Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

Cortactin interacts with αDystrobrevin-1 and regulates murine neuromuscular junction morphology.

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.

Plasticity and structural alterations of mitochondria and sarcoplasmic organelles in muscles of mice deficient in α-dystrobrevin, a component of the dystrophin-glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) plays a crucial role in maintaining the structural integrity of the plasma membrane and the neuromuscular junction. In this study, we investigated the impact of the deficiency of α-dystrobrevin (αdbn), a component of the DGC, on the homeostasis of intracellular organelles, specifically mitochondria and the sarcoplasmic reticulum (SR). In αdbn deficient muscles, we observed a significant increase in the membrane-bound ATP synthase complex levels, a marker for mitochondria in oxidative muscle fiber types compared to wild-type. Furthermore, examination of muscle fibers deficient in αdbn using electron microscopy revealed profound alterations in the organization of mitochondria and the SR within certain myofibrils of muscle fibers. This included the formation of hyper-branched intermyofibrillar mitochondria with extended connections, an extensive network spanning several myofibrils, and a substantial increase in the number/density of subsarcolemmal mitochondria. Concurrently, in some cases, we observed significant structural alterations in mitochondria, such as cristae loss, fragmentation, swelling, and the formation of vacuoles and inclusions within the mitochondrial matrix cristae. Muscles deficient in αdbn also displayed notable alterations in the morphology of the SR, along with the formation of distinct anomalous concentric SR structures known as whorls. These whorls were prevalent in αdbn-deficient mice but were absent in wild-type muscles. These results suggest a crucial role of the DGC αdbn in regulating intracellular organelles, particularly mitochondria and the SR, within muscle cells. The remodeling of the SR and the formation of whorls may represent a novel mechanism of the unfolded protein response (UPR) in muscle cells.

Unraveling Hermansky-Pudlak syndrome type 7: a case report and comprehensive literature review on the identification of DTNBP1 variants.

PURPOSE: We report a case of Hermansky-Pudlak Syndrome type 7 (HPS-7) caused by a homozygous variant in the dystrobrevin-binding protein 1 gene (DTNBP1) and highlight the genetic challenges associated with this rare disorder. METHODS: Case report. Literature review was performed by searching PubMed on May 2023, without language or date restriction, using the following terms: Hermansky-Pudlak syndrome, Hermansky-Pudlak syndrome type 7, and dystrobrevin-binding protein 1 gene. RESULTS: We report a case of a 69-year-old Portuguese female who presented for ophthalmic evaluation with long-standing severe visual impairment, pronounced photophobia, right-eye esotropia, and bilateral pendular nystagmus. Anterior segment examination revealed iris transillumination defects, while the ocular fundus showed hypopigmentation and the absence of the foveal reflex. The patient had a history of oculocutaneous albinism (OCA) and recurrent epistaxis. Her family history was positive for first-degree consanguineous parents and a deceased sister at young age who also exhibited OCA and recurrent epistaxis. Genetic testing identified a homozygous pathogenic nonsense variant in the DTNBP1, c.307C>T p.(Gln103*). The patient's clinical features and genetic testing support the diagnosis of HPS-7. The identified variant has been previously reported in the literature, in adult patients of Portuguese descent. CONCLUSION: This work highlights the genetic complexity of HPS-7 and emphasizes the importance of genetic testing in the diagnosis of this rare disorder. The identification of a rare pathogenic variant expands our understanding of HPS-7 genetics and suggests a possible founder effect in the Portuguese population.

Seguridad y eficacia de ataluren en pacientes con diagnóstico de distrofia muscular de duchenne portadores de una mutación sin sentido en el gen de distrofina / Safety and efficacy of ataluren in patients diagnosed with duchenne muscular dystrophy who carry a nonsense mutation in the dystrophin gene

INTRODUCCIÓN: Antecedentes: El Instituto de Evaluación de Tecnologías en Salud e Investigación ha recibido la solicitud de evaluar el uso de Ataluren para su uso en Pacientes ambulantes mayores de 5 años con diagnóstico de distrofia muscular de Duchenne debida a una mutación sin sentido en el gen de la distrofina dentro del sistema de EsSalud, indicación actualmente no contemplada en el petitorio de medicamentos. Esta acción sigue lo estipulado en la Directiva N° 002-IETSI-ESSALUD-2015 y el objetivo final es determinar el estado del arte sobre la eficacia y seguridad de ataluren. Tecnología Sanitaria de Interés: Ataluren: La PTC124 (3-(5-(2-fluorofeni)-1, 2,4-oxadiazol-3-y1)-ácido benzoico), también conocida como Ataluren (TranslarnaTM) es una molécula pequeña de oxadiazol cuyo mecanismo de acción consiste en cominuar la traducción de ARNm sobre los codones de terminación prematuros causados por la mutación sin sentido, permitiendo la síntesis de distrofina completa y funcional. METODOLOGIA: Estrategia de Busqueda: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de Ataluren para el tratamiento de la DMD en las bases de datos de MEDLINE, EMBASE, CENTRAL, DARE y TRIPDATABASE. Se hizo una búsqueda adicional en www.clinicaltrials.gov, para poder identificar ensayos clínicos aún en elaboración o que no hayan sido publicados. Adicionalmente, se hizo una búsqueda dentro de la información generada por las principales instituciones internacionales oncológicas y agencias de tecnologías sanitarias que realizan revisiones sistemáticas (RS), evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC). RESULTADOS:Sinopses de la Evidencia: Se realizó la búsqueda bibliográfica y de evidencia científica que sustente el uso de ataluren en DMD según la pregunta PICO establecida. Para el presente documento se seleccionó el siguiente cuerpo de evidencia que es resumido a continuación: Guías Clínicas: Se identificó una única guía práctica realizada en Colombia que hizo mención a este tratamiento. Evaluaciones de tecnología sanitaria: Se identificó una ETS del Reino Unido. Revisiones sistemáticas: No se identificaron revisiones sistemáticas. Estudios de calidad de vida: No se identificaron estudios que evaluaran calidad de vida. Ensayos clínicos: Se identificaron dos ECAs correspondientes a las fases 2a y fase 2 b. Ensayos clínicos en curso: se identificó el registro correspondiente a un estudio de fase III pendiente de publicar sus resultados. CONCLUSIONES: se evidencia que ataluren es un medicamento aún en estudio que no ha demostrado al momento ser diferente a placebo en el tratamiento de la DMD con mutación sin sentido. De hecho, la evidencia disponible que el ataluren no es mejor que el placebo en mejorar indicadores clínicos importantes en el manejo de esta enfermedad, como la DC6M, considerada como desenlace principal en enfermedades raras con compromiso neuromuscular. Ataluren tampoco mostró ser diferente al placebo en mejorar la calidad de vida de los pacientes, ni disminuye los tiempos para realizar tareas motoras como subir o bajar escalones, correr o caminar 10 metros y levantarse desde la posición supina. El Instituto de Evaluación de Tecnología en Salud e investigación ­ IETSI, no aprueba el uso de ataluren para el tratamiento de la DMD con mutación sin sentido del gen de la distrofina.