Functional and genetic analysis of sexual reproduction in the thermodimorphic fungal pathogen Paracoccidioides spp

Tese de Doutoramento em Ciências da Vida e SaúdeParacoccidioides brasiliensis e Paracoccidioides lutzii são fungos multinucleares e termo dimórficos, sendo também os agentes etiológicos da Paracoccidioidomicose (PCM), uma micose que é endémica da América do Sul. Estima-se que a PCM afeta 10 milhões de pessoas em países da América Latina, sendo que o Brasil apresenta cerca de 80% dos casos relatados. Este fungo sofre uma transição morfológica da sua forma nãopatogénica, micelar e conídeos, existentes em temperaturas ambientes, para a forma de levedura que é patogénica na temperatura corporal dos mamíferos. Paracoccidioides apresenta um ciclo de vida assexuado. Entretanto, estudos recentes apoiam a existência de ciclo sexual em Paracoccidioides, pela: i) a existência no seu genoma de praticamente todos os genes homólogos necessários para a reprodução sexual em Ascomicetos, incluindo recetores de feromonas, membros da via de sinalização para reprodução sexuada e isoformas dos loci MAT1-1/MAT1-2, e ii) identificação de eventos de recombinação genética entre isolados do meio ambiente. Apesar destas evidências, a reprodução sexuada no género Paracoccidioides nunca foi observada na natureza ou em condições laboratoriais; contrariamente ao observado em espécies de géneros filogeneticamente próximos como Histoplasma e Aspergillus. Nesta tese, estudámos o processo de reprodução sexuada em Paracoccidioides sob diversos aspetos do processo de reprodução sexuada em Ascomicetos. Os nossos resultados mostram a baixa expressão dos genes que expressam os recetores das feromonas a e α (PREA e PREB, receptivamente), o gene da feromona α (PBα) e os genes MAT, nas formas de levedura e micélio em vários isolados de Paracoccidioides. O ensaio de indução realizado com a feromona sintética Pbα nas estirpes MAT1-2 de Paracoccidioides tinha como objetivo avaliar a resposta transcricional dos genes relacionados com a reprodução sexuada, entretanto a presença de Pbα falhou e não serviu como estímulo para a resposta da ativação da transcrição dos genes MAT1-2, PREB e STE12, sugerindo que as estirpes testadas são insensíveis à Pbα. As funcionalidades dos genes relacionados com a reprodução sexuada em Paracoccidioides foram analisadas através da expressão heteróloga desses genes em estipes mutantes de Saccharomyces cerevisiae que não possuíam os genes homólogos correspondentes. Nós demonstramos que as estirpes de S. cerevisiae que expressavam de forma heteróloga PREB respondiam à Pbα sintética, tanto como à Pbα extraída do sobrenadante de culturas de Paracoccidioides através da formação de shmoos e pela paragem de crescimento e ciclo celular. Estes resultados nos levaram a concluir que as estirpes de Paracoccidioides secretam a feromona alfa ativa no meio de cultura, que é capaz de ativar seu recetor cognitivo. Em A. nidulans e A. fumigatus, um grupo específico de proteínas reguladoras que respondem à presença da luz, denominadas proteínas Velvet, apresentam um papel crucial no ciclo sexual na ausência de luz. Através de análises in silico, nós demonstramos que Paracoccidioides tem proteínas que apresentam os domínios Velvet, demonstrando elevada homologia com os domínios das proteínas de Aspergillus. Além disso, o regulador negativo GprD e os reguladores positivos NsdD e LaeA da reprodução sexuada apresentam funções importantes durante o desenvolvimento sexual. Homólogos destes componentes também estão presentes no género Paracoccidioides. Neste estudo, nós analisamos os padrões de expressão génica destes genes na presença e ausência de luz em diferentes estirpes de Paracoccidioides. Nós concluímos que a expressão do MAT locus, dos genes Velvet, e dos reguladores LAEA, GPRD e NSD, ocorrem independente da presença de luz. Neste trabalho, nós também realizamos ensaios de reprodução sexuada com isolados de Paracoccidioides. Nós delineamos nossos ensaios, através das informações previamente publicadas relacionadas com os ensaios de reprodução sexuada em Aspergillus e Paracoccidioides, também usamos estirpes MAT1-1 e MAT1-2 de Paracoccidioides que expressam genes responsáveis pela resistência à dois antibióticos diferentes. Após oito meses de incubação em diferentes meios e no escuro, analisamos as células recolhidas da intersecção das duas estirpes de MAT locus opostos, co-cultivadas que eram resistentes à ambos antibióticos. Através de análise microscópica, encontramos estruturas semelhantes à esporulação, ondulação de hifas, típicas em estágios iniciais da reprodução sexuada, e ascomas revestidos por células Hülle. As morfologias observadas nessas estruturas são comuns entre Ascomicetos durante o desenvolvimento sexual. O cultivo de ascósporos das estruturas semelhantes à ascomata para posterior análise não foi possível ser realizado. Também efetuamos análise por PCR das células que se desenvolveram em meio seletivo com ambos antibióticos de forma a confirmar a recombinação genética resultante da reprodução sexuada, que seria demonstrada pela presença de genes parentais e dos genes responsáveis pela resistência aos antibióticos, derivados das estirpes parentais. Contudo, os resultados dessa análise não foram conclusivos. De forma geral, nossos resultados fornecem novos conhecimentos relativos à existência de um sistema de reprodução sexuada em Paracoccidioides, através da demonstração da ativação da via de sinalização da reprodução sexuada pelo reconhecimento de feromona-recetor, no sistema heterólogo. Também identificamos as proteínas Velvet e a homologia de seus domínios, assim como demonstramos que a expressão de tais genes é independente da luz. Finalmente, em nossos ensaios de reprodução sexuada fomos capazes de obter em Paracoccidioides estruturas comuns ao início da reprodução sexuada. Estes resultados apresentam novas indicações para: i) a identificação de experimentos que possuam as condições ideais para a reprodução sexuada de Paracoccidioides em condições laboratoriais; ii) descobrir os mecanismos moleculares e os componentes envolvidos neste processo.Paracoccidioides brasiliensis and Paracoccidioides lutzii are multinuclear thermodimorphic fungi and the etiological agents of paracoccidioidomycosis (PCM), a prevalent mycosis in South America. PCM is estimated to affect 10 million individuals in countries from Latin America, and Brazil present 80% of the diagnosed disease. The fungus undergoes a morphological transition from a non-pathogenic mycelial or conidial form at environmental temperatures to a multiple-budding pathogenic yeast form at the mammalian host temperature. Paracoccidioides is known to present an asexual cycle, but sexual reproduction in the Paracoccidioides genus was never observed, neither in nature nor under laboratory conditions. However, recent studies have revealed indications that support the existence of a sexual cycle in Paracoccidioides isolates. The first indication is that this fungus harbors homologues of most of the genes necessary for sexual reproduction in Ascomycetes, including pheromone receptors, mating signaling pathway components and MAT1-1/MAT1-2 idiomorphs. A second indication came from the discovery of recombination events in Paracoccidioides strains isolated from nature. This information supports the idea of the occurrence of sexual reproduction in the Paracoccidioides genus. Despite the presence of mating loci and all essential mating-pheromone signaling pathway genes and the discovery of such a mode of reproduction in the closely-related phylogenetic genera Histoplasma and Aspergillus, sexual reproduction in Paracoccidioides was never uncovered. In this thesis, we studied several aspects that are relevant for sexual reproduction in Ascomycetes. We present data showing low expression levels of the a- and α-pheromone receptor genes (PREA and PREB), the α-pheromone gene (PBα) and the MAT genes, in yeast and mycelia forms of Paracoccidioides isolates. Induction assays with synthetic α-pheromone were performed in P. brasiliensis MAT1-2 strains to assess the transcriptional response of mating-related genes, however Pbα failed to elicit transcriptional activation of MAT1-2, PREB and STE12, suggesting that the strains tested are insensitive to Pbα. Functionality of Paracoccidioides mating genes was further assessed by their heterologous expression in the corresponding Saccharomyces cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pbα either isolated from Paracoccidioides culture supernatants or in its synthetic form, by shmoo formation and by growth and cell cycle arrests. This led us to conclude that Paracoccidioides species secrete an active α-pheromone into the culture medium that is capable to activate its cognate receptor. In A. nidulans and A. fumigatus, a particular group of light-responsive regulatory proteins, named Velvet proteins, play a crucial role in the sexual cycle in the absence of light. In addition, the negative regulator GprD and positive regulator NsdD of sexual reproduction play an important role during the sexual development. Homologues of these components are also present in the Paracoccidioides genus. In this study, we therefore analyzed the gene expression patterns of these genes in the presence and absence of light in different Paracoccidioides strains. Through in silico analysis, we show that Paracoccidioides Velvet proteins harbor the Velvet domains present in the homologous proteins from Aspergillus. We conclude that expression of the mating locus (MAT), the Velvet genes, and regulators LAEA, GPRD and NSDD, occurs in a light-independent manner. Based on the presence and expression of these genes in Paracoccidioides isolates and their protein interactions as described in A. nidulans, a protein interaction model was also proposed for Paracoccidioides, that may be uncovered by further functional genomics and protein analysis. In this work, we also performed mating assays with Paracoccidioides isolates. We designed our mating assays taking advantage of previous information regarding mating assays in Aspergillus and Paracoccidioides, and used MAT1-1 and MAT1-2 Paracoccidioides strains that were marked with two different antibiotic resistance genes. After eight months of incubation on different media in the dark, we analyzed cells from the intersection of the two co-cultivated opposite mating types that were resistant to both antibiotics. By microscopic analysis we found structures that are similar to sporulation, the hyphae loops classical in early stages of sexual reproduction, and the ascoma structures surrounded by Hülle cells. The morphology observed in these structures is common among ascomycetes during sexual reproduction development. Cultivation of ascospores from the ascomata-like structures for further analysis however could not be achieved. We also performed PCR analysis on cells that had developed on both selective antibiotics in order to confirm genetic recombination resulting from sexual reproduction, as would be indicated by the presence of parental and antibiotic genes derived from both mating strains. However, the result of this analysis was not conclusive. Overall, our results provide new insights into the existence of a functional mating system in Paracoccidioides, by demonstrating signal activation by the mating α-pheromone-receptor system in a yeast model. Moreover, we identified Velvet proteins motifs in Paracoccidioides and showed that their gene expression is independent of light. Finally, we also achieved the development of early-stage sexual structures in Paracoccidioides mating assays. These data therefore provide novel directions for the experimental identification of the ideal conditions for Paracoccidioides sexual reproduction in laboratory and consequently uncover the molecular mechanisms and the components involved in this process

Functionality of the paracoccidioides mating α-pheromone-receptor system

Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the a-pheromone (PBa) gene, and the a- and apheromone receptor (PREB and PREA) genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic a pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to a-pheromone. In order to further evaluate the biological functionality of the pair a-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pba pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active a-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBa in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides a-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.MHJS and JFM were supported by Fundacão para a Ciência e Tecnologia (FCT) grants. This work was supported by a grant from FCT (PTDC/BIA-MIC/ 108309/2008)

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (

Age-Related Metabolic Pathways Changes in Dental Follicles: A Pilot Study

Aging is not a matter of choice; it is our fate. The “time-dependent functional decline that affects most living organisms” is coupled with several alterations in cellular processes, such as cell senescence, epigenetic alterations, genomic instability, stem cell exhaustion, among others. Age-related morphological changes in dental follicles have been investigated for decades, mainly motivated by the fact that cysts and tumors may arise in association with unerupted and/or impacted teeth. The more we understand the physiology of dental follicles, the more we are able to contextualize biological events that can be associated with the occurrence of odontogenic lesions, whose incidence increases with age. Thus, our objective was to assess age-related changes in metabolic pathways of dental follicles associated with unerupted/impacted mandibular third molars from young and adult individuals. For this purpose, a convenience sample of formalin-fixed paraffin-embedded (FFPE) dental follicles from young (<16 y.o., n = 13) and adult (>26 y.o., n = 7) individuals was selected. Samples were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based untargeted metabolomics. Multivariate and univariate analyses were conducted, and the prediction of altered pathways was performed by mummichog and Gene Set Enrichment Analysis (GSEA) approaches. Dental follicles from young and older individuals showed differences in pathways related to C21-steroid hormone biosynthesis, bile acid biosynthesis, galactose metabolism, androgen and estrogen biosynthesis, starch and sucrose metabolism, and lipoate metabolism. We conclude that metabolic pathways differences related to aging were observed between dental follicles from young and adult individuals. Our findings support that similar to other human tissues, dental follicles associated with unerupted tooth show alterations at a metabolic level with aging, which can pave the way for further studies on oral pathology, oral biology, and physiology

P. brasiliensis virulence is affected by SconC, the negative regulator of inorganic sulfur assimilation

Conidia/mycelium-to-yeast transition of Paracoccidioidesbrasiliensis is a critical step for the establishment of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Thus, knowledge of the factors that mediate this transition is of major importance for the design of intervention strategies. So far, the only known pre-requisites for the accomplishment of the morphological transition are the temperature shift to 37°C and the availability of organic sulfur compounds. In this study, we investigated the auxotrophic nature to organic sulfur of the yeast phase of Paracoccidioides, with special attention to P. brasiliensis species. For this, we addressed the role of SconCp, the negative regulator of the inorganic sulfur assimilation pathway, in the dimorphism and virulence of this pathogen. We show that down-regulation of SCONC allows initial steps of mycelium-to-yeast transition in the absence of organic sulfur compounds, contrarily to the wild-type fungus that cannot undergo mycelium-to-yeast transition under such conditions. However, SCONC down-regulated transformants were unable to sustain yeast growth using inorganic sulfur compounds only. Moreover, pulses with inorganic sulfur in SCONC down-regulated transformants triggered an increase of the inorganic sulfur metabolism, which culminated in a drastic reduction of the ATP and NADPH cellular levels and in higher oxidative stress. Importantly, the down-regulation of SCONC resulted in a decreased virulence of P. brasiliensis, as validated in an in vivo model of infection. Overall, our findings shed light on the inability of P. brasiliensis yeast to rely on inorganic sulfur compounds, correlating its metabolism with cellular energy and redox imbalances. Furthermore, the data herein presented reveal SconCp as a novel virulence determinant of P. brasiliensis.J.F.M. and J.G.R. were supported by a PhD grant from Fundacao para a Ciencia e Tecnologia (FCT). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Sturme. and M. Saraiva are Ciencia 2008 fellows. The authors would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

TLR9 activation dampens the early inflammatory response to paracoccidioides brasiliensis, Impacting host survival

Background: Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear.J.F. Menino was supported by a grant from Fundacao para a Ciencia e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Saraiva is a Ciencia 2007 fellow and M. Sturme is a Ciencia 2008 fellow. We would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Regulação e caracterização de transportes de ácidos carboxílicos em Saccharomyces cerevisiae e Candida glabrata

Dissertação de mestrado em Genética MolecularOs estudos de endocitose e tráfego intracelular de proteínas da membrana plasmática, tais como transportadores e receptores em células eucariontes são de grande importância, visto que são processos altamente regulados que possuem grande relevância na fisiologia celular. Alterações nas condições ambientais (nutrientes e substratos) podem conduzir à endocitose de transportadores indesejados. O transportador de monocarboxilatos Jen1 em Saccharomyces cerevisiae tem-se revelado um excelente modelo eucarionte para dissecar geneticamente os mecanismos que regulam o tráfego intracelular de transportadores membranares, de acordo com alterações fisiológicas. Trabalhos anteriores realizados pelo nosso grupo demonstraram que a glucose induz a endocitose de Jen1, apenas em minutos, um processo dependente quer de fosforilação por Yck1, quer de ubiquitinação a cargo da ubiquitina ligase Rsp5. Este trabalho teve como objectivo estudar os mecanismos e sinais que regulam a endocitose de Jen1 em S. cerevisiae. Verificou-se que, para além da glucose, outros açúcares como a frutose e sacarose, conduziam a uma rápida internalização e degradação do transportador. Demonstrámos que a proteína Art4, pertencente a família dos adaptadores Arrestin-related trafficking (Art), é crucial para a internalização e perda de actividade de Jen1, induzida por glucose. Adicionalmente, tentámos identificar qual a via de sinalização que regula a endocitose de Jen1 após um pulso de glucose, a células crescidas em meio contendo ácido láctico. Verificou-se que Reg1, um componente da fosfatase Glc7 é essencial para a diminuição na actividade de Jen1, ao contrário do que se verifica para o sensor de glucose Gpr1. Numa outra vertente deste trabalho investigámos a capacidade de Candida glabrata, a segunda levedura patogénica mais prevalente em humanos, crescer em meios com diferentes fontes de carbono, nomeadamente meios contendo ácidos carboxílicos. Estes estudos fisiológicos foram estendidos a duas estirpes interrompidas em genes potencialmente codificantes para transportadores de ácido acético. Os resultados obtidos mostraram que a fonte preferencial de carbono em C. glabrata é a glucose e que todas as estirpes estudadas possuem dificuldade de crescimento em meio contendo ácido acético a pH 5,0. Esta caracterização constituiu uma base essencial para trabalhos futuros nesta levedura.Studies on the endocytosis and intracellular traffic of plasma membrane proteins such as transporters and receptors in eukaryotes cells are very important since they are highly regulated processes and very relevant to the cell physiology. The monocarboxylate transporter Jen1 of Saccharomyces cerevisiae, has proven to be an excellent eukaryotic model for genetically dissecting mechanisms that regulate intracellular trafficking of plasma membrane proteins, in response to physiological constraints. Previous work carried out by our group, demonstrated that glucose induces downregulation of Jen1 within minutes, a process dependent both on phosphorylation by Yck1 and ubiquitylation, carried out by the ubiquitin ligase Rsp5. One of the main goals of this work was to study the mechanisms and signals that regulate Jen1 endocytosis in S. cerevisiae. It was demonstrated that, in addition to glucose, other sugars such as fructose and sucrose trigger a rapid internalization and degradation of the transporter. We demonstrated that the protein Art4, belonging to the family of Arrestin-related trafficking proteins (Art) is crucial for Jen1 glucose induced downregulation. Additionally, we attempted to identify the signaling pathway that regulates Jen1 endocytosis, after a pulse of glucose to cells grown in a lactic acid containing media. We have shown that Reg1, a component of the Glc7 phosphatase, is essential for Jen1 downregulation, in contrast to what is observed for the glucose sensor Gpr1. In another scope of this work, we have studied the ability of Candida glabrata, the second most prevalent pathogenic yeast in humans, to grow in media containing different carbon sources, namely in media containing carboxylic acids. These physiological studies were extended to two other strains deleted in genes potentially coding for acetic acid transporters. The results obtained show that glucose is the preferential carbon source in C. glabrata and that all the studied strains have difficulties in growing in media containing acetic acid at pH 5,0, as the sole carbon and energy source. This physiological characterization is an essential base of study for future work

Nursing infant with acquired toxoplasmosis in the first months of life – a case report

Toxoplasmosis is caused by Toxoplasma gondii and the probability of this infection occurring in the first months of life is usually low because its transmission is related to eating habits. A 6-month-old nursing infant was diagnosed with acute toxoplasmosis, which was identified through anti- T. gondii IgA, IgM and low-avidity IgG serologic assays, polymerase chain reaction (PCR) and mouse bioassay test although its mother was seronegative. This serological divergence between mother and child led us to interview the mother regarding epidemiological factors. During this interview, she reported that she had given her 2-month-old baby a piece of undercooked beef to suck on. After some time, the baby presented fever and cervical lymphadenitis. This report emphasizes the importance of serological surveys of toxoplasmosis in nursing infants presenting with fever and lymphadenitis, in view of the possible acquisition of toxoplasmosis in the first months of life