Paracoccidioides brasiliensis : factores de virulência e susceptibilidade do hospedeiro

Tese de doutoramento em Ciências da SaúdeIn the last decades, significant advances were accomplished on the study of neglected fungal diseases, mainly in defining the pathogen genetics and host risk factors. Paracoccidioidomycosis, a mycosis caused by Paracoccidioides species, is one of these diseases, and it is estimated to affect 10 million individuals in countries from Latin America. Over the last years, genomic and transcriptome studies on this fungus raised important questions, especially on the consequences that the multinucleated nature and morphological heterogeneity of Paracoccidioides brasiliensis can have on triggering host defence mechanisms. However, these aspects were never addressed. Moreover, the lack of efficient molecular tools to further explore P. brasiliensis has been hindering the identification of genetic factors that govern virulence and the polymorphic nature of the yeast pathogenic phase, as well as the mechanisms underlying its dimorphic behaviour. The highly heterogeneous yeast morphology and budding patterns are considered a hallmark of this fungus, and previous genetic studies have highlighted the relevance of the cell division cycle 42 (CDC42) expression on such phenotype. Nevertheless, the morphological trends followed by each Paracoccidioides isolate were never evaluated. Similarly, a possible association between genetic factors determining the fungus morphology and the morphological pattern of each isolate was never addressed. To address these issues, a detailed morphogenetic evaluation was carried out in the yeast-form of 11 clinical and environmental Paracoccidioides isolates from the different groups of P. brasiliensis and Paracoccidioides lutzii species. We found that each phylogenetic group does not follow any characteristic morphologic profile, whereas bud area and shape of each isolate reveals to be highly dependent on the mother cell, indicating a high level of conservation of these traits throughout cell progeny. Importantly, we also found strong correlations between PbCDC42 expression (a molecule known to control the morphological behavior of the yeast phase) and both the shape of mother and bud cells and the area of the buds. Altogether, these findings further explore the polymorphic nature of P. brasiliensis, providing information on the trends followed by P. brasiliensis. During the conidia/mycelium-to–yeast transition, a known requirement for the pathogenesis of this fungus, several morphological and phenotypical alterations occur. Among these alterations, the auxotrophy to organic sulfur compounds that is associated to the yeast phase of P. brasiliensis is particularly striking. Although this yeast-phase related nutritional requirement is shared with other dimorphic fungi, the molecular bases underlying it are yet to be uncovered. In this sense, we addressed the role of SconCp, the negative regulator of the inorganic sulfur assimilation pathway, in the fungus dimorphism and virulence. By means of genetic down-regulation of SCONC, we show that P. brasiliensis can overcome its yeast-auxotrophy to organic sulfur compounds, being able to assimilate inorganic sulfur. However, this revealed to drastically reduce the ATP and NADPH cellular levels, leading to increased oxidative cellular stress. This redox imbalance, consequence of the activation of the inorganic sulfur assimilatory pathway, probably led to the decreased virulence of the knock-down isolates, as we show using a mouse model of infection. Our data provides relevant insights on the mechanisms controlling P. brasiliensis dimorphism, revealing SconCp as a novel virulence determinant. P. brasiliensis yeast form is also characterized by its multinucleated nature. Thus, knowing that during the infection fungal cell death is likely to result in the release of large amounts of DNA, one could expect the triggering of innate immune mechanisms of the host via Toll-Like Receptor 9 (TLR9). This molecule is the member of the TLR family known to recognize unmethylated CpG sequences in DNA molecules. Nevertheless, TLR9 role during P. brasiliensis infections was never assessed. We herein demonstrate that activation of this receptor upon recognition of P. brasiliensis yeast cells is an event that seems to be crucial in early-times of infection. Lack of this receptor caused the premature death of the hosts (in a mouse model of infection with P. brasiliensis yeast cells), associated with signs of organ-pathology and high production of pro-inflammatory cytokines. One possible explanation for this profile can be the abnormal neutrophilia observed in TLR9-depleted infected mice. Overall, we show that TLR9 activation is immuno-protective in early stages of P. brasiliensis infections, playing an important role on the development of a controlled cell-mediated response. The work developed throughout this thesis provides new data on the morphological traits followed by the pathogenic fungus P. brasiliensis, and further establishes the relevance of CDC42 on the heterogeneity of cell shape. Moreover, we present new data on the yeast-phase metabolism, further clarifying how dimorphism impacts sulfur metabolism and its relevance for pathogenesis. We also provide relevant data for the elucidation of the mechanisms prompted by the host for the development of an appropriate immune response against P. brasiliensis infections, bringing together the multinucleated nature of this pathogen with the protective activation of the pattern recognition receptor TLR9.Nas últimas décadas obtiveram-se avanços significativos no estudo de doenças negligenciadas, nomeadamente no que concerne à elucidação da genética do organismo patogénico e de factores de risco do hospedeiro. A paracoccidioidomicose, uma micose causada por espécies de Paracoccidioides, é um desses casos, estimando-se afectar cerca de 10 milhões de indivíduos em países da América Latina. Diversos estudos, nomeadamente aqueles focados na genómica e transcriptómica de isolados de Paracoccidioides brasiliensis, têm levantado questões importantes, particularmente no que respeita ao efeito que a sua natureza multinucleada e variabilidade morfológica possa ter na activação de mecanismos de defesa do hospedeiro, um aspecto nunca abordado até à data. Além destes aspectos, a ausência de ferramentas moleculares é um factor que tem vindo a dificultar tanto a identificação de factores genéticos envolvidos na virulência e natureza polimórfica da fase leveduriforme do fungo, bem como a elucidação dos mecanismos subjacentes ao seu comportamento dimórfico. Uma particularidade da fase leveduriforme destas espécies é a sua heterogeneidade morfológica, assim como os seus padrões de gemulação. Estudos anteriores salientam a importância do gene CDC42 (cell division cycle 42) na definição destas características, no entanto, estas nunca foram determinadas individualmente para cada isolado de Paracoccidioides. Outro aspecto nunca abordado é a identificação de possíveis associações entre factores genéticos que determinem a morfologia do fungo e o padrão morfológico de cada isolado. De forma a elucidar estes aspectos, realizou-se uma detalhada análise morfogenética da fase leveduriforme de 11 isolados clínicos e ambientais de Paracoccidioides pertencentes a espécies de P. brasiliensis e Paracoccidioides lutzii. Os nossos resultados demonstram a ausência de um perfil morfológico característico para cada uma das linhagens destas espécies. No entanto, a área e forma das gémulas de cada isolado revelou-se altamente dependente da célula-mãe, sugerindo um elevado nível de conservação destas características ao longo das várias gerações. De referir também as fortes correlações encontradas entre a expressão do gene PbCDC42, uma peça importante no controlo do comportamento morfológico da fase leveduriforme, e a forma das células-mãe e gémulas, assim como com a área das gémulas. Estes dados permitem ampliar o conhecimento actual referente à natureza polimórfica de P. brasiliensis, nomeadamente no que respeita à clarificação das suas características morfológicas. Em P. brasiliensis, diversas alterações morfológicas e fenotípicas ocorrem durante o processo de transição da fase de conídeo/micélio para a fase leveduriforme, um processo fundamental inerente à patogenicidade do fungo. Para além destas alterações, a auxotrofia da fase leveduriforme para compostos de enxofre orgânico é também uma característica marcante. Embora este requisito nutricional seja comum a outros fungos dimórficos, as suas bases moleculares nunca foram clarificadas. Neste contexto, procedeu-se à avaliação do papel da proteína SconC, um regulador negativo da via de assimilação do enxofre inorgânico, no dimorfismo e virulência do fungo. Através da redução de expressão do gene SCONC por manipulação genética demonstrou-se que, nestas circunstâncias, a fase leveduriforme das células de P. brasiliensis consegue ultrapassar a sua auxotrofia a compostos de enxofre orgânico, utilizando fontes de enxofre inorgânico. No entanto, esta alteração metabólica levou a uma redução drástica dos níveis celulares de ATP e NADPH, provocando um aumento significativo do stress oxidativo. Este facto pode explicar a diminuição de virulência observada nas estirpes de P. brasiliensis reduzidas na expressão do gene SCONC, como verificado experimentalmente em ensaios com ratinhos. Os resultados aqui apresentados fornecem informações relevantes relativas ao controlo dos mecanismos subjacentes ao processo dimórfico de P. brasiliensis, evidenciando SconCp como uma molécula determinante no processo de virulência. A forma leveduriforme de P. brasiliensis é também caracterizada pela sua natureza multinucleada. Durante o processo de infecção, a morte celular do fungo pode resultar na libertação de elevadas quantidades de DNA, levando eventualmente à activação de mecanismo de imunidade inata via receptores Toll-like 9 (TLR9) por parte do hospedeiro. O TLR9 é a molécula da família dos receptores Toll-like responsável pelo reconhecimento de sequências CpG não-metiladas em moléculas de DNA. No entanto, o papel deste receptor no contexto de infecções por P. brasiliensis nunca foi explorado. Neste trabalho demostrou-se que a activação deste receptor é um processo que parece ser crucial durante as fases iniciais de infecção com P. brasiliensis. Utilizando como modelo ratinhos deletados em TLR9, verificou-se uma morte prematura do hospedeiro aquando da sua infecção com células de P. brasiliensis, associada a sinais de patologia em vários órgãos e elevada produção de citocinas pró-inflamatórias. Este perfil pode estar associado ao elevado recrutamento de neutrófilos para o local de infecção. Em suma, demonstrou-se que a activação de TLR9 é imuno-protectora em fases iniciais de infecções por P. brasiliensis, tendo um papel preponderante no controlo do desenvolvimento de uma resposta imune mediada por células. O trabalho desenvolvido no âmbito desta tese acrescenta novos conhecimentos relativamente à morfologia de P. brasiliensis, relevando-se a importância dos níveis de expressão do gene CDC42 no perfil morfológico da fase leveduriforme do fungo. São também apresentados novos dados sobre o metabolismo da fase leveduriforme de P. brasiliensis, permitindo esclarecer o impacto do dimorfismo celular no metabolismo de enxofre e sua relevância na patogenicidade do fungo. São também apresentados dados essenciais que permitem novos conhecimentos sobre os mecanismos desenvolvidos pelo hospedeiro numa resposta imune apropriada a infecções por P. brasiliensis, associando-se a natureza multinucleada do fungo com a activação de mecanismos de defesa via TLR9

Gene knockdown in Paracoccidioides brasiliensis using antisense RNA

Paracoccidioides brasiliensis is a thermal dimorphic fungus which at host environment exhibits a multinucleated and multibudding yeast form. The cellular and molecular mechanisms underlying these phenotypes remain to be clarified, mostly due to the absence of efficient classical genetic and molecular techniques. Here we describe a method for gene expression knockdown in P. brasiliensis by antisense RNA (aRNA) technology taking advantage of an Agrobacterium tumefaciens-mediated transformation (ATMT) system. Together, these techniques represent a reliable toolbox that can be employed for functional genetic analysis of putative virulence factors and morphogenic regulators, aiming to the identification of new potential drug targets.Fundação para a Ciência e a Tecnologia, Portugal (FCT) - Ref. PTDC/BIA-MIC/108309/2008, Ref. SFRH/BD/33446/2008)

Matrix Metalloproteinases as Plasma Indicators of Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle and has a high economic impact on farmers. COD is caused by an endocrine imbalance within the hypothalamic-pituitary-ovarian axis which prevents the mature Graafian follicle from ovulating. The cause at the molecular level is not well understood. Our research investigated the possibility of using plasma concentrations of matrix metalloproteinases (MMP) -2 and -9 and their natural tissue inhibitors (TIMP) -1 and -2 as prognostic indicators of COD. Plasma samples from cystic and non-cystic dairy cows were analyzed using ELISA. Although plasma concentrations of MMP-2 and -9 were greater and TIMP-2 was lower in non-cystic compared to cystic cows, no significant differences were observed in MMP-2 and -9 and TIMP-1 and -2 due to cyst status. However, the TIMP-1:MMP-9 and TIMP-2:MMP-2 molar ratios were greater, (P=0.099) and (P=0.038), respectively, in cystic compared to non-cystic cows, suggesting a proteolytic insufficiency in cows with COD that may be a contributing factor to the anovulatory pathology. These data may provide the groundwork for future research and development of tools for dairy farmers to selectively choose replacement heifers less likely to develop COD

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)

Functional characterization of a xylose transporter in Aspergillus nidulans

BACKGROUND: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. RESULTS: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. CONCLUSIONS: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.We would like to thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) for providing financial support. We also thank Dr Eckardt Boles for providing the EBY.VW4000 yeast strain, Dr Ronald Hector for providing the plasmids pRH274 and pRH195, Dr Michel Flipphi for providing the Delta creA4 strain, and the two anonymous reviewers for their comments and suggestions. We also acknowledge the Program project grant GM068087 (PI Jay Dunlap) for providing the deletion cassettes

Clinical Epidemiology of Buruli ulcer from Benin (2005-2013): effect of time-delay to diagnosis on clinical forms and severe phenotypes

Buruli Ulcer (BU) is a neglected infectious disease caused by Mycobacterium ulcerans that is responsible for severe necrotizing cutaneous lesions that may be associated with bone involvement. Clinical presentations of BU lesions are classically classified as papules, nodules, plaques and edematous infiltration, ulcer or osteomyelitis. Within these different clinical forms, lesions can be further classified as severe forms based on focality (multiple lesions), lesions' size (>15 cm diameter) or WHO Category (WHO Category 3 lesions). There are studies reporting an association between delay in seeking medical care and the development of ulcerative forms of BU or osteomyelitis, but the effect of time-delay on the emergence of lesions classified as severe has not been addressed. To address both issues, and in a cohort of laboratory-confirmed BU cases, 476 patients from a medical center in Allada, Benin, were studied. In this laboratory-confirmed cohort, we validated previous observations, demonstrating that time-delay is statistically related to the clinical form of BU. Indeed, for non-ulcerated forms (nodule, edema, and plaque) the median time-delay was 32.5 days (IQR 30.0-67.5), while for ulcerated forms it was 60 days (IQR 20.0-120.0) (p = 0.009), and for bone lesions, 365 days (IQR 228.0-548.0). On the other hand, we show here that time-delay is not associated with the more severe phenotypes of BU, such as multi-focal lesions (median 90 days; IQR 56-217.5; p = 0.09), larger lesions (diameter >15 cm) (median 60 days; IQR 30-120; p = 0.92) or category 3 WHO classification (median 60 days; IQR 30-150; p = 0.20), when compared with unifocal (median 60 days; IQR 30-90), small lesions (diameter =15 cm) (median 60 days; IQR 30-90), or WHO category 1+2 lesions (median 60 days; IQR 30-90), respectively. Our results demonstrate that after an initial period of progression towards ulceration or bone involvement, BU lesions become stable regarding size and focal/multi-focal progression. Therefore, in future studies on BU epidemiology, severe clinical forms should be systematically considered as distinct phenotypes of the same disease and thus subjected to specific risk factor investigation.The research leading to these results received funding from the Health Services of the Fundacao Calouste Gulbenkian under the grant Proc. No94776 LJ; from the Fundacao para a Ciecia e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte); from the Quadro de Referencia Estrategico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). A. G. Fraga received an individual FCT fellowship (SFRH/BPD/68547/2010) and J. Menino received an individual QREN fellowship (UMINHO/BPD/14/2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Prospecção de pesquisa e inovação em sistemas de produção de mangaba cultivada.

ODS: 2 Fome zero e agricultura sustentável; 17 Parcerias e meios de implementação

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

GO-BIOCHORUME: sistemas biológicos de utilização do chorume proveniente de explorações de pecuária intensiva de bovinos leiteiros

info:eu-repo/semantics/publishedVersio