Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different ...mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.
Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A
P. brasiliensis cDNA expression library ...was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown
P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-
l-amino-acid decarboxylase (
Pbddc), lumazine synthase (
Pbls) and a homologue of the high affinity copper transporter (
Pbctr3) were obtained. Recombinant proteins
PbDDC and
PbLS were obtained; a peptide was synthesized for
PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RT-PCR demonstrated high transcript levels of
Pbddc,
Pbls and
Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of
P. brasiliensis.
The fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic granulomatous mycosis prevalent in Latin America. In an effort to elucidate the molecular mechanisms involved in ...fungus cell wall assembly and morphogenesis, β-1,3-glucanosyltransferase 3 (PbGel3p) is presented here. PbGel3p presented functional similarity to the glucan-elongating/glycophospholipid-anchored surface/pH-regulated /essential for pseudohyphal development protein families, which are involved in fungal cell wall biosynthesis and morphogenesis. The full-length cDNA and gene were obtained. Southern blot and in silico analysis suggested that there is one copy of the gene in P. brasiliensis. The recombinant PbGel3p was overexpressed in Escherichia coli, and a polyclonal antibody was obtained. The PbGEL3 mRNA, as well as the protein, was detected at the highest level in the mycelium phase. The protein was immunolocalized at the surface in both the mycelium and the yeast phases. We addressed the potential role of PbGel3p in cell wall biosynthesis and morphogenesis by assessing its ability to rescue the phenotype of the Saccharomyces cerevisiae gas1Δ mutant. The results indicated that PbGel3p is a cell wall-associated protein that probably works as a β-1,3-glucan elongase capable of mediating fungal cell wall integrity.
Zinc plays a critical role in a diverse array of biochemical processes. However, an excess of zinc is deleterious to cells; therefore, cells require finely tuned homeostatic mechanisms to balance the ...uptake and the storage of zinc. There is also increasing evidence supporting the importance of zinc during infection. To understand better how Paracoccidioides adapts to zinc deprivation, we compared the two-dimensional (2D) gel protein profile of yeast cells during zinc starvation to yeast cells grown in a zinc rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity, as determined by image analysis. In response to zinc deprivation, a total of 423 out of 845 protein spots showed a significant change in abundance. Quantitative RT-qPCR analysis of RNA from Paracoccidioides grown under zinc restricted conditions validated the correlation between the differentially regulated proteins and transcripts. According to the proteomic data, zinc deficiency may be a stressor to Paracoccidioides, as suggested by the upregulation of a number of proteins related to stress response, cell rescue, and virulence. Other process induced by zinc deprivation included gluconeogenesis. Conversely, the methylcitrate cycle was downregulated. Overall, the results indicate a remodelling of the Paracoccidioides response to the probable oxidative stress induced during zinc deprivation.
•Paracoccidiodes behaviour during zinc deprivation.•Metabolic and cellular processes are reorganized during zinc deprivation.•The major response affected by zinc restriction was related to oxidative stress.
Paracoccidioidomycosis is caused by the dimorphic fungus Paracoccidioides brasiliensis. The extracellular matrix (ECM) plays an important role in regulation of cell adhesion, differentiation, ...migration and proliferation of cells. An in vitro binding assay of P. brasiliensis yeast cells adhering to type I collagen and fibronectin was performed in order to identify novel adhesins. Representational difference analysis (RDA) was employed to identify genes upregulated under adhesion-inducing conditions. Expressed sequence tags (ESTs) from cDNA libraries generated by the RDA technique were analyzed. Genes related to functional categories, such as metabolism, transcription, energy, protein synthesis and fate, cellular transport and biogenesis of cellular components were upregulated. Transcripts encoding the P. brasiliensis protein enolase (PbEno) and the high-affinity cooper transporter (PbCtr3) were identified and further characterized. The recombinant enolase (rPbEno) and a synthetic peptide designed for PbCtr3 were obtained and demonstrated to be able to bind ECM components. Immunofluorescence assays demonstrated that rPbEno specifically binds to the macrophage surface, reinforcing the role of this molecule in the P. brasiliensis interaction with host cells. In addition, upregulation of selected genes was demonstrated by qRT-PCR. In synthesis, the strategy can be useful in characterization of potential P. brasiliensis adhesins.
Metal Acquisition and Homeostasis in Fungi Bailão, Elisa Flávia Luiz Cardoso; Parente, Ana Flávia Alves; Parente, Juliana Alves ...
Current fungal infection reports,
12/2012, Letnik:
6, Številka:
4
Journal Article
Recenzirano
Transition metals, particularly iron, zinc and copper, have multiple biological roles and are essential elements in biological processes. Among other micronutrients, these metals are frequently ...available to cells in only limited amounts, thus organisms have evolved highly regulated mechanisms to cope and to compete with their scarcity. The homeostasis of such metals within the animal hosts requires the integration of multiple signals producing depleted environments that restrict the growth of microorganisms, acting as a barrier to infection. As the hosts sequester the necessary transition metals from invading pathogens, some, as is the case of fungi, have evolved elaborate mechanisms to allow their survival and development to establish infection. Metalloregulatory factors allow fungal cells to sense and to adapt to the scarce metal availability in the environment, such as in host tissues. Here we review recent advances in the identification and function of molecules that drive the acquisition and homeostasis of iron, copper and zinc in pathogenic fungi.
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The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the
most frequent systemic mycosis in Latin America. In humans, infection starts by
inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate
into the yeast parasitic phase. Here we describe the characterization of a proline-rich
protein (PRA/Ag2) homologue of P. brasiliensis, a predictable cell wall protein, first
identified in Coccidioides immitis. The protein, the cDNA and genomic sequences were
analyzed. Southern blot analysis suggested that there is one copy of the gene in P.
brasiliensis. The cloned cDNA was expressed in Escherichia coli and the purified
rPbPRA/Ag2 was used to obtain polyclonal antibody. The purified recombinant protein
was recognized by sera of patients with proven paracoccidioidomycosis and not by sera
of healthy individuals. Immunoelectron microscopy and biochemical studies
demonstrated the presence of PbPRA/Ag2 in the fungal cell wall, linked through a GPIanchor.
The expression of the Pbpra/ag2 gene was analyzed by real time PCR and
results demonstrated developmental regulation in phases of P. brasiliensis, with a
higher expression in the mycelium saprobic phase. The protein expression analyses
corroborate the transcript levels.
O fungo dimórfico Paracoccidioides brasiliensis é o agente causador da micose sistêmica mais prevalente na América Latina. Em humanos, a infecção se dá pela inalação de propágulos do fungo, que ao alcançarem o epitélio pulmonar transformam se na fase leveduriforme, parasitária, do fungo P. brasiliensis. Neste trabalho,descrevemos a caracterização de um homólogo de PRA/Ag2 (Antígeno rico em prolina), uma proteína predita de parede celular, primeiramente identificada em Coccidioides immitis. A proteína, o cDNA e a seqüência genômica foram analisados.
Análises através de Southern blot sugerem que há somente uma cópia do gene em
P.brasiliensis. O cDNA clonado foi expresso em Escherichia coli e a rPbPRA/Ag2
purificada foi utilizada para obtenção do anticorpo policlonal. A proteína recombinante
purificada foi reconhecida pelo soro de pacientes com paracoccidioidomicose
comprovada e não foi reconhecida pelo soro de pacientes saudáveis. Microscopia
eletrônica e estudos bioquímicos demonstraram a presença do PbPRA/Ag2 na parede
celular de P. brasiliensis, ligada através de uma âncora de GPI. A expressão do gene
que codifica Pbpra/ag2 foi analisada através de PCR em Tempo Real e os resultados
demonstraram regulação da expressão nas fases de P. brasiliensis, com maior nível de
expressão na fase miceliana. Análises da expressão protéica corroboram os resultados
da RT-PCR em Tempo Real.
Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2012.
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O fungo Paracoccidioides é um patógeno humano com ampla distribuição na América Latina. A infecção se inicia por inalação de conídios e ou propágulos da forma miceliana que ao atingirem o pulmão do hospedeiro se diferenciam na fase leveduriforme. Embora o perfil de expressão gênica em Paracoccidioides venha sendo estudado, pouco se conhece sobre o padrão de expressão de genes desta espécie durante o processo infeccioso. Durante a infecção, patógenos regulam o metabolismo em resposta a diferentes tipos de estresse e quantidades variadas de nutrientes disponíveis ;
no hospedeiro, presumivelmente permitindo a sua adaptação e sobrevivência. Os íons ;
metálicos são elementos essenciais para a manutenção de importantes vias metabólicas, ;
e por esse motivo, a capacidade de adquiri-los pelos patógenos, a partir de fontes ;
disponíveis no organismo hospedeiro, é considerada um fator de virulência. O zinco é ;
considerado um íon metálico essencial para todos os organismos, por ser requerido para ;
transcrição, tradução, replicação, resistência ao estresse oxidativo e virulência. Transportadores para este íons são altamente expressos em condições de infecção por Paracoccidioides. O presente trabalho descreve a análise de genes e proteínas diferencialmente expressos em células leveduriformes de Paracoccidioides em ;
condições que mimetizam a infecção. Foi realizada a comparação do perfil proteômico ;
de Paracoccidioides durante a privação de zinco e na disponibilidade desse metal com o ;
objetivo de se descrever os mecanismos utilizados pelo fungo para sobreviver no ;
ambiente com baixa disponibilidade de zinco, mimetizando a condição encontrada pelo ;
patógeno durante a infecção. Adicionalmente, foi realizada a análise transcricional de ;
Paracoccidioides recuperado de animais infectados. Os dados revelaram que proteínas ;
relacionadas com resposta ao estresse, metabolismo de aminoácidos, gliconeogênese e ;
ciclo do metilcitrato foram regulados durante a privação de zinco. Os transcritos ;
induzidos durante a infecção no pulmão foram aqueles predominantemente relacionados ;
com metabolismo de lipídios, ácidos graxos e isoprenóides, metabolismo de aminoácidos, resposta ao estresse e virulência e metabolismo de carboidratos. A expressão diferencial dos genes e proteínas identificados foi confirmada por ensaios de ;
qRT-PCR. Os dados gerados podem facilitar estudos funcionais de novos genes e ;
proteínas os quais podem ser importantes para estratégias de sobrevivência e adaptação ;
do Paracoccidioides no hospedeiro. ______________________________________________________________________________ ABSTRACT
Paracoccidioides is a fungal human pathogen with a wide distribution in Latin America. The fungus causes infection through host inhalation of airborne propagules of ;
the mycelial phase of the organism. These particles reach the lungs and differ in the ;
yeast phase. Although gene expression in Paracoccidioides had been studied, little is ;
known about the genome sequences expressed by this species during the infection ;
process. During infection, pathogens regulate the metabolism in response to different ;
types of stress and varying amounts of nutrients available in the host, presumably ;
allowing its adaptation and survival. Metal ions are essential elements for the ;
maintenance of important metabolic pathways, and therefore, the ability to acquire them ;
by pathogens from sources available in the host organism, is considered a virulence ;
factor. Zinc is considered a metal ion essential for all organisms, being required for transcription, translation, replication, resistance to oxidative stress and virulence. Here we describe the analysis of genes and proteins differentially expressed in yeast cells of Paracoccidioides under conditions mimicking of infection. It was performed a ;
comparison of the proteomic profile of Paracoccidioides during zinc deprivation and in the availability of the metal in order to describe the mechanisms used by the fungus to ;
survive in the environment with low vailability of zinc, mimicking the condition ;
encountered by the pathogen during infection. Farther, was performed transcriptional ;
analysis of Paracoccidioides recovered from lung of infected animals. The data ;
revealed that proteins related to stress response, amino acid metabolism, ;
gluconeogenesis and methylcitrate cycle were regulated during deprivation of zinc. The ;
transcripts induced during infection in the lung were predominantly those related to ;
lipid metabolism, fatty acid and isoprenoid, amino acid metabolism, carbohydrate ;
metabolism and stress response and virulence. The differential expression of genes and ;
proteins identified was confirmed by qRT-PCR assays. The data generated can facilitate ;
functional studies of novel genes and proteins which may be important for survival ;
strategies and adaptation of Paracoccidioides in the host.
The quality of sugarcane broth can be affected by soil management. In compacted soils the productivity is reduced, and the raw material is poor. This research aimed to evaluate productivity and ...quality of sugarcane for four soil management types: (CT) heavy harrow + light harrow; (CTI) Subsoiler + light harrow; (MT) Subsoiler and (NT) no soil movement. The variables investigated were resistance to soil penetration (SPR), the chemical raw material quality (broth) and crop yield. In crop rows, SPR did not reach severe levels up to 0.3 m for sugarcane root development. However, below this layer, MT, NT, and CTI reached SPR limiting values of 2.50, 2.35 and 1.95 MPa, respectively. In inter-crop rows, compaction was concentrated in soil surface layers (0–0.3 m). In addition, all adopted managements presented SPR above the critical value (2 MPa). The soil preparation forms qualitatively affected the sugarcane broth, showing higher fiber and protein contents in NT, MT, and CT. The PS, Brix, TRS, and Pol were not affected by soil management. Still, higher absolute values were found in the NT, indicating an increase in broth quality when applying conservation management. The highest yields were obtained by reduced tillage (MT), surpassing the lowest yield management (NT) with an increase of 10.5 Mg ha−1.
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