Transcriptional analysis of the accession process by Paracoccidioides brasiliensis and functional characterization of adhesins

Made available in DSpace on 2014-07-29T15:26:19Z (GMT). No. of bitstreams: 1 Tese Sarah Veloso Nogueira.pdf: 4968384 bytes, checksum: 1a3070a4c2260fba10919cd4f6ccd4f4 (MD5) Previous issue date: 2010-03-11Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis, prevalent in Latin America. Extracellular matrix (ECM) is a complex net where collagens, laminin and fibronectin can be found and, when exposed, is the first site for the fungus adhesion. Our aim was to study genes involved in the adhesion process using Representational Difference Analysis (RDA). RDA is a PCR-coupled subtractive method that allows the isolation of genes differentially expressed in two different cDNA populations. Hence, cDNAs were synthesized from RNAs extracted from P. brasiliensis yeast cells adhered to collagen and fibronectin to identify overexpressed genes. Genes involved in a wide range of cellular process were found and PbCtr3 (cooper transporter) and enolase (PbEno) were chosen to further studies. A synthetic peptide (PbCTR3) and the recombinant enolase (rPbEno) were utilized together with the anti-rPbEno polyclonal antibody in functional analysis with ECM components and plasminogen. The studies suggest that P. brasiliensis enolase, in the surface, is able to generate plasmin from plasminogen by plasminogen activator. Therefore, it was also demonstrated that this protein is secreted and able to promote fungus adhesion and invasion to cells. These findings clearly establish the role of enolase in the patogenicity of P. brasiliensis.Paraccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM), uma micose sistêmica, prevalente na América Latina. A matriz extracelular (MEC) é uma rede complexa formada por colágeno, laminina, fibronectina, entre outros componentes, que, quando exposta, é o local inicial de adesão do fungo. Nosso objetivo foi estudar genes envolvidos nesse processo de adesão utilizando Análise Diferencial Representacional (RDA). RDA é um método de subtração acoplado a PCR que permite o isolamento de genes diferencialmente expressos entre duas populações de cDNAs diferentes. Assim, cDNAs foram sintetizados a partir de RNAs extraídos de células leveduriformes de P. brasiliensis aderidos à colágeno e fibronectina para identificar genes super-expressos nestas condições. Genes envolvidos com vários processos celulares foram observados e PbCtr3 (transportador de cobre) e enolase (PbEno) foram escolhidos para análises adicionais. Um peptídeo sintético (PbCTR3) e a proteína recombinante (rPbEno) foram utilizados, juntamente com o anticorpo policlonal antirPbEno em análises funcionais com componentes da MEC e plasminogênio. Os estudos sugerem que a enolase de P. brasiliensis, localizada na parede celular, é capaz de gerar plasmina a partir do plasminogênio mediada pelo ativador de plasminôgenio. Além disso, foi também demonstrado que esta proteína é secretada sendo capaz de promover a adesão e invasão do fungo a células. Esses estudos claramente estabelecem o papel da enolase na patogenicidade de P. brasiliensis

Recombinant BCG: innovations on an old vaccine. Scope of BCG strains and strategies to improve long-lasting memory

Submitted by Jaqueline Silva ([email protected]) on 2018-11-01T18:35:48Z No. of bitstreams: 2 Artigo - Adeliane Castro da Costa - 2014.pdf: 461993 bytes, checksum: d6bacb2d38d85ebaeed76b4cbdae2f0f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Rejected by Luciana Ferreira ([email protected]), reason: No Lattes o nome está : Ana Paula Junqueira-Kipnis on 2018-11-05T09:27:14Z (GMT)Submitted by Jaqueline Silva ([email protected]) on 2018-11-06T12:02:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Adeliane Castro da Costa - 2014.pdf: 461993 bytes, checksum: d6bacb2d38d85ebaeed76b4cbdae2f0f (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-11-07T09:55:04Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Adeliane Castro da Costa - 2014.pdf: 461993 bytes, checksum: d6bacb2d38d85ebaeed76b4cbdae2f0f (MD5)Made available in DSpace on 2018-11-07T09:55:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Adeliane Castro da Costa - 2014.pdf: 461993 bytes, checksum: d6bacb2d38d85ebaeed76b4cbdae2f0f (MD5) Previous issue date: 2014-04-07Bacille Calmette–Guérin (BCG), an attenuated vaccine derived from Mycobacterium bovis, is the current vaccine of choice against tuberculosis (TB). Despite its protection against activeTB in children, BCG has failed to protect adults againstTB infection and active disease development, especially in developing countries where the disease is endemic. Currently, there is a significant effort toward the development of a newTB vaccine.This review article aims to address publications on recombinant BCG (rBCG) published in the last 5 years, to highlight the strategies used to develop rBCG, with a focus on the criteria used to improve immunological memory and protection compared with BCG. The literature review was done in April 2013, using the key words TB, rBCG vaccine, and memory. This review discusses the BCG strains and strategies currently used for the modification of BCG, including: overexpression of Mycobacterium tuberculosis (Mtb) immunodominant antigens already present in BCG; gene insertion of immunodominant antigens from Mtb absent in the BCG vaccine; combination of introduction and overexpression of genes that are lost during the attenuation process of BCG; BCG modifications for the induction of CD8CT-cell immune responses and cytokines expressing rBCG. Among the vaccines discussed,VPM1002, also called rBCGDureC:hly, is currently in human clinical trials. Much progress has been made in the effort to improve BCG, with some promising candidates, but considerable work is still required to address functional long-lasting memory

Leptospira interrogans enolase is secreted extracellularly and interacts with plasminogen

Ko, Albert Icksang “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-07-14T13:52:40Z No. of bitstreams: 1 Nogueira SV leptospira interrogans enolase....pdf: 596360 bytes, checksum: 25c1e60a4d8ada1452d8e816c20ca1ec (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-07-14T14:11:43Z (GMT) No. of bitstreams: 1 Nogueira SV leptospira interrogans enolase....pdf: 596360 bytes, checksum: 25c1e60a4d8ada1452d8e816c20ca1ec (MD5)Made available in DSpace on 2017-07-14T14:11:43Z (GMT). No. of bitstreams: 1 Nogueira SV leptospira interrogans enolase....pdf: 596360 bytes, checksum: 25c1e60a4d8ada1452d8e816c20ca1ec (MD5) Previous issue date: 2013National Institutes of Health (AI080615, AI088752 and AI052473)University of Maryland. Virginia Maryland Regional College of Veterinary Medicine. Department of Veterinary Medicine. Maryland, USAUniversity of Maryland. Virginia Maryland Regional College of Veterinary Medicine. Department of Veterinary Medicine. Maryland, USAUniversity of Maryland. Virginia Maryland Regional College of Veterinary Medicine. Department of Veterinary Medicine. Maryland, USAUniversity of Maryland. Virginia Maryland Regional College of Veterinary Medicine. Department of Veterinary Medicine. Maryland, USAYale University School of Public Health. Department of Epidemiology of Microbial Diseases. New Haven, Connecticut, USAYale University School of Public Health. Department of Epidemiology of Microbial Diseases. New Haven, Connecticut, USAUniversity of Maryland. Virginia Maryland Regional College of Veterinary Medicine. Department of Veterinary Medicine. Maryland, USALeptospira interrogans is the agent for leptospirosis, an important zoonosis in humans and animals across the globe. Surface proteins of invading pathogens, such as L. interrogans, are thought to be responsible for successful microbial persistence in vivo via interaction with specific host components. In particular, a number of invasive infectious agents exploit host proteolytic pathways, such as one involving plasminogen (Pg), which aid in efficient pathogen dissemination within the host. Here we show that L. interrogans serovar Lai binds host Pg and that the leptospiral gene product LA1951, annotated as enolase, is involved in this interaction. Interestingly, unlike in related pathogenic Spirochetes, such as Borrelia burgdorferi, LA1951 is not readily detectable in the L. interrogans outer membrane. We show that the antigen is indeed secreted extracellularly; however, it can reassociate with the pathogen surface, where it displays Pg-binding and measurable enzymatic activity. Hamsters infected with L. interrogans also develop readily detectable antibody responses against enolase. Taken together, our results suggest that the L. interrogans enolase has evolved to play a role in pathogen interaction with host molecules, which may contribute to the pathogenesis of leptospirosis

Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis , is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis . Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S -transferase (GST) fusion protein (recombinant P. brasiliensis enolase [r Pb Eno]). The P. brasiliensis native enolase ( Pb Eno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-r Pb Eno antibody. Immobilized purified r Pb Eno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and r Pb Eno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between Pb Eno and plasminogen was lysine dependent. In competition experiments, purified r Pb Eno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis , suggesting that this interaction required surface-localized Pb Eno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of r Pb Eno. The expression of Pb Eno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated Pb Eno may contribute to the pathogenesis of P. brasiliensis

Plasminogen (Pg) binds <i>L. interrogans</i> enolase and activates plasmin.

<p><u>The error bars indicate the standard deviations from three independent experiments performed in triplicate</u>. * <i><u>P</u></i><u> < 0.05</u>. (A) <u>Pg binds to <i>L. interrogans</i> in a concentration-dependent manner. Microtiter plates were coated with fixed cells and incubated with various concentrations of human plasminogen (hPg)</u>. (B) <u><i>L. interrogans</i> converts Pg into plasmin in the presence of tissue plasminogen (tPA). Well-bound <i>L. interrogans</i> cells were incubated with hPg (1µg) and a chromogenic substrate (D-valyl-L-lysyl-<i>p</i>-nitroaniline hydrochloride) in the presence or absence of tPA and a known Pg inhibitor, a lysine analogue, ε-ACA</u>. (C) <u>A Pg inhibitor</u> blocks <i>L. interrogans</i>-hPg interaction. Microtiter plates were coated with fixed <i>L. interrogans</i> and increasing concentrations (0 to 100 mM) of ε-ACA were incubated with a fixed amount (1µg) of hPg. (D) Fibrinolytic activity of Pg-bound <i>L. interrogans</i>. Panels represent <i>L. interrogans</i> cells in the presence or absence of hPg and tPA. Arrow denotes fibrinolytic activity of spirochetes only in the presence of both hPg and tPA.</p

Cellular localization of <i>L. interrogans</i> enolase.

<p>(A) Subcellular localization of enolase. <i>L. interrogans</i> whole cells (WC) were separated into outer membrane (OM) and protoplasmic cylinder (PC) fractions, resolved by SDS-PAGE, and immunoblotted with antibodies specific for enolase or proteins known to localize in the OM (LipL32) or in the inner membrane (LipL31). (B) Enolase is secreted extracellularly. Viability of the <i>L. interrogans</i> culture was determined by microscopy, and the supernatant was collected from a culture of intact viable cells. The samples were filtered, concentrated, and analyzed by 2D gel electrophoresis followed by immunoblotting assays using antibodies against enolase (upper panel) or a subcellular protein LipL31 (lower panel). </p

Enolase can be detected on <i>L. interrogans</i> surfaceand specifically interacts with outer membrane proteins.

<p>(<b>A</b>) Detection of enolase on the surface of intact <i>L. interrogans</i>. Microtiter plates were coated in the absence or presence of intact or lysed <i>L. interrogans</i> (10⁹/ml) and incubated with enolase antibody. Bound antibody was detected using HRP-labeled secondary antibodies and TMB substrate for color development, which was recorded at <i>A</i>₄₅₀. A known surface-exposed and outer membrane protein, LipL32, and a subsurface inner membrane protein, LipL31, were used as controls. (<b>B</b>) Interaction of enolase with OM proteins. A fixed amount (1µg) of solubilized proteins from isolated OM vesicles were coated on microtiter plates and assessed for binding with increasing amount of recombinant enolase, as described in panel B. The binding of enolase to immobilized OM proteins reached saturation at 5 µg, as there is a significant increase (P < 0.001) in individual OD values between 0-5 µg, while the difference between 5 and 10 µg values is nonsignificant (P > 0.05). (<b>C</b>) Recognition of recombinant enolase by infected hamster serum as assessed by immunoblotting. Recombinant enolase was probed with antibodies produced in immunized mice or antiserum collected from hamster infected with <i>L. interrogans</i>. The arrow indicates the position of enolase. Migration of protein standards is shown to the left.</p

Enzymatic activities of recombinant and native surface-associated <i>L. interrogans</i> enolase.

<p>(A) Enolase activity of the recombinant enolase is highly saturable over time. Enzyme activity was measured by recoding the catalysis of 2- phosphoglycerate to phosphoenolpyruvate for a period of 20 min using 1.6 µg of recombinant enolase. (B) Substrate-dependent saturation of enzymatic activities of recombinant enolase. Increasing concentrations of the substrate (2- phosphoglycerate) were incubated with a fixed amount (4 µg) of enolase. (C) Enolase activity is detectable on the surface of intact <i>L. interrogans</i>. Conversion of 2- phosphoglycerate to phosphoenolpyruvate was used to measure enolase activity in the presence of intact <i>L. interrogans</i> or <i>E. coli</i> cells. The error bars indicate the standard deviations from three independent experiments performed in triplicates, * P <0.05.</p

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pl 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.FAPESPConselho Nacional de Desenvolvirnento Cientifico e Tecnologico (CNPq)CAPESFCF-UNES

A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

<div><p>Tuberculosis (TB) is an infectious disease caused by <i>Mycobacterium tuberculosis</i> (Mtb) that is a major public health problem. The vaccine used for TB prevention is <i>Mycobacterium bovis</i> bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine <i>in vivo</i> was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.</p></div