Evidências de seleção diversificante mediada por células T em genes de Mycobacterium tuberculosis expressos in vivo

Dissertação de Mestrado em Ciências da SaúdeA tuberculose, uma doença devastadora causada por bactérias do complexo Mycobacterium tuberculosis (MTBC), mantém-se vastamente fora de controlo. Embora a imunidade mediada por células T positivas para cluster de diferenciação 4 (T CD4+) seja crítica para o controlo da infeção, não existe um conhecimento profundo sobre as características das respostas protetoras mediadas por células T bem como dos fatores que têm impacto no seu estabelecimento. Neste contexto, a diversidade genética do MTBC tem sido subestimada, visto que a maioria dos antigénios conhecidos de M. tuberculosis são hiperconservados. A existência de epítopos de células T variáveis foi investigada através da análise da diversidade, evolução e imunogenicidade de uma ilha genómica expressa in vivo (iVEGI) em 270 genomas do MTBC. A iVEGI apresentou um nível de diversidade nucleotídica acima da média do genoma, devido à presença de 21 genes altamente diversos. A análise computacional de evolução molecular previu 11 codões de nove genes como estando sob seleção diversificante. De particular interesse foram três substituições nos genes accD2, Rv0987 e Rv0988, que alteraram significativamente o número de péptidos codificados com elevada afinidade de ligação prevista para diferentes moléculas de antigénio leucocitário humano (HLA) de classe II. Ensaios in vitro realizados com o HLA DRB1*01:01 corroboraram a existência de péptidos com elevada afinidade de ligação e revelaram diferenças de mais de 58% na afinidade de ligação entre péptidos wild-type e mutante que sobrepõem AccD2 R233L e Rv0987 V169L. Notavelmente, o péptido AccD2228-241 demonstrou níveis de afinidade e estabilidade tão elevados como o controlo positivo ESAT-63-17, sendo assim um forte candidato para um epítopo de célula T CD4+ restrito a uma linhagem de M. tuberculosis. Durante a longa coevolução entre M. tuberculosis e populações humanas, a diversidade genética em epítopos específicos de células T pode ter sido selecionada de modo a induzir respostas imunes vantajosas para o agente patogénico. Revelar a trajetória evolutiva de M. tuberculosis em resposta à pressão imunológica pode oferecer ferramentas sem precedentes para explorar os seus alvos moleculares em favor do hospedeiro, nomeadamente para o desenvolvimento de vacinas mais eficientes.Tuberculosis, a devastating disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC), remains vastly uncontrolled. Although cluster of differentiation 4 positive (CD4+) T cell-mediated immunity is critical for infection control, a deep knowledge on the characteristics of the protective T cell responses and on the factors impacting their establishment is lacking. In this context, the MTBC genetic diversity has been underappreciated, as most of the known M. tuberculosis antigens are hyperconserved. The existence of varying T cell epitopes was investigated by analysing the diversity, evolution and immunogenicity of an in vivo-expressed genomic island across 270 MTBC genomes. The in vivo-expressed genomic island displayed a level of nucleotide diversity above the whole-genome average due to the presence of 21 highly diverse genes. Computational molecular evolution analysis predicted 11 codons from nine genes to be under diversifying selection. Of particular interest were three substitutions in the genes accD2, Rv0987 and Rv0988, which significantly altered the number of encoded peptides with predicted high binding affinity to class II human leukocyte antigen (HLA) molecules. In vitro assays performed with HLA DRB1*01:01 corroborated the existence of high binding affinity peptides and revealed differences of more than 58% in the binding affinity between wild-type and variant peptides overlapping AccD2 R233L and Rv0987 V169L. Notably, the AccD2228-241 peptide showed affinity and stability levels as high as the positive control ESAT-63-17, thus being a strong candidate for an M. tuberculosis lineage-restricted CD4+ T cell epitope. During the long co-evolution of M. tuberculosis and human populations, genetic diversity in specific T cell epitopes might have been selected to induce immune responses advantageous for the pathogen. Unveiling the evolutionary path of M. tuberculosis in response to immune pressure might offer unprecedented tools to exploit its molecular targets in favour of the host, namely for the development of more efficient vaccines

Evolutionary genetics of Mycobacterium tuberculosis and HIV-1: “The Tortoise and the Hare”

The already enormous burden caused by Mycobacterium tuberculosis and Human Immunodeficiency Virus type 1 (HIV-1) alone is aggravated by co-infection. Despite obvious differences in the rate of evolution comparing these two human pathogens, genetic diversity plays an important role in the success of both. The extreme evolutionary dynamics of HIV-1 is in the basis of a robust capacity to evade immune responses, to generate drug-resistance and to diversify the populationlevel reservoir of M group viral subtypes. Compared to HIV-1 and other retroviruses, M. tuberculosis generates minute levels of genetic diversity within the host. However, emerging whole-genome sequencing data show that the M. tuberculosis complex contains at least nine human-adapted phylogenetic lineages. This level of genetic diversity results in differences in M. tuberculosis interactions with the host immune system, virulence and drug resistance propensity. In co-infected individuals, HIV-1 and M. tuberculosis are likely to co-colonize host cells. However, the evolutionary impact of the interaction between the host, the slowly evolving M. tuberculosis bacteria and the HIV-1 viral “mutant cloud” is poorly understood. These evolutionary dynamics, at the cellular niche of monocytes/macrophages, are also discussed and proposed as a relevant future research topic in the context of single-cell sequencing.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020; by the projects NORTE-01- 0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by Gilead Génese PGG/009/2017. ASP, CM, and PMMA were funded by FCT PhD scholarships PD/BD/127827/2016, SFRH/BD/132797/2017 and PDE/BDE/113599/2015, respectively

Mycobacterium tuberculosis Complex Exhibits Lineage-Specific Variations Affecting Protein Ductility and Epitope Recognition

The advent of whole-genome sequencing has provided an unprecedented detail about the evolution and genetic significance of species-specific variations across the whole Mycobacterium tuberculosis Complex. However, little attention has been focused on understanding the functional roles of these variations in the protein coding sequences. In this work, we compare the coding sequences from 74 sequenced mycobacterial species including M. africanum, M. bovis, M. canettii, M. caprae, M. orygis, and M. tuberculosis. Results show that albeit protein variations affect all functional classes, those proteins involved in lipid and intermediary metabolism and respiration have accumulated mutations during evolution. To understand the impact of these mutations on protein functionality, we explored their implications on protein ductility/disorder, a yet unexplored feature of mycobacterial pro-teomes. In agreement with previous studies, we found that a Gly71Ile substitution in the PhoPR virulence system severely affects the ductility of its nearby region in M. africanum and animal-adapted species. In the same line of evidence, the SmtB transcriptional regulator shows amino acid variations specific to the Beijing lineage, which affects the flexibility of the N-terminal trans-activation domain. Furthermore, despite the fact that MTBC epitopes are evolutionary hyperconserved, we identify strain-and lineag-especific amino acid mutations affecting previously known T-cell epitopes such as EsxH and FbpA (Ag85A). Interestingly, in silico studies reveal that these variations result in differential interaction of epitopes with the main HLA haplogroups.Gobierno de Aragón (DGA-GC B18 and B25), the Spanish Ministry of Science and Competitiveness (BIO2014-52580P, CSIC13-4E-2490), Instituto de Salud Carlos III (PI12/01970) and the European Commission Horizon 2020 (H2020-PHC-643381). Some of these grants were partially financed by the EU FEDER Program. This work was also supported by Fundação para a Ciência e Tecnologia, Portugal (IF/00474/2014) and cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER)info:eu-repo/semantics/publishedVersio

Photodeposition of silver on Zinc/Calcium ferrite nanoparticles: A contribution to efficient effluent remediation and catalyst reutilization

The efficient photodegradation of textile dyes is still a challenge, especially considering resistant azo dyes. In this work, zinc/calcium mixed ferrite nanoparticles prepared by the sol-gel method were coupled with silver by a photodeposition method to enhance the photocatalytic potency. The obtained zinc/calcium ferrites are mainly cubic-shaped nanoparticles sized 15 ± 2 nm determined from TEM and XRD and an optical bandgap of 1.6 eV. Magnetic measurements indicate a superparamagnetic behavior with saturation magnetizations of 44.22 emu/g and 27.97 emu/g, respectively, for Zn/Ca ferrite and Zn/Ca ferrite with photodeposited silver. The zinc/calcium ferrite nanoparticles with photodeposited silver showed efficient photodegradation of the textile azo dyes C.I. Reactive Blue 250 and C.I. Reactive Yellow 145. Subsequent cycles of the use of the photocatalyst indicate the possibility of magnetic recovery and reutilization without a significant loss of efficiency.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of CF-UM-UP (UIDB/04650/2020) and research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020) funded by FCT, FEDER, PORTUGAL2020, and COMPETE2020

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran\u27s eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R$^{2}$ = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R$^{2}$ = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Aim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types