Lorena da Silveira Derengowski

University of Brasília, Brasília, Federal, Brazil

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Publications (14)35.34 Total impact

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    ABSTRACT: Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro. Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-α, receptor-interacting protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen. Copyright © 2015 by The American Association of Immunologists, Inc.
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    ABSTRACT: Background: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. Results: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style.
    BMC Genomics 10/2014; 15:943. DOI:10.1186/1471-2164-15-943 · 4.04 Impact Factor
  • Mycoses 05/2014; 57:96-97. · 1.81 Impact Factor
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    ABSTRACT: Antimicrobial peptides (AMPs) are natural antibiotics produced by various organisms such as mammals, arthropods, plants, and bacteria. In addition to antimicrobial activity, AMPs can induce chemokine production, accelerate angiogenesis, and wound healing and modulate apoptosis in multicellular organisms. Originally, their antimicrobial mechanism of action was thought to consist solely of an increase in pathogen cell membrane permeability, but it has already been shown that several AMPs do not modulate membrane permeability in the minimal lethal concentration. Instead, they exert their effects by inhibiting processes such as protein and cell wall synthesis, as well as enzyme activity, among others. Although resistance to these molecules is uncommon several pathogens developed different strategies to overcome AMPs killing such as surface modification, expression of efflux pumps, and secretion of proteases among others. This review describes the various mechanisms of action of AMPs and how pathogens evolve resistance to them.
    Frontiers in Microbiology 12/2013; 4:353. DOI:10.3389/fmicb.2013.00353 · 3.94 Impact Factor
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    ABSTRACT: Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoa predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least two-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on the ORF CNAG_05662, which was potentially related to sugar transport but with no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (Polyol Transporter Protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungal-protozoal interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host.
    Eukaryotic Cell 03/2013; DOI:10.1128/EC.00073-13 · 3.18 Impact Factor
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    ABSTRACT: Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen.
    PLoS Neglected Tropical Diseases 01/2012; 6(1):e1459. DOI:10.1371/journal.pntd.0001459 · 4.49 Impact Factor
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    ABSTRACT: A unique aspect of the interaction of the fungus Cryptococcus neoformans with macrophages is the phenomenon of nonlytic exocytosis, also referred to as “vomocytosis” or phagosome extrusion/expulsion, which involves the escape of fungal cells from the phagocyte with the survival of both cell types. This phenomenon has been observed only in vitro using subjective and time-consuming microscopic techniques. In spite of recent advances in our knowledge about its mechanisms, a major question still remaining is whether this phenomenon also occurs in vivo. In this study, we describe a novel flow cytometric method that resulted in a substantial gain in throughput for studying phagocytosis and nonlytic exocytosis in vitro and used it to explore the occurrence of this phenomenon in a mouse model of infection. Furthermore, we tested the hypothesis that host cell phagosomal pH affected nonlytic exocytosis. The addition of the weak bases ammonium chloride and chloroquine resulted in a significant increase of nonlytic exocytosis events, whereas the vacuolar ATPase inhibitor bafilomycin A1 had the opposite effect. Although all three agents are known to neutralize phagosomal acidity, their disparate effects suggest that phagosomal pH is an important and complex variable in this process. Our experiments established that nonlytic exocytosis occurred in vivo with a frequency that is possibly much higher than that observed in vitro. These results in turn suggest that nonlytic exocytosis has a potential role in the pathogenesis of cryptococcosis.
    mBio 06/2011; 2(4). DOI:10.1128/mBio.00167-11 · 6.88 Impact Factor
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    ABSTRACT: Thermodimorphic fungi include most causative agents of systemic mycoses, but the molecular mechanisms that underlie their defining trait, i.e. the ability to shift between mould and yeast on temperature change alone, remain poorly understood. We hypothesised that the heat shock factor (Hsf), a protein that evolved to sense thermal stimuli quickly, might play a role in this process in addition to the known regulator Drk1 and the Ryp proteins. To test this hypothesis, we characterised the Hsf from the thermodimorph Paracoccidioides lutzii (formerly Paracoccidioides brasiliensis isolate 01). We show in the present work that PlHsf possesses regulatory domains that are exclusive of the Eurotiomycetidae family, suggesting evolutionary specialisation; that it can successfully rescue the otherwise lethal loss of the native protein of Saccharomyces cerevisiae; and that its DNA-binding domain is able to recognise regulatory elements from the promoters of both Drk1 and Ryp1. An in silico screening of all 1 kb sequences upstream of P. lutzii ORFs revealed that 7% of them possess a heat shock element. This is the first description of a heat shock factor in a thermodimorphic fungus.
    Fungal Genetics and Biology 06/2011; 48(10):947-55. DOI:10.1016/j.fgb.2011.06.005 · 3.26 Impact Factor
  • International journal of antimicrobial agents 10/2009; 34(6):619-21. DOI:10.1016/j.ijantimicag.2009.08.010 · 4.26 Impact Factor
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    ABSTRACT: Farnesol is a sesquiterpene alcohol produced by many organisms, and also found in several essential oils. Its role as a quorum sensing molecule and as a virulence factor of Candida albicans has been well described. Studies revealed that farnesol affect the growth of a number of bacteria and fungi, pointing to a potential role as an antimicrobial agent. Growth assays of Paracoccidioides brasiliensis cells incubated in the presence of different concentrations of farnesol were performed by measuring the optical density of the cultures. The viability of fungal cells was determined by MTT assay and by counting the colony forming units, after each farnesol treatment. The effects of farnesol on P. brasiliensis dimorphism were also evaluated by optical microscopy. The ultrastructural morphology of farnesol-treated P. brasiliensis yeast cells was evaluated by transmission and scanning electron microscopy. In this study, the effects of farnesol on Paracoccidioides brasiliensis growth and dimorphism were described. Concentrations of this isoprenoid ranging from 25 to 300 microM strongly inhibited P. brasiliensis growth. We have estimated that the MIC of farnesol for P. brasiliensis is 25 microM, while the MLC is around 30 microM. When employing levels which don't compromise cell viability (5 to 15 microM), it was shown that farnesol also affected the morphogenesis of this fungus. We observed about 60% of inhibition in hyphal development following P. brasiliensis yeast cells treatment with 15 microM of farnesol for 48 h. At these farnesol concentrations we also observed a significant hyphal shortening. Electron microscopy experiments showed that, despite of a remaining intact cell wall, P. brasiliensis cells treated with farnesol concentrations above 25 microM exhibited a fully cytoplasmic degeneration. Our data indicate that farnesol acts as a potent antimicrobial agent against P. brasiliensis. The fungicide activity of farnesol against this pathogen is probably associated to cytoplasmic degeneration. In concentrations that do not affect fungal viability, farnesol retards the germ-tube formation of P. brasiliensis, suggesting that the morphogenesis of this fungal is controlled by environmental conditions.
    Annals of Clinical Microbiology and Antimicrobials 05/2009; 8:13. DOI:10.1186/1476-0711-8-13 · 1.51 Impact Factor
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    Annals of Clinical Microbiology and Antimicrobials 04/2009; 8:13. · 1.51 Impact Factor
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    ABSTRACT: related sequences plus a number of randomly chosen ascomycete outgroups. This showed the isolate to be within the Pyrenochaeta romeroi clade. In April 2007, treatment with posaconazole 400 mg b.i.d. was started. Random posaconazole serum levels measured by bioassay were 0.9–2.1 mg/L. Following 17 months of posaconazole ther-apy there was nearly complete disappearance of local pain and decreased local swelling. MRI in August 2008 of the patient's foot showed extensive bony and soft tissue infection. Posaconazole was discontinued in March 2009 and further swelling was observed over the subsequent 6 months. Pyrenochaeta romeroi is widely distributed worldwide, where it grows on soil and vegetation, particularly in tropical climates. It has been reported as a rare cause of mycetoma in humans [5,6]. Clinical trials have shown successful treatment of eumyce-toma with ketoconazole and itraconazole [1]. However, our patient responded poorly or not at all to itraconazole, corroborating in vitro results. There are no reports on the use of voriconazole in P. romeroi infections. In our patient, voriconazole was minimally effective and response was best classed as a failure. The difference in voriconazole susceptibility results observed is presumably due to the change in methodology, as on both occasions the same isolate was tested. Unresponsiveness to treatment with voriconazole in our patient corroborates the susceptibility results using the modified EUCAST method. Posaconazole has been shown to have comparable or improved in vitro activity compared with itraconazole against agents causing mycetoma [7]. A literature search revealed no reports on the clinical efficacy of posaconazole in Pyrenochaeta infections. A modest response at best was seen in our patient with posaconazole initially, with clinical evidence of breakthrough more recently. Considering the fact that our patient had worked barefoot in direct contact with cotton imported from mycetoma-endemic countries with possible microscopic injuries to his feet, as well as the absence of any known preceding injuries, this seems the probable route of infection. We believe that this case is the first description of this mode of mycetoma acquisition. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Ahmed AOA, van Leeuwen W, Fahal A, van de Sande W, Verbrugh H, van Belkum A. Mycetoma caused by Madurella mycetomatis: a neglected infectious burden. Lancet Infect Dis 2004;4:566–74. [2] Hay RJ, Mackenzie DW. Mycetoma (madura foot) in the United Kingdom—a sur-vey of forty-four cases. Clin Exp Dermatol 1983;8:553–62. [3]
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    ABSTRACT: Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.
    Medical Mycology 04/2008; 46(2):125-34. DOI:10.1080/13693780701670509 · 2.26 Impact Factor
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    ABSTRACT: Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.
    Medical biology 01/2008; 46(2):125-134.
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    Lorena da Silveira Derengowski
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    ABSTRACT: Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2006. O fungo Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose, a micose sistêmica de maior prevalência na América Latina. O mecanismo mais importante de defesa do hospedeiro contra infecções por P. brasiliensis é a resposta imunológica celular. Esse fungo é capaz de sobreviver e se replicar no interior de macrófagos murinos e humanos, revelando a existência de mecanismos que permitam a sobrevivência no ambiente inóspito do fagossomo da célula hospedeira. Em concordância com essa habilidade de P. brasiliensis em sobreviver nesse ambiente de carência nutricional, estresse oxidativo e baixo pH, análises do transcriptoma desse fungo revelam ortólogos a potenciais genes de virulência de outros microrganismos patogênicos. Nesse contexto, esse trabalho descreve a análise da expressão de potenciais genes de virulência de P. brasiliensis por RT-PCR. Visando a avaliar a expressão dos genes icl1 (isocitrato liase) e mls1 (malato sintase), que codificam as enzimas do ciclo do glioxalato, leveduras de P. brasiliensis foram crescidas em condições que potencialmente mimetizam às encontradas no interior do fagossomo, substituindo-se glicose por acetato como única fonte de carbono. Os resultados sugerem um significativo aumento na expressão de icl1 e mls1 quando do cultivo do fungo em meio contendo apenas acetato. A mesma análise foi feita para os genes ady2, que codifica uma permease de acetato, e icl2, que parece codificar outra isoforma da enzima isocitrato liase. Entretanto, os resultados indicam que os genes ady2 e icl2 de P. brasiliensis são regulados independentemente das fontes de carbono utilizadas nesse trabalho. Ainda, a expressão de ure1 (urease) foi analisada quando do cultivo de P. brasiliensis em pH ácido. Esses experimentos não mostraram uma regulação do gene ure1 desse fungo dependente de pH. Adicionalmente, esse trabalho descreve, pela primeira vez, experimentos de RT-PCR utilizando RNA extraído de leveduras de P. brasiliensis após 6 horas de co-cultura com macrófagos murinos. Os resultados mostram um aumento no nível de expressão dos genes icl1, mls1 e ady2, quando comparada à expressão por células crescidas em meio convencional. Essas observações indicam claramente que P. brasiliensis utiliza o ciclo do glioxalato como uma importante via metabólica alternativa para produção de energia, como observado para outros fungos. ______________________________________________________________________________ ABSTRACT The fungus Paracoccidioides brasiliensis is the ethiologic agent of paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. The most important mechanism of host defense against P. brasiliensis infection is cell-mediated immune response. This fungus is able to survive and replicate within the phagossome of murine and human macrophages, revealing that it have evolved mechanisms allowing its survival within the phagocytic cells, which are considered an inhospitable habitat. In agreement with the ability to survive in this nutritionally poor, oxidative stress and low pH environment, the analysis of P. brasiliensis transcriptome revealed several putative orthologs to virulence genes of other human facultative intracellular pathogenic microorganisms. In this context, this work describes the analysis of putative virulence genes expression by RT-PCR. In order to evaluate the expression of glyoxylate cycle genes, icl1 (isocitrate lyase) and mls1 (malate synthase), the yeast form of P. brasiliensis was grown in media mimicking the phagossome millieu, in which glucose was replaced by acetate as a sole carbon source. The results suggests a significantly increase in the icl1 and mls1 expression when this fungus was grown in media with acetate as the only carbon source. The same analyzis was performed for ady2 gene, that encode an acetate permease, and icl2 gene, that probably encodes another putative isoform of isocitrate lyase enzyme. However, the results indicate that ady2 and icl2 genes of P. brasiliensis are not regulated by these different carbon sources used in this work. On the other hand, the expression of ure1 (urease) was analyzed in this work when P. brasiliensis yeast cells were grown in acid pH, mimicking the phagossome environment. These experiments don’t show a pHdependent regulation by ure1 gene of this fungus. In addition, this work describes for the first time RT-PCR experiments using RNA extracted from yeast cells recovered from murine macrophages after 6 hours of co-culture. The results show the higher levels of icl1, mls1 and ady2 gene expression when compared to cells grown in conventional media. These observations clearly indicates that P. brasiliensis uses the glyoxylate cycle as an important alternative energetic metabolism pathway, as observed for other fungi.

Publication Stats

114 Citations
35.34 Total Impact Points

Institutions

  • 2008–2015
    • University of Brasília
      • Department of Cell Biology
      Brasília, Federal, Brazil
  • 2014
    • Universidade Católica de Brasília
      • Pós-Graduação em Ciências Genômicas e Biotecnologia
      Brasília, Federal, Brazil
  • 2011
    • Albert Einstein College of Medicine
      • Department of Microbiology & Immunology
      New York, New York, United States