RESUMEN
Staphylococcus argenteus is a recently described member of the Staphylococcus aureus complex (SAC) and is associated with human disease. The frequency and intensity of infections caused by S. argenteus are similar to those of Staphylococcus aureus. S. argenteus can harbor antibiotic resistance genes and a variety of virulence factors analogous to methicillin-resistant S. aureus (MRSA). The aim of our study was to analyze a collection of isolates in the Dutch national MRSA surveillance from January 2008 until March 2021 that were nontypeable by multilocus variable-number tandem-repeat analysis (MLVA). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) was used for identifying the S. argenteus isolates, and whole-genome sequencing and SeqSphere were used to generate an in-house whole-genome multilocus sequence typing (wgMLST) scheme for typing the isolates. Furthermore, the presence of antibiotic resistance genes, replicons, and virulence genes was determined. Of 52,467 isolates submitted as MRSA from January 2008 until March 2021, 64 isolates (0.12%) were nontypeable with MLVA, and 54 of them were identified with mass spectrometry (MALDI-ToF MS) as S. argenteus. It appeared in retrospect that the first methicillin-resistant S. argenteus (MRSArg) was already submitted in 2008. An in-house-developed S. argenteus wgMLST scheme revealed that S. argenteus isolates clustered in 5 genomic groups which were characterized by distinct MLST types, resistomes, plasmid replicon families, and virulence factors. All but one isolate carried the staphylococcal chromosomal cassette mec (SCCmec) type IV harboring the methicillin resistance gene mecA and represent MRSArg. Most of the isolates with SCCmec subtype IVc(2B) had a trimethoprim resistance gene, dfrG, and harbored a blaZ-carrying plasmid, and most MRSArg isolates have the immune-modulating genes scn and sak. Nine of the 47 isolates carried enterotoxin-encoding genes seg, sei, sem, seo, and seu, which might be able to cause food poisoning. In some persons there was long-term persistence of MRSArg, and there were several genetically related MRSArg isolates in people living in close proximity, suggesting direct human-human transmission. IMPORTANCE We show that MRSArg has been circulating in the Netherlands since at least 2008. Although MRSArg is distinct from MRSA, it has a comparable population structure and carries similar resistance and virulence genes. The Dutch national MRSA surveillance has been expanded to include other methicillin-resistant members of the S. aureus complex, such as S. argenteus and Staphylococcus schweitzeri.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Resistencia a la Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus/genética , Tipificación de Secuencias Multilocus , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/epidemiología , Antibacterianos/farmacología , Enterotoxinas , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Surveillance is the cornerstone of surgical site infection prevention programs. The validity of the data collection and awareness of vulnerability to inter-rater variation is crucial for correct interpretation and use of surveillance data. The aim of this study was to investigate the reliability and validity of surgical site infection (SSI) surveillance after colorectal surgery in the Netherlands. METHODS: In this multicentre prospective observational study, seven Dutch hospitals performed SSI surveillance after colorectal surgeries performed in 2018 and/or 2019. When executing the surveillance, a local case assessment was performed to calculate the overall percentage agreement between raters within hospitals. Additionally, two case-vignette assessments were performed to estimate intra-rater and inter-rater reliability by calculating a weighted Cohen's Kappa and Fleiss' Kappa coefficient. To estimate the validity, answers of the two case-vignettes questionnaires were compared with the answers of an external medical panel. RESULTS: 1111 colorectal surgeries were included in this study with an overall SSI incidence of 8.8% (n = 98). From the local case assessment it was estimated that the overall percent agreement between raters within a hospital was good (mean 95%, range 90-100%). The Cohen's Kappa estimated for the intra-rater reliability of case-vignette review varied from 0.73 to 1.00, indicating substantial to perfect agreement. The inter-rater reliability within hospitals showed more variation, with Kappa estimates ranging between 0.61 and 0.94. In total, 87.9% of the answers given by the raters were in accordance with the medical panel. CONCLUSIONS: This study showed that raters were consistent in their SSI-ascertainment (good reliability), but improvements can be made regarding the accuracy (moderate validity). Accuracy of surveillance may be improved by providing regular training, adapting definitions to reduce subjectivity, and by supporting surveillance through automation.
Asunto(s)
Cirugía Colorrectal/estadística & datos numéricos , Monitoreo Epidemiológico , Infección de la Herida Quirúrgica/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Estudios Prospectivos , Reproducibilidad de los Resultados , Infección de la Herida Quirúrgica/microbiologíaAsunto(s)
Antibacterianos/farmacología , Carbapenémicos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Selección Genética , Resistencia betalactámica , beta-Lactamas/farmacología , Ertapenem , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.
Asunto(s)
Glucósidos/metabolismo , Leishmania/genética , Leishmania/metabolismo , Transcripción Genética , Uracilo/análogos & derivados , Técnicas de Inactivación de Genes , ARN Polimerasa II/metabolismo , ARN Bicatenario/metabolismo , Uracilo/metabolismoRESUMEN
RATIONALE: The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. OBJECTIVE: Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. METHODS AND RESULTS: In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. CONCLUSIONS: Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.
Asunto(s)
Actinas/metabolismo , Endosomas/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteolisis , Animales , Transporte Biológico , Células Cultivadas , Colesterol/metabolismo , Lisosomas/metabolismo , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Modelos Animales , Orgánulos/metabolismoRESUMEN
The genomic DNA of kinetoplastid parasites contains a unique modified base, beta-d-glucosyl-hydroxymethyluracil or base J. We recently reported that two proteins, called J-binding protein (JBP) 1 and 2, which regulate the levels of J in the genome, display features of the family of Fe(II)-2-oxoglutarate dependent dioxygenases and are likely to be the enzymes catalyzing the first step in J biosynthesis. In this study, we examine the effects of replacing the four conserved residues critical for the activity of this class of enzymes on the function of Leishmania tarentolae JBP2. The results show that each of these four residues is indispensable for the ability of JBP2 to stimulate J synthesis, while mutating non-conserved residues has no consequences. We conclude that JBP2, like JBP1, is in all probability a thymidine hydroxylase involved in the biosynthesis of base J.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glucósidos/biosíntesis , Leishmania/enzimología , Oxigenasas de Función Mixta/metabolismo , Proteínas Protozoarias/metabolismo , Timidina/metabolismo , Uracilo/análogos & derivados , Sustitución de Aminoácidos/genética , Animales , ADN Protozoario/química , ADN Protozoario/genética , Proteínas de Unión al ADN/genética , Leishmania/genética , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Protozoarias/genética , Análisis de Secuencia de ADN , Uracilo/biosíntesisRESUMEN
Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergent-resistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging approximately 70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.
Asunto(s)
Colesterol/química , Desmosterol/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Albúminas/metabolismo , Animales , Células CHO , Cricetinae , Detergentes , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Insulina/metabolismo , Insulina/farmacología , Radioisótopos de Yodo , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Fracciones Subcelulares/químicaRESUMEN
Niemann-Pick type C (NPC) disease is a neuro-visceral cholesterol storage disorder caused by mutations in the NPC-1 or NPC-2 gene. In the present paper, we studied IR (insulin receptor) activation and the plasma-membrane lipid assembly in primary hepatocytes from control and NPC1-/- mice. We have previously reported that, in hepatocytes, IR activation is dependent on cholesterol-sphingolipid rafts [Vainio, Heino, Mansson, Fredman, Kuismanen, Vaarala and Ikonen (2002) EMBO Rep. 3, 95-100]. We found that, in NPC hepatocytes, IR levels were up-regulated and the receptor activation was compromised. Defective IR activation was reproduced in isolated NPC plasma-membrane preparations, which displayed an increased cholesterol content and saturation of major phospholipids. The NPC plasma membranes were less fluid than control membranes as indicated by increased DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence anisotropy values. Both in NPC hepatocytes and plasma-membrane fractions, the association of IR with low-density DRMs (detergent-resistant membranes) was increased. Moreover, the detergent resistance of both cholesterol and phosphatidylcholine were increased in NPC membranes. Finally, cholesterol removal inhibited IR activation in control membranes but restored IR activation in NPC membranes. Taken together, the results reveal a lipid imbalance in the NPC hepatocyte, which increases lipid ordering in the plasma membrane, alters the properties of lipid rafts and interferes with the function of a raft-associated plasma-membrane receptor. Such a mechanism may participate in the pathogenesis of NPC disease and contribute to insulin resistance in other disorders of lipid metabolism.
Asunto(s)
Hepatocitos/metabolismo , Microdominios de Membrana/metabolismo , Enfermedades de Niemann-Pick/metabolismo , Enfermedades de Niemann-Pick/patología , Proteínas/genética , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Proteína Niemann-Pick C1 , Enfermedades de Niemann-Pick/genética , Proteínas/metabolismo , Transducción de SeñalRESUMEN
The potential contribution of lipids to insulin signaling has excited interest because of the notion that cholesterol and sphingolipids form functional microdomains-lipid rafts-in cell membranes and that these domains may affect signal transduction. In this Perspective, we discuss the evidence suggesting that cholesterol-sphingolipid rafts play a role in the pathogenesis of insulin resistance. The data relating insulin signaling to lipid rafts in the main insulin target tissues are briefly summarized, including partially controversial findings on the role of caveolae versus other types of rafts. In addition, recent results pointing toward the importance of raft perturbations in the pathogenesis of insulin resistance are discussed. Notably, several studies suggest a correlation between membrane lipid composition and insulin sensitivity. We put forward the idea that the dyslipidemic changes typically associated with insulin resistance and metabolic syndrome may impair the functionality of rafts in insulin target cells, thereby promoting insulin resistance.
Asunto(s)
Resistencia a la Insulina/fisiología , Lípidos de la Membrana/fisiología , Microdominios de Membrana/fisiología , Adipocitos/fisiología , Animales , Células CHO , Caveolas/fisiología , Compartimento Celular , Cricetinae , Cricetulus , Endocitosis , Hepatocitos/fisiología , Humanos , Islotes Pancreáticos/fisiopatología , Lípidos de la Membrana/química , Microdominios de Membrana/química , Síndrome Metabólico/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Células Musculares/fisiología , Receptor de Insulina/fisiología , Transducción de Señal , Esfingolípidos/química , Esfingolípidos/fisiologíaRESUMEN
Mammalian cells have evolved complex feedback mechanisms to ensure sufficient supply of cholesterol and to prevent its excessive accumulation. During the process of atherosclerosis, these homeostatic mechanisms fail in macrophages. Uncontrolled cholesterol deposition is promoted by scavenger functions of the macrophages and the adaptive mechanisms elicited are not sufficient to process the lipid load. Consequently, a lipid-laden 'foam cell' is formed. In this review, we summarize key aspects of intracellular cholesterol processing in the special case of macrophages, including mechanisms of lipoprotein cholesterol uptake, fate of the internalized cholesterol and mechanisms implicated in cholesterol efflux. The importance of inflammatory cues, the cellular compartmentalization of cholesterol homeostatic responses and the increasing information on the transcriptional control of cholesterol balance are discussed.
Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana , Receptores de Lipoproteína , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteínas E/biosíntesis , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ésteres del Colesterol/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Espumosas/metabolismo , Humanos , Inflamación/metabolismo , Receptores X del Hígado , Receptores Nucleares Huérfanos , Fragmentos de Péptidos/biosíntesis , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Inmunológicos/metabolismo , Receptores de LDL/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Factores de Transcripción/biosíntesisRESUMEN
Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.