Molecular and functional characterization of the quality control Valosin-containing protein VCP/p97 and of its co-factors in Leishmania

Leishmania est un parasite protozoaire eucaryote unicellulaire qui infecte plus de 1.6 millions de personnes chaque année dans plus de 98 pays. Aucun vaccin humain est actuellement disponible et peu de traitements efficaces sont utillisés pour lutter contre le large spectre de pathologies causées par Leishmania. Récemment, l’étude du contrôle de la qualité des protéines chez Leishmania infantum a révélé que DDX3, une DEAD-box hélicase à ARN dotée de multiples fonctions dans le métabolisme de l’ARN et la signalisation cellulaire, joue un rôle central dans le contrôle de qualité des protéines dans la mitochondrie. Une étude plus approfondie de ce mécanisme a révélé des interactions potentielles de DDX3 avec des composantes clés de la réponse cellulaire au stress, en particulier avec une protéine de la famille des AAA + ATPases, VCP/p97/Cdc48. Comme VCP/p97/Cdc48 participe à de multiples étapes dans le contrôle de qualité des protéines en utilisant son hydrolyse de l’ATP pour séparer les protéines ubiquitinées de leurs partenaires et les acheminer au protéasome 26S pour dégradation, nous avons émis l’hypothèse que l’homologue très conservé chez Leishmania, LiVCP, pourrait agir de la même façon. Cette étude a permis la caractérisation fonctionnelle de l’homologue VCP chez Leishmania, son rôle dans la réponse du parasite au stress et sa survie dans les macrophages, ses interactions potentielles avec d’autres partenaires dont des cofacteurs clés, ainsi que la modélisation 3D des interactions LiVCP-cofacteurs. En utilisant des mutants génétiquement générés ayant moins de copies du gène LiVCP ou des mutants dominants négatifs avec une activité VCP altérée, nous avons démontré que LiVCP est un gène essentiel et que les mutants VCP sont incapables de survivre sous le shock de la chaleur et présentent un déficit de croissance très marqué chez les amastigotes. De plus, nous avons montré une forte accumulation de protéines polyubiquitinées et une sensibilité accrue au stress protéotoxique chez ces mutants, soutenant la fonction de chaperone sélective de l'ubiquitine de LiVCP. Grâce à des analyses in silico et à la «protéomique en réseau» en utilisant des études de co-immunoprécipitation et de spectrométrie de masse (LC-MS / MS), nous avons établi le premier réseau protéique de VCP chez les parasites protozoaires et déterminé que p47, FAF2, UFD1, PUB1 et l’hétérodimère NPL4-UFD1 étaient les principaux cofacteurs de LiVCP. Enfin, nos travaux nous ont permis de faire progresser nos connaissances générales sur la protéine essentielle VCP et le contrôle de la qualité des protéines chez Leishmania et d’indiquer quelques perspectives intéressantes pour approfondir notre compréhension sur ces mécanismes importants non seulement chez Leishmania mais aussi chez d’autres trypanosomatides.Leishmania is a unicellular eukaryotic protozoan parasite that infects over 1.6 million people each year in more than 98 countries. No human vaccine is currently available and few effective treatments are used to combat the broad spectrum of diseases caused by Leishmania. Recently, studies on Protein Quality Control in Leishmania infantum revealed that the multifunctional DEAD-box RNA helicase DDX3 involved among others in RNA metabolism and cell signaling plays a central role in mitochondrial protein quality control. Further studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly with the conserved AAA+ ATPase VCP/ p97/Cdc48. As VCP is associated with many ubiquitin-dependent cellular pathways that are central to protein quality control in other eukaryotic systems using its ATP hydrolysis to separate ubiquitinated proteins from their partners and bring them to the 26S proteasome for degradation, we hypothesized that the Leishmania highly conserved counterpart, LiVCP, might act in similar way. This study enabled the functional characterization of the Leishmania VCP homolog, its role in the parasite's response to stress and survival inside macrophages, its potential interactions with other partners including key VCP cofactors, and the homology 3D modeling of LiVCP-cofactor interactions. Using genetically engineered mutants with fewer copies of the LiVCP gene or dominant negative mutants with altered VCP activity, we demonstrated that LiVCP is an essential gene and that VCP mutants are unable to survive under heat stress and exhibit a very marked growth defect in amastigotes. In addition, we showed a high accumulation of polyubiquitinated proteins and increased susceptibility to proteotoxic stress in these mutants, supporting that LiVCP has an ubiquitin selective chaperone function. Using "network proteomics" analyses by co-immunoprecipitation and mass spectrometry (LC-MS/MS) studies, we established the first VCP protein network in protozoan parasites and determined p47, FAF2, UFD1, PUB1 and the NPL4-UFD1 heterodimer as the major cofactors of LiVCP. Overall, our work allowed us to advance general knowledge of the essential role of VCP in Leishmania protein quality control and to propose some interesting perspectives to deepen our understanding of these important pathways not only in Leishmania but also in other trypanosomatids

The COVID-19 pandemic and plans for economic reopening in Brazil: a documental analysis

BACKGROUND: In March 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. In Brazil, the high rate of dissemination made it necessary to adopt restrictive measures nationwide with the discussion regarding the resumption of economic activities starting in April. In mid-December the country had 6,970,034 cases diagnosed and 182,799 deaths from COVID-19. OBJECTIVE: To analyze the content and characteristics of official documents, which guided the period of transition and resumption of economic activities in Brazil based on health indicators. METHODS: This is a documental research, carried out between May and July 2020, using official websites and publications from the state governments of the 27 federative units in Brazil as sources. In the study, only documents that used epidemiological and health indicators were included as determining criteria for decision making in relation to the easing, permanence or regression of social isolation measures adopted during the COVID-19 pandemic. FINDINGS: Plans, decrees and technical notes were identified for 18 Brazilian federal units. In most documents, the scientific team was made up exclusively of technicians (n = 10). The number of indicators found ranged from 2 to 11, being stratified into 5 categories: frequency and distribution of the disease; social and collective adherence; installed capacity or service profile; productive potential; and availability of supplies. MAIN CONCLUSIONS: Knowing governmental strategies, adopted in the easing of restrictive measures, in the face of the coronavirus (SARS-CoV-2) pandemic based on indicators and with the possibility of comparison between different federative units, provides subsidies for understanding the outcome of the disease by place of occurrence, allowing the construction of a panorama pathology in the country. The appropriation of the findings of this study by Brazil and other countries also serves as an instrument for reflection and planning of policies adopted during the COVID-19 pandemic

estudio descriptivo, 2020-2021

Publisher Copyright: © 2022, Ministry of Health. All rights reserved.Objective: To describe the profile and temporal variation of hospital admissions and deaths due to severe acute respiratory syndrome (SARS) caused by COVID-19 in Piauí, Brazil, according to place of hospitalization. Methods: We performed a descriptive study using data from the Influenza Surveillance Information System between 2020 and 2021. Case fatality ratio among hospital records with outcome and respective 95% confidence intervals (95%CI) were calculated. Results: We included 12,649 individuals who were mostly male (57.1%), Black (61.2%) and had one or two comorbidities (30.5%). Case fatality ratio among hospital records with outcome was higher in the state’s interior region than in its capital, with proportion of 44.1% (95%CI 42.0;46.3) for those who were hospitalized, 82.3% (95%CI 79.7;84.8) for those admitted to intensive care units and 96.6% (95%CI 94.9;97.8) for those undergoing invasive mechanical ventilation. Conclusion: The study enabled characterization of the profile of SARS hospitalizations due to COVID-19 in Piauí and demonstrated high case fatality ratio, among hospital records with outcome, which remained high during the study period, especially in the interior of the state.publishersversionpublishe

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

Introduction Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification. Results The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis