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1.
Proc Natl Acad Sci U S A ; 120(30): e2301538120, 2023 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-37459522

RESUMEN

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.


Asunto(s)
Oxigenasas de Función Mixta , Neumonía , Humanos , Ratones , Animales , Oxigenasas de Función Mixta/metabolismo , Pseudomonas aeruginosa/metabolismo , Polisacáridos/metabolismo , Inmunización
2.
J Transl Med ; 15(1): 245, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29202780

RESUMEN

BACKGROUND: Chronic fatigue syndrome (CFS) is a prevalent and disabling condition among adolescent. The disease mechanisms are unknown. Previous studies have suggested elevated plasma levels of several cytokines, but a recent meta-analysis of 38 articles found that of 77 different cytokines measured in plasma, transforming growth factor beta (TGF-ß) was the only one that was elevated in patients compared to controls in a sufficient number of articles. In the present study we therefore compared the plasma levels of the three TGF-ß isoforms in adolescent CFS patients and healthy controls. In addition, the study explored associations between TGF-ß levels, neuroendocrine markers, clinical markers and differentially expressed genes within the CFS group. METHODS: CFS patients aged 12-18 years (n = 120) were recruited nation-wide to a single referral center as part of the NorCAPITAL project (ClinicalTrials ID: NCT01040429). A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls (n = 68) were recruited from local schools. The three isoforms of TGF-ß (TGF-ß1, TGF-ß2, TGF-ß3) were assayed using multiplex technology. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. Whole blood gene expression was assessed by RNA sequencing in a subgroup of patients (n = 29) and controls (n = 18). RESULTS: Plasma levels of all three isoforms of TGF-ß were equal in the CFS patients and the healthy controls. Subgrouping according to the Fukuda and Canada 2003 criteria of CFS did not reveal differential results. Within the CFS group, all isoforms of TGF-ß were associated with plasma cortisol, urine norepinephrine and urine epinephrine, and this association pattern was related to fatigue score. Also, TGF-ß3 was related to expression of the B cell annotated genes TNFRSF13C and CXCR5. CONCLUSIONS: Plasma levels of all TGF-ß isoforms were not altered in adolescent CFS. However, the TGF-ß isoforms were associated with neuroendocrine markers, an association related to fatigue score. Furthermore, TGF-ß3 might partly mediate an association between plasma cortisol and B cell gene expression. Trial registration Clinical Trials NCT01040429.


Asunto(s)
Síndrome de Fatiga Crónica/sangre , Factor de Crecimiento Transformador beta/sangre , Adolescente , Biomarcadores/metabolismo , Estudios de Casos y Controles , Demografía , Femenino , Humanos , Modelos Lineales , Masculino , Sistemas Neurosecretores/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
3.
Nat Commun ; 12(1): 1230, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33623002

RESUMEN

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Oxigenasas de Función Mixta/metabolismo , Polisacáridos/metabolismo , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Animales , Proteínas Bacterianas/química , Proteínas Portadoras/química , Muerte Celular , Proteínas del Sistema Complemento/metabolismo , Humanos , Ratones , Viabilidad Microbiana , Oxidación-Reducción , Dominios Proteicos , Proteoma/metabolismo , Proteómica , Infecciones por Pseudomonas/sangre , Especificidad por Sustrato , Transcripción Genética , Virulencia , Factores de Virulencia/metabolismo
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