Obligate intracellular bacteria: Evasion and adaptative tactics shaping the host-pathogen interface

Funding Information: Work in IS laboratory was funded by the European Regional Development Fund (ERDF), through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, under project[s] POCI-01-0145-FEDER-029592 (PTDC/SAU-INF/29592/2017) (UNDOHIJACK), UIDB/04539/2020, and UIDP/04539/2020 (CIBB). Work in LJM laboratory has been supported by FCT through grants PTDC/BIA-MIC/28503/2017 (to LJM), UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO), and LA/P/0140/2020 (i4HB).publishersversionpublishe

Estudo das diferentes estratégias empregues numa tarefa com ou sem privação de visão

Inexistent

Macrophages Infected by a Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Reveal Differential Reprogramming Signatures Early in Infection

Despite their high degree of genomic similarity, different spotted fever group (SFG) Rickettsia are often associated with very different clinical presentations. For example, Rickettsia conorii causes Mediterranean spotted fever, a life-threatening disease for humans, whereas Rickettsia montanensis is associated with limited or no pathogenicity to humans. However, the molecular basis responsible for the different pathogenicity attributes are still not understood. Although killing microbes is a critical function of macrophages, the ability to survive and/or proliferate within phagocytic cells seems to be a phenotypic feature of several intracellular pathogens. We have previously shown that R. conorii and R. montanensis exhibit different intracellular fates within macrophage-like cells. By evaluating early macrophage responses upon insult with each of these rickettsial species, herein we demonstrate that infection with R. conorii results in a profound reprogramming of host gene expression profiles. Transcriptional programs generated upon infection with this pathogenic bacteria point toward a sophisticated ability to evade innate immune signals, by modulating the expression of several anti-inflammatory molecules. Moreover, R. conorii induce the expression of several pro-survival genes, which may result in the ability to prolong host cell survival, thus protecting its replicative niche. Remarkably, R. conorii-infection promoted a robust modulation of different transcription factors, suggesting that an early manipulation of the host gene expression machinery may be key to R. conorii proliferation in THP-1 macrophages. This work provides new insights into the early molecular processes hijacked by a pathogenic SFG Rickettsia to establish a replicative niche in macrophages, opening several avenues of research in host-rickettsiae interactions

Molecular analysis of the interaction between cardosin A and phospholipase Dα

Cardosin A is an RGD-containing aspartic proteinase from the stigmatic papillae of Cynara cardunculus L. A putative cardosin A-binding protein has previously been isolated from pollen suggesting its potential involvement in pollen2013pistil interaction [Faro C, Ramalho-Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J & Pires E (1999) J Biol Chem274, 28724201328729]. Here we report the identification of phospholipase D03B1 as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase D03B1 constructs were performed. Results revealed that the C2 domain of phospholipase D03B1 contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase D03B1 can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains

A Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Trigger Differential Proteome Signatures in Macrophages

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid β-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage

SEQUELAS DA COVID-19: IMPORTÂNCIA DA CONSULTA MULTIDISCIPLINAR NOS CUIDADOS DE SAÚDE PRIMÁRIOS

Objetivo: Identificar quais as sequelas da doença COVID-19 predominantes, para implementação de uma consulta multidisciplinar, nos Cuidados de Saúde Primários, para acompanhamento dos utentes que apresentam sequelas da doença COVID-19. Métodos: Trata-se de um estudo descritivo, assente na metodologia do planeamento em saúde. Dos 145 utentes residentes na área geográfica de abrangência da Unidade de Cuidados na Comunidade, que estiveram infetados pelo vírus SARS-CoV-2, nos meses de janeiro, fevereiro e março de 2021, foi selecionada uma amostra por conveniência de 49 utentes. A estes, foi feita telefonicamente uma entrevista, suportada numa checklist validada por profissionais de saúde, experts nesta temática. Após a recolha da informação, foi feita a análise descritiva, com recurso ao SPSS (Statistical Package for the Social Sciences) versão 27 para Windows. Resultados: Da amostra de 49 indivíduos, 56,3% são do género masculino e 43,8% do género feminino. A média de idades é de 45,2 anos, variando entre um mínimo de 19 e um máximo de 75 anos. Quanto à situação profissional, 77,1% encontram-se empregados, 10,4% desempregados, 8,3% estão reformados e 4,2% são estudantes. Relativamente à presença de sequelas da doença COVID-19, destes 49 utentes, 28 referiram ter tido sequelas, sendo a sequela mais referida o cansaço (47,9%) e as menos frequentes as cefaleias, ansiedade/depressão, entre outras, num valor percentual de 2,1%. Dos 28 indivíduos que referiram ter tido sequelas, 53,6% mencionaram que as mesmas alteraram a sua qualidade de vida. Conclusões: Atendendo a que cerca de metade dos indivíduos que estiveram infetados mencionaram ter tido sequelas da doença, e destes, também cerca de metade referiram comprometimento da sua qualidade de vida, parece essencial a implementação de uma consulta multidisciplinar, nos Cuidados de Saúde Primários, que permita atuar sobre as sequelas de forma eficaz, contribuindo para a recuperação da sua saúde e qualidade de vida

Molecular cloning and characterization of procirsin, an active aspartic protease precursor from <i>Cirsium vulgare</i> (Asteraceae)

Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are wellknown milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards j-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.Centro de Investigación de Proteínas Vegetale

PLD[alfa] is a cardosin A binding protein : identification and characterization of the interaction reveals KGE as a novel protein recognition sequence

Tese de doutoramento em Bioquímica (Enzimologia) apresentada à Fac. de Ciências e Tecnologia de Coimbr

Pragmática da língua literária em Fernando Pessoa : ler e analisar Fernando Pessoa em tempos de pragmática da literatura e da linguagem

Dissertação de mestrado em Literatura de Língua Portuguesa : Investigação e Ensino (2º ciclo), apresentada à Faculdade de Letras da Universidade de CoimbraA leitura e análise da poesia ortónima de Fernando Pessoa no âmbito de uma perspectivação pragmática da língua literária, enquanto objecto do nosso trabalho, articulam campos vários que se complementam entre si e influenciam o entendimento da polissemia do seu texto poético plural. A riqueza semântica e estética reconhecida no estudo da poesia ortónima pessoana decorre dos elementos linguísticos e translinguísticos que a sustentam e dos efeitos que, numa visão pragmática do fenómeno literário, a arte verbal da sua construção semiótica provoca e/ou evoca no leitor e no investigador (enquanto instâncias co-construtivas das múltiplas concretizações literárias do seu texto, num modelo bi-activo da leitura, derivado das concepções de Ingarden, da Teoria da Recepção e da Crítica da Resposta do Leitor). Socorrendo-nos do inúmero material já existente sobre a obra e a complexa personalidade de Fernando Pessoa, reexplorámos de forma contextual a teia de dados amplamente conhecidos do seu percurso existencial, na tentativa de compreender e criar novos sentidos na análise do seu corpus textual (fundamentos e pragmática dos seus biografemas). O estudo da poesia ortónima pessoana não pode fazer-se prescindindo da individualização (croceanamente intuitiva, artística) da língua literária no ideolecto do poeta criador, capaz de mascarar o seu mundo subjectivo numa aventura estética pelo seu elevado grau de intelectualidade (imaginação criativa sobre o signo linguístico como expressão dum não-eu). De forma muito peculiar, a confissão do poeta sobre a fragmentação da personalidade em múltiplos “eus” está directamente relacionada com a sua teoria do fingimento poético, evocando uma maneira muito própria de estar na poesia e para ela, enquanto realidade mentalmente construída e singular projecção de escrita intelectualizada, um fenómeno interseccionista a nível psicológico e discursivo. A poesia pessoana enquanto ficção lírica simula o poeta, a vida, o homem, suscitando de forma excêntrica e dramática um ser permanentemente em crise existencial pela vocação persistente de pensar e sonhar, incapaz de se integrar plenamente na realidade do mundo empírico e circunstancial. As palavras na linguagem literária de Fernando Pessoa evocam imagens de um ser torturado num complexo rigor de expressão, pela análise lúcida e irónica das coisas que representam. Poesia do real e do sonho, da consciência e da inconsciência, do sentir e do pensar, do absoluto e do relativo, enquanto arte verbal fluída do conhecimento estético da língua, constrói-se na atitude ambígua (plena e vazia) de Pessoa na sua relação existencial com a realidade interior e exterior, consequente da busca inquietante de respostas ( sempre bloquedas ou minadas) ao mistério da vida e suas implicações

Moonlighting in Rickettsiales: Expanding Virulence Landscape

The order Rickettsiales includes species that cause a range of human diseases such as human granulocytic anaplasmosis (Anaplasma phagocytophilum), human monocytic ehrlichiosis (Ehrlichia chaffeensis), scrub typhus (Orientia tsutsugamushi), epidemic typhus (Rickettsia prowazekii), murine typhus (R. typhi), Mediterranean spotted fever (R. conorii), or Rocky Mountain spotted fever (R. rickettsii). These diseases are gaining a new momentum given their resurgence patterns and geographical expansion due to the overall rise in temperature and other human-induced pressure, thereby remaining a major public health concern. As obligate intracellular bacteria, Rickettsiales are characterized by their small genome sizes due to reductive evolution. Many pathogens employ moonlighting/multitasking proteins as virulence factors to interfere with multiple cellular processes, in different compartments, at different times during infection, augmenting their virulence. The utilization of this multitasking phenomenon by Rickettsiales as a strategy to maximize the use of their reduced protein repertoire is an emerging theme. Here, we provide an overview of the role of various moonlighting proteins in the pathogenicity of these species. Despite the challenges that lie ahead to determine the multiple potential faces of every single protein in Rickettsiales, the available examples anticipate this multifunctionality as an essential and intrinsic feature of these obligates and should be integrated into available moonlighting repositories