Transcriptome profile of the response of paracoccidioides spp. to a camphene thiosemicarbazide derivative

ABSTARCT: Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment

Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MSE technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite–host relationship and pathogenesis of PCM

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database - the quality controlled standard tool for routine identification of human and animal pathogenic fungi

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.This study was supported by an National Health and Medical Research Council of Australia (NH&MRC) grant [#APP1031952] to W Meyer, S Chen, V Robert, and D Ellis; CNPq [350338/2000-0] and FAPERJ [E-26/103.157/2011] grants to RM Zancope-Oliveira; CNPq [308011/2010-4] and FAPESP [2007/08575-1] Fundacao de Amparo Pesquisa do Estado de So Paulo (FAPESP) grants to AL Colombo; PEst-OE/BIA/UI4050/2014 from Fundacao para a Ciencia e Tecnologia (FCT) to C Pais; the Belgian Science Policy Office (Belspo) to BCCM/IHEM; the MEXBOL program of CONACyT-Mexico, [ref. number: 1228961 to ML Taylor and [122481] to C Toriello; the Institut Pasteur and Institut de Veil le Sanitaire to F Dromer and D Garcia-Hermoso; and the grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) to CM de Almeida Soares and JA Parente Rocha. I Arthur would like to thank G Cherian, A Higgins and the staff of the Molecular Diagnostics Laboratory, Division of Microbiology and Infectious Diseases, Path West, QEII Medial Centre. Dromer would like to thank for the technical help of the sequencing facility and specifically that of I, Diancourt, A-S Delannoy-Vieillard, J-M Thiberge (Genotyping of Pathogens and Public Health, Institut Pasteur). RM Zancope-Oliveira would like to thank the Genomic/DNA Sequencing Platform at Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ [RPT01A], Brazil for the sequencing. B Robbertse and CL Schoch acknowledge support from the Intramural Research Program of the NIH, National Library of Medicine. T Sorrell's work is funded by the NH&MRC of Australia; she is a Sydney Medical School Foundation Fellow.info:eu-repo/semantics/publishedVersio

Caracterização filogenética de Babesia canis vogeli em cães do estado de Goiás, Brasil

Submitted by Luciana Ferreira ([email protected]) on 2020-02-10T12:44:39Z No. of bitstreams: 2 Artigo - Sabrina Castilho Duarte - 2011.pdf: 553655 bytes, checksum: e8c565073864859af3033f4b8e3541d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2020-02-11T10:39:50Z (GMT) No. of bitstreams: 2 Artigo - Sabrina Castilho Duarte - 2011.pdf: 553655 bytes, checksum: e8c565073864859af3033f4b8e3541d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2020-02-11T10:39:51Z (GMT). No. of bitstreams: 2 Artigo - Sabrina Castilho Duarte - 2011.pdf: 553655 bytes, checksum: e8c565073864859af3033f4b8e3541d1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2011-12O gênero Babesia compreende protozoários causadores de enfermidades denominadas babesioses. Cães geralmente são acometidos por Babesia canis ou Babesia gibsoni, sendo a primeira classificada em subespécies Babesia canis canis, Babesia canis vogeli e Babesia canis rossi. Entre essas, Babesia canis vogeli predomina no Brasil. O objetivo desse trabalho foi realizar estudo filogenético de amostras de Babesia em cães, em Goiânia, Goiás. Amostras de sangue foram obtidas de 890 cães atendidos no Hospital Veterinário de Goiás, apresentando sinais clínicos de babesiose. Somente amostras com presença de parasitos intraeritrocitários típicos foram utilizadas. Estas foram submetidas a extração de DNA e amplificação de fragmento do gene 18S rRNA pela PCR. Os produtos de PCR foram purificados e sequenciados. Foram sequenciadas 35 amostras, das quais apenas 17 foram mantidas após avaliação de qualidade. A análise de similaridade fornecida pelo BLASTn demonstrou que as 17 sequências deste estudo eram correspondentes a Babesia canis vogeli. Pela utilização do programa Mega4, foi possível verificar que as amostras de Babesia canis vogeli, provenientes de cães da cidade de Goiânia, apresentam, alto grau de similaridade molecular (99,2 a 100%) com isolados de referência de outras regiões do Brasil e do mundo, depositados em GenBank.The genus Babesia comprises protozoa that cause diseases known as babesiosis. Dogs are commonly affected by Babesia canis or Babesia gibsoni. Babesia canis is divided into the subspecies Babesia canis canis, Babesia canis vogeli and Babesia canis rossi. Among these, Babesia canis vogeli predominates in Brazil. The objective of this study was to conduct a phylogenetic analysis on Babesia isolates from dogs in Goiânia, Goiás. Blood samples were obtained from 890 dogs presenting clinical signs suggestive of canine babesiosis that were attended at a veterinary hospital of Goiás. Only samples presenting typical intraerythrocytic parasites were used in the study. These were subjected to DNA extraction and amplification of a fragment of the 18S rRNA, by means of PCR. The PCR products were purified and sequenced. Sequences were obtained from 35 samples but only 17 of these were kept after quality assessment. Similarity analysis using BLASTn demonstrated that all 17 sequences corresponded to B. canis vogeli. Analysis using the Mega4 software showed that the isolates of B. canis vogeli from dogs in Goiânia present a high degree of molecular similarity (99.2 to 100%) in comparison with other reference isolates from other regions of Brazil and worldwide, deposited in GenBank

Antifungal Resistance, Metabolic Routes as Drug Targets, and New Antifungal Agents: An Overview about Endemic Dimorphic Fungi

Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs

Interaction of <i>P</i>. <i>brasiliensis</i> yeast cells with macrophages and evaluation of phagolysosome maturation.

<p>The interaction assay was performed using two <i>P</i>. <i>brasiliensis</i> isolates (<i>Pb</i>18 and <i>Pb</i>339) and are shown in the lanes named macrophages + <i>Pb</i>18 yeast cells and macrophages + <i>Pb</i>339 yeast cells, respectively. The pictures were taken in bright field (shown in the bright field column), at 395/420 nm for Calcofluor probe (shown in the Calcofluor white column) and at 579/599 nm for Lysotracker probe (shown in the Lysotracker column). Fungal control cells are shown in the lanes named <i>Pb</i>18 yeast cells and <i>Pb</i>339 yeast cells. Control macrophage cells were also performed and are shown in the lane named macrophage (control). All representative pictures were taken using an Axioscope microscope (Carl Zeiss) and magnified 1000X.</p