrated significant reductions in their mean NRS pain scores within 60 min following the first administration of IV ketamine. There were no differences in the change in pain scores between both groups (-2.2 vs -2.6, mean difference 0.4, 95% CI -0.4 to 1.1, p = 0.34). Use of rescue analgesics (40.7% vs 36.5%, p = 0.43) and adverse effects were similar between groups, including early discontinuation of the ketamine infusion (37.2% vs. 37.3%, p = 0.99). Overall, the most common adverse effects were agitation (7.3%) and nausea (7.0%). CONCLUSION: The analgesic efficacy and safety of high-dose sub-dissociative ketamine (≥0.3 mg/kg) was not superior to low-dose (< 0.3 mg/kg) for the management of acute pain in the ED. Low-dose ketamine <0.3 mg/kg is an effective and safe pain management strategy in this population.
Asunto(s)
Dolor Agudo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ketamina , Adulto , Humanos , Dolor Agudo/tratamiento farmacológico , Estudios Retrospectivos , Resultado del Tratamiento , Método Doble Ciego , Dimensión del Dolor , Analgésicos/uso terapéutico , Analgésicos Opioides/uso terapéutico , Servicio de Urgencia en HospitalRESUMEN
We describe clinical details, including novel findings, of two further children with the newly defined TLK2-related disorder. One patient was recruited to the Deciphering Developmental Delay (DDD) Study to identify underlying etiology of global developmental delay. The other was detected on whole-exome sequencing as part of second line investigations following normal microarray. Both patients were found to have de novo heterozygous pathogenic TLK2 variants. A novel c.6del p.(Glu3Lysfs*) loss-of-function frameshift variant was found in Patient 1. A c.1121+1G>A splice-donor variant was detected in Patient 2. TLK2-related neurodevelopmental disorder is a specific syndrome that has been recently described. Global developmental delay, behavioral problems, gastrointestinal disorders, and typical facial dysmorphism are common features. Neuropsychiatric disorders, ophthalmic, musculoskeletal and cranial abnormalities, as well as short stature, have also all been described. The novel findings we describe include sleep disturbance, nondifferentiation of lateral semi-circular canals (where asymmetric semi-circular canals were a feature in the previous cohort), vesico-ureteric reflux, and bilateral periauricular skin tags. Here, we report a novel TLK2 variant and previously undescribed features of TLK2-related disorder, to expand the clinical phenotype and provide further genotype-phenotype correlation.
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Discapacidad Intelectual , Trastornos del Neurodesarrollo , Niño , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Estudios de Asociación Genética , Heterocigoto , Humanos , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Fenotipo , Secuenciación del ExomaRESUMEN
TAB2 is a gene located on chromosome 6q25.1 and plays a key role in development of the heart. Existing literature describes congenital heart disease as a common recognized phenotype of TAB2 gene variants, with evidence of a distinct syndromic phenotype also existing beyond this. Here we describe 14 newly identified individuals with nine novel, pathogenic TAB2 variants. The majority of individuals were identified through the Deciphering Developmental Disorders study through trio whole exome sequencing. Eight individuals had de novo variants, the other six individuals were found to have maternally inherited, or likely maternally inherited, variants. Five individuals from the same family were identified following cardiac disease gene panel in the proband and subsequent targeted familial gene sequencing. The clinical features of this cohort were compared to the existing literature. Common clinical features include distinctive facial features, growth abnormalities, joint hypermobility, hypotonia, and developmental delay. Newly identified features included feeding difficulties, sleep problems, visual problems, genitourinary abnormality, and other anatomical variations. Here we report 14 new individuals, including novel TAB2 variants, in order to expand the emerging syndromic clinical phenotype and provide further genotype-phenotype correlation.
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Cardiopatías Congénitas , Discapacidad Intelectual , Proteínas Adaptadoras Transductoras de Señales/genética , Niño , Discapacidades del Desarrollo/genética , Estudios de Asociación Genética , Cardiopatías Congénitas/genética , Humanos , Discapacidad Intelectual/genética , Fenotipo , Secuenciación del ExomaRESUMEN
Accurate diagnosis of acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical for appropriate management of patients with this disease. We examined the possible complementary role of laboratory-developed class-specific clinical serology in assessing SARS-CoV-2 infection in hospitalized patients. Serological tests for immunoglobulin G (IgG), IgA, and IgM antibodies against the receptor binding domain (RBD) of SARS-CoV-2 were evaluated using samples from real-time reverse transcription-quantitative PCR (qRT-PCR)-confirmed inpatient coronavirus disease 2019 (COVID-19) cases. We analyzed the influence of timing and clinical severity on the diagnostic value of class-specific COVID-19 serology testing. Cross-sectional analysis revealed higher sensitivity and specificity at lower optical density cutoffs for IgA in hospitalized patients than for IgG and IgM serology (IgG area under the curve [AUC] of 0.91 [95% confidence interval {CI}, 0.89 to 0.93] versus IgA AUC of 0.97 [95% CI, 0.96 to 0.98] versus IgM AUC of 0.95 [95% CI, 0.92 to 0.97]). The enhanced performance of IgA serology was apparent in the first 2 weeks after symptom onset and the first week after PCR testing. In patients requiring intubation, all three tests exhibit enhanced sensitivity. Among PCR-negative patients under investigation for SARS-CoV-2 infection, 2 out of 61 showed clear evidence of seroconversion IgG, IgA, and IgM. Suspected false-positive results in the latter population were most frequently observed in IgG and IgM serology tests. Our findings suggest the potential utility of IgA serology in the acute setting and explore the benefits and limitations of class-specific serology as a complementary diagnostic tool to PCR for COVID-19 in the acute setting.
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COVID-19 , SARS-CoV-2 , Anticuerpos Antivirales , Estudios Transversales , Humanos , Inmunoglobulina M , Sensibilidad y EspecificidadRESUMEN
To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection.
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Adaptación Fisiológica/genética , Clostridioides difficile/fisiología , Regulación Bacteriana de la Expresión Génica/genética , Evasión Inmune/genética , Operón/genética , Animales , Péptidos Catiónicos Antimicrobianos/inmunología , Infecciones por Clostridium/genética , Infecciones por Clostridium/inmunología , Cricetinae , Femenino , Genes Bacterianos/genética , Interacciones Huésped-Patógeno/fisiología , Masculino , Mesocricetus , Ratones , Ratones Endogámicos C57BL , CatelicidinasRESUMEN
BACKGROUND: Palliative care has been identified as an area of low outpatient referral from our emergency department, yet palliative care has been shown to improve the quality of patient's lives. AIM: This study investigates both provider and patient perspectives on palliative care for the purpose of identifying barriers to increased palliative care utilization within our healthcare system. DESIGN: Two surveys were developed, one for patients/caregivers and one for healthcare providers. SETTING/PARTICIPANTS: This was a single-center study completed at a quaternary academic emergency department. A survey was sent to emergency medicine providers with 47% response rate. Research staff approached Emergency Department patients who had been identified to be high risk to fill out paper surveys with 76% response rate. RESULTS: Only 28% of patients had already undergone palliative care, with an additional 25% interested in palliative care. Nearly half of the patients felt that they needed more resources to prevent hospital visits. Patients identified low understanding of palliative care and difficulty accessing appointments as barriers to consultation. Among providers, 98% indicated that they had patients who would benefit from palliative care. A majority of providers highlighted patient understanding of palliative care and access to appointments as barriers to palliative care. Notably, 52% of providers reported that emergency medicine provider knowledge was a barrier to palliative care consultation. CONCLUSIONS: Despite emergency department patients' self-identified need for resources and emergency medicine providers' recognition of patients who would benefit from palliative care, few patients receive palliative care consultation.
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Servicio de Urgencia en Hospital , Personal de Salud , Cuidados Paliativos , Satisfacción del Paciente , Servicio de Urgencia en Hospital/estadística & datos numéricos , Personal de Salud/estadística & datos numéricos , Enfermería de Cuidados Paliativos al Final de la Vida/estadística & datos numéricos , Humanos , Satisfacción del Paciente/estadística & datos numéricos , Derivación y Consulta , Encuestas y CuestionariosRESUMEN
Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to ß-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to ß-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of ß-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a ß-lactamase, which is a common ß-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a ß-lactamase that was greatly induced in response to several classes of ß-lactam antibiotics. An in-frame deletion of the operon abolished ß-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the ß-lactam ampicillin. We found that the activity of this ß-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to ß-lactams. These results provide support for the function of a ß-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile ß-lactamase activity.
Asunto(s)
Clostridioides difficile/patogenicidad , beta-Lactamasas/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Lipoproteínas/genética , Lipoproteínas/metabolismo , beta-Lactamasas/genética , beta-Lactamas/farmacologíaRESUMEN
Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.
Asunto(s)
Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Etanolamina Amoníaco-Liasa/genética , Etanolamina/metabolismo , Animales , Clostridioides difficile/patogenicidad , Cricetinae , Intestinos/microbiología , Nutrientes , Virulencia , Factores de VirulenciaRESUMEN
Clostridioides (Clostridium) difficile causes severe diarrheal disease that is directly associated with antibiotic use and resistance. Although C. difficile demonstrates intrinsic resistance to many antimicrobials, few genetic mechanisms of resistance have been characterized in this pathogen. In this study, we investigated the putative resistance factor, CD1240 (VanZ1), an ortholog of the teicoplanin resistance factor, VanZ, of Enterococcus faecium. In C. difficile, the vanZ1 gene is located within the skin element of the sporulation factor σK, which is excised from the mother cell compartment during sporulation. This unique localization enabled us to create a vanZ1 deletion mutant by inducing excision of the skin element. The Δskin mutant exhibited moderately decreased resistance to teicoplanin and had small effects on growth in some other cell-surface antimicrobials tested. Examination of vanZ1 expression revealed induction of vanZ1 transcription by the antimicrobial peptide LL-37; however, LL-37 resistance was not impacted by VanZ1, and none of the other tested antimicrobials induced vanZ1 expression. Further, expression of vanZ1 via an inducible promoter in the Δskin mutant restored growth in teicoplanin. These results demonstrate that like the E. faecium VanZ, C. difficile VanZ1 contributes to low-level teicoplanin resistance through an undefined mechanism.
Asunto(s)
Antibacterianos/farmacología , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Farmacorresistencia Bacteriana , Teicoplanina/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Orden Génico , Homología de Secuencia , CatelicidinasRESUMEN
Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σ(V) does not induce dlt expression in response to lysozyme, indicating that σ(V) is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5' RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σ(V)-dependent manner and that this pathway impacts virulence in vivo.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/inmunología , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Muramidasa/inmunología , Factor sigma/inmunología , Alanina/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Pared Celular/genética , Pared Celular/inmunología , Clostridioides difficile/genética , Clostridioides difficile/inmunología , Cricetulus , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/genética , Enterocolitis Seudomembranosa/microbiología , Femenino , Muramidasa/genética , Mutación , Operón , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Factor sigma/genética , Transducción de Señal , Estereoisomerismo , Ácidos Teicoicos/metabolismo , VirulenciaRESUMEN
The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species.
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Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Esporas Bacterianas/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/microbiología , Cricetinae , Femenino , Regulación Bacteriana de la Expresión Génica/fisiología , Mutación , Polimorfismo de Nucleótido Simple , VirulenciaRESUMEN
Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.
Asunto(s)
Fosfatasa Alcalina/genética , Clostridioides difficile/genética , Expresión Génica , Genes Reporteros , Activación Enzimática , Orden Génico , Plásmidos/genética , Transcripción GenéticaRESUMEN
Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.
RESUMEN
Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.