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1.
Nature ; 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34670266

RESUMO

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.

2.
3.
Geroscience ; 43(4): 1975-1993, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34117600

RESUMO

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels.


Assuntos
Cádmio , Metalotioneína , Envelhecimento/genética , Animais , Cádmio/toxicidade , Rim , Fígado , Metalotioneína/genética , Camundongos
4.
Blood ; 138(21): 2093-2105, 2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34125889

RESUMO

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes common in persons aged ≥80 years, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 persons aged ≥80 years and investigated the relationships between CHIP and associated pathologies. Mutations were observed in one-third of persons aged ≥80 years and were associated with reduced survival. Mutations in JAK2 and splicing genes, multiple mutations (DNMT3A, TET2, and ASXL1 with additional genetic lesions), and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1, or JAK2 were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was common in persons aged ≥80 years, with the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of subjects aged ≥80 years with cytopenia had presumptive evidence of myeloid neoplasm. In summary, specific mutational patterns define different risk of developing myeloid neoplasms vs inflammatory-associated diseases in persons aged ≥80 years. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.

6.
Nucleic Acids Res ; 49(W1): W199-W206, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34038548

RESUMO

Methylage is an epigenetic marker of biological age that exploits the correlation between the methylation state of specific CG dinucleotides (CpGs) and chronological age (in years), gestational age (in weeks), cellular age (in cell cycles or as telomere length, in kilobases). Using DNA methylation data, methylage is measurable via the so called epigenetic clocks. Importantly, alterations of the correlation between methylage and age (age acceleration or deceleration) have been stably associated with pathological states and occur long before clinical signs of diseases become overt, making epigenetic clocks a potentially disruptive tool in preventive, diagnostic and also in forensic applications. Nevertheless, methylage dependency from CpGs selection, mathematical modelling, tissue specificity and age range, still makes the potential of this biomarker limited. In order to enhance model comparisons, interchange, availability, robustness and standardization, we organized a selected set of clocks within a hub webservice, EstimAge (Estimate of methylation Age, http://estimage.iac.rm.cnr.it), which intuitively and informatively enables quick identification, computation and comparison of available clocks, with the support of standard statistics.


Assuntos
Metilação de DNA , Software , Ilhas de CpG , Epigênese Genética , Internet , Fatores de Tempo
7.
Elife ; 102021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33941312

RESUMO

Extreme longevity is the paradigm of healthy aging as individuals who reached the extreme decades of human life avoided or largely postponed all major age-related diseases. In this study, we sequenced at high coverage (90X) the whole genome of 81 semi-supercentenarians and supercentenarians [105+/110+] (mean age: 106.6 ± 1.6) and of 36 healthy unrelated geographically matched controls (mean age 68.0 ± 5.9) recruited in Italy. The results showed that 105+/110+ are characterized by a peculiar genetic background associated with efficient DNA repair mechanisms, as evidenced by both germline data (common and rare variants) and somatic mutations patterns (lower mutation load if compared to younger healthy controls). Results were replicated in a second independent cohort of 333 Italian centenarians and 358 geographically matched controls. The genetics of 105+/110+ identified DNA repair and clonal haematopoiesis as crucial players for healthy aging and for the protection from cardiovascular events.


Assuntos
Hematopoiese Clonal/genética , Reparo do DNA , Longevidade/genética , Sequenciamento Completo do Genoma/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Patrimônio Genético , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Mutação , Sequenciamento Completo do Genoma/métodos
8.
Front Aging Neurosci ; 13: 639428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790779

RESUMO

Alzheimer's disease (AD) is characterized by specific alterations of brain DNA methylation (DNAm) patterns. Age and sex, two major risk factors for AD, are also known to largely affect the epigenetic profiles in brain, but their contribution to AD-associated DNAm changes has been poorly investigated. In this study we considered publicly available DNAm datasets of four brain regions (temporal, frontal, entorhinal cortex, and cerebellum) from healthy adult subjects and AD patients, and performed a meta-analysis to identify sex-, age-, and AD-associated epigenetic profiles. In one of these datasets it was also possible to distinguish 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles. We showed that DNAm differences between males and females tend to be shared between the four brain regions, while aging differently affects cortical regions compared to cerebellum. We found that the proportion of sex-dependent probes whose methylation is modified also during aging is higher than expected, but that differences between males and females tend to be maintained, with only a few probes showing age-by-sex interaction. We did not find significant overlaps between AD- and sex-associated probes, nor disease-by-sex interaction effects. On the contrary, we found that AD-related epigenetic modifications are significantly enriched in probes whose DNAm varies with age and that there is a high concordance between the direction of changes (hyper or hypo-methylation) in aging and AD, supporting accelerated epigenetic aging in the disease. In summary, our results suggest that age-associated DNAm patterns concur to the epigenetic deregulation observed in AD, providing new insights on how advanced age enables neurodegeneration.

9.
Cell ; 184(7): 1821-1835.e16, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33667349

RESUMO

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Linfócitos B/imunologia , COVID-19 , Convalescença , Células 3T3 , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Linfócitos B/citologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/terapia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Masculino , Camundongos , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
10.
J Clin Oncol ; 39(11): 1223-1233, 2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33539200

RESUMO

PURPOSE: Recurrently mutated genes and chromosomal abnormalities have been identified in myelodysplastic syndromes (MDS). We aim to integrate these genomic features into disease classification and prognostication. METHODS: We retrospectively enrolled 2,043 patients. Using Bayesian networks and Dirichlet processes, we combined mutations in 47 genes with cytogenetic abnormalities to identify genetic associations and subgroups. Random-effects Cox proportional hazards multistate modeling was used for developing prognostic models. An independent validation on 318 cases was performed. RESULTS: We identify eight MDS groups (clusters) according to specific genomic features. In five groups, dominant genomic features include splicing gene mutations (SF3B1, SRSF2, and U2AF1) that occur early in disease history, determine specific phenotypes, and drive disease evolution. These groups display different prognosis (groups with SF3B1 mutations being associated with better survival). Specific co-mutation patterns account for clinical heterogeneity within SF3B1- and SRSF2-related MDS. MDS with complex karyotype and/or TP53 gene abnormalities and MDS with acute leukemia-like mutations show poorest prognosis. MDS with 5q deletion are clustered into two distinct groups according to the number of mutated genes and/or presence of TP53 mutations. By integrating 63 clinical and genomic variables, we define a novel prognostic model that generates personally tailored predictions of survival. The predicted and observed outcomes correlate well in internal cross-validation and in an independent external cohort. This model substantially improves predictive accuracy of currently available prognostic tools. We have created a Web portal that allows outcome predictions to be generated for user-defined constellations of genomic and clinical features. CONCLUSION: Genomic landscape in MDS reveals distinct subgroups associated with specific clinical features and discrete patterns of evolution, providing a proof of concept for next-generation disease classification and prognosis.


Assuntos
Genômica/métodos , Síndromes Mielodisplásicas/classificação , Feminino , Humanos , Masculino , Síndromes Mielodisplásicas/genética , Prognóstico , Estudos Retrospectivos
11.
BMC Bioinformatics ; 22(1): 60, 2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563206

RESUMO

BACKGROUND: Current high-throughput technologies-i.e. whole genome sequencing, RNA-Seq, ChIP-Seq, etc.-generate huge amounts of data and their usage gets more widespread with each passing year. Complex analysis pipelines involving several computationally-intensive steps have to be applied on an increasing number of samples. Workflow management systems allow parallelization and a more efficient usage of computational power. Nevertheless, this mostly happens by assigning the available cores to a single or few samples' pipeline at a time. We refer to this approach as naive parallel strategy (NPS). Here, we discuss an alternative approach, which we refer to as concurrent execution strategy (CES), which equally distributes the available processors across every sample's pipeline. RESULTS: Theoretically, we show that the CES results, under loose conditions, in a substantial speedup, with an ideal gain range spanning from 1 to the number of samples. Also, we observe that the CES yields even faster executions since parallelly computable tasks scale sub-linearly. Practically, we tested both strategies on a whole exome sequencing pipeline applied to three publicly available matched tumour-normal sample pairs of gastrointestinal stromal tumour. The CES achieved speedups in latency up to 2-2.4 compared to the NPS. CONCLUSIONS: Our results hint that if resources distribution is further tailored to fit specific situations, an even greater gain in performance of multiple samples pipelines execution could be achieved. For this to be feasible, a benchmarking of the tools included in the pipeline would be necessary. It is our opinion these benchmarks should be consistently performed by the tools' developers. Finally, these results suggest that concurrent strategies might also lead to energy and cost savings by making feasible the usage of low power machine clusters.


Assuntos
Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Software , Sequenciamento Completo do Exoma , Sequenciamento de Cromatina por Imunoprecipitação , Biologia Computacional/métodos , Sequenciamento Completo do Exoma/normas , Fluxo de Trabalho
12.
Sci Rep ; 11(1): 3030, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542369

RESUMO

In this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.


Assuntos
Bactérias/genética , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenoma/genética , Bactérias/classificação , Humanos , Metagenômica , Microbiota/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
13.
Mech Ageing Dev ; 194: 111426, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33385396

RESUMO

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naïve and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development.


Assuntos
Envelhecimento/metabolismo , Pesquisa Biomédica , Encéfalo/metabolismo , Geriatria , Mediadores da Inflamação/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Envelhecimento/patologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Estudos de Casos e Controles , Europa (Continente) , Feminino , Genômica , Humanos , Masculino , Metabolômica , Atividade Motora , Degeneração Neural , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Projetos de Pesquisa , Transdução de Sinais , Estudos em Gêmeos como Assunto
15.
Nature ; 589(7840): 131-136, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33239787

RESUMO

The liver connects the intestinal portal vasculature with the general circulation, using a diverse array of immune cells to protect from pathogens that translocate from the gut1. In liver lobules, blood flows from portal triads that are situated in periportal lobular regions to the central vein via a polarized sinusoidal network. Despite this asymmetry, resident immune cells in the liver are considered to be broadly dispersed across the lobule. This differs from lymphoid organs, in which immune cells adopt spatially biased positions to promote effective host defence2,3. Here we used quantitative multiplex imaging, genetic perturbations, transcriptomics, infection-based assays and mathematical modelling to reassess the relationship between the localization of immune cells in the liver and host protection. We found that myeloid and lymphoid resident immune cells concentrate around periportal regions. This asymmetric localization was not developmentally controlled, but resulted from sustained MYD88-dependent signalling induced by commensal bacteria in liver sinusoidal endothelial cells, which in turn regulated the composition of the pericellular matrix involved in the formation of chemokine gradients. In vivo experiments and modelling showed that this immune spatial polarization was more efficient than a uniform distribution in protecting against systemic bacterial dissemination. Together, these data reveal that liver sinusoidal endothelial cells sense the microbiome, actively orchestrating the localization of immune cells, to optimize host defence.


Assuntos
Microbioma Gastrointestinal/imunologia , Fígado/imunologia , Fígado/microbiologia , Simbiose/imunologia , Animais , Bactérias/imunologia , Bactérias/isolamento & purificação , Separação Celular , Quimiocina CXCL9/imunologia , Células Endoteliais/citologia , Células Endoteliais/imunologia , Feminino , Humanos , Macrófagos do Fígado/citologia , Macrófagos do Fígado/imunologia , Macrófagos do Fígado/metabolismo , Fígado/irrigação sanguínea , Fígado/citologia , Linfócitos/imunologia , Masculino , Camundongos , Modelos Imunológicos , Imagem Molecular , Células Mieloides/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Simbiose/genética , Transcriptoma
16.
Microorganisms ; 8(12)2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33255715

RESUMO

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

17.
Curr Opin Immunol ; 65: 102-106, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-33289646

RESUMO

Life expectancy has grown tremendously. This incredible achievement for mankind has been obtained mostly thanks to three pillars: hygiene, antibiotics and vaccines. They represent one of the most effective forms of medical intervention. From Jenner's work to new vaccines, immunization has reduced the consequences of infectious diseases. In the last years antimicrobial resistance (AMR) as well as emerging infectious diseases have been rated as major threats for our society, as their toll is forecasted to drastically impinge on human health and economies. Indeed, recently, the whole world has experienced such problems because of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of Covid-19. Herein, we propose an excursus through the three main pillars (hygiene, antibiotics and vaccination) that contributed to improving life expectancy, their clinical and economic impact and the role of vaccines to fight AMR-related diseases and emerging infectious diseases like Covid-19.


Assuntos
Vacinas contra COVID-19 , COVID-19/prevenção & controle , Doenças Transmissíveis Emergentes/prevenção & controle , Resistência Microbiana a Medicamentos , Vacinas , Antibacterianos/uso terapêutico , COVID-19/epidemiologia , COVID-19/imunologia , Humanos , Higiene , Pandemias/prevenção & controle , SARS-CoV-2/imunologia , Vacinação
18.
Rev Fac Cien Med Univ Nac Cordoba ; 77(4): 301-306, 2020 12 09.
Artigo em Espanhol | MEDLINE | ID: mdl-33351372

RESUMO

Introduction: Acromegaly prevalence is 35-70 / million. Transsphenoidal surgery is the first-line treatment, with a remission rate of 80% for microadenomas and 50% for macroadenomas. Our aim was to evaluate the surgical results in Córdoba and determine predictive remission factors due to the lack of records. Methods: Retrospective-descriptive study of patients with surgery as the first therapeutic line. Remission criteria: IGF1 normalization for age/sex, with GH ≤1.0 g/L. Test X2 and Fisher's exact test with p<0.05. Results: 38 patients were included: 61% women and 39% men; Average age 45 years. Most frequent chief complaint: headache and acral growth (26%), visual disturbances (20%). Macroadenomas were the 84% of the tumors. Of 37 patients, 54% underwent microscopic surgery, 38% endoscopic and 8% transcranial. The 29% of patients showed post-operative complications and diabetes insipidus was the most frequent (10%). The percentage of them was: 33% transcranial surgery, 29% endoscopic and 25% microscopic (p = 0.557). The biochemical remission at 6 months was 34% and at 12 months 55% (p= 0.0001). No significant differences between the endoscopic and microscopic approach (p = 0.071). Of 36 patients, 31% showed complete tumor resection. The subjective clinical improvement was 88%. There weren´t predictive remission factors with significant differences. Conclusion: The surgical biochemical remission was similar to the bibliography. We didn´t find predictive remission factors but a larger number of patients could modify these results.


Assuntos
Acromegalia , Acromegalia/cirurgia , Adenoma/cirurgia , Feminino , Adenoma Hipofisário Secretor de Hormônio do Crescimento , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/cirurgia , Estudos Retrospectivos , Osso Esfenoide , Resultado do Tratamento
19.
Front Microbiol ; 11: 586285, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193236

RESUMO

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

20.
Front Microbiol ; 11: 575377, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33250869

RESUMO

Metagenomics-based high-throughput sequencing (HTS) enables comprehensive detection of all species comprised in a sample with a single assay and is becoming a standard method for outbreak investigation. However, unlike real-time PCR or serological assays, HTS datasets generated for pathogen detection do not easily provide yes/no answers. Rather, results of the taxonomic read assignment need to be assessed by trained personnel to gain information thereof. Proficiency tests are important instruments of validation, harmonization, and standardization. Within the European Union funded project COMPARE [COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe], we conducted a proficiency test to scrutinize the ability to assess diagnostic metagenomics data. An artificial dataset resembling shotgun sequencing of RNA from a sample of contaminated trout was provided to 12 participants with the request to provide a table with per-read taxonomic assignments at species level and a report with a summary and assessment of their findings, considering different categories like pathogen, background, or contaminations. Analysis of the read assignment tables showed that the software used reliably classified the reads taxonomically overall. However, usage of incomplete reference databases or inappropriate data pre-processing caused difficulties. From the combination of the participants' reports with their read assignments, we conclude that, although most species were detected, a number of important taxa were not or not correctly categorized. This implies that knowledge of and awareness for potentially dangerous species and contaminations need to be improved, hence, capacity building for the interpretation of diagnostic metagenomics datasets is necessary.

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