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

ABSTRACT 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 [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. 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 rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.

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A 32-Kilodalton Hydrolase Plays an Important Role in Paracoccidioides brasiliensis Adherence to Host Cells and Influences Pathogenicity

Infection and Immunity ◽

10.1128/iai.00692-10 ◽

2010 ◽

Vol 78 (12) ◽

pp. 5280-5286 ◽

Cited By ~ 35

Author(s):

Orville Hernández ◽

Agostinho J. Almeida ◽

Angel Gonzalez ◽

Ana Maria Garcia ◽

Diana Tamayo ◽

...

Keyword(s):

Epithelial Cells ◽

Paracoccidioides Brasiliensis ◽

Protein A ◽

Host Cells ◽

Yeast Cells ◽

Dimorphic Fungus ◽

Morphological Alterations ◽

Cell Vitality ◽

Initial Interaction ◽

Mouse Model Of Infection

ABSTRACT One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.003830-0 ◽

2009 ◽

Vol 58 (6) ◽

pp. 706-713 ◽

Cited By ~ 50

Author(s):

Fabiana Cristina Donofrio ◽

Ana Carolina Alvarez Calil ◽

Elaine Toscano Miranda ◽

Ana Marisa Fusco Almeida ◽

Gil Benard ◽

...

Keyword(s):

Epithelial Cells ◽

Solid Medium ◽

Binding Protein ◽

Paracoccidioides Brasiliensis ◽

Yeast Cells ◽

Blood Agar ◽

Isolation And Identification ◽

Cell Extracts ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

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 pI 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.

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Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei

Scientific Reports ◽

10.1038/s41598-020-78178-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kritsada Pruksaphon ◽

Mc Millan Nicol Ching ◽

Joshua D. Nosanchuk ◽

Anna Kaltsas ◽

Kavi Ratanabanangkoon ◽

...

Keyword(s):

Phase Transition ◽

Therapeutic Potential ◽

Specific Antigen ◽

Human Monocyte ◽

Host Cells ◽

Yeast Cells ◽

Fungal Pathogenesis ◽

Dimorphic Fungus ◽

Talaromyces Marneffei

AbstractTalaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T.marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells’ transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T.marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

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BsaB, a Novel Adherence Factor of Group B Streptococcus

Infection and Immunity ◽

10.1128/iai.01014-13 ◽

2013 ◽

Vol 82 (3) ◽

pp. 1007-1016 ◽

Cited By ~ 21

Author(s):

Shengmei Jiang ◽

Michael R. Wessels

Keyword(s):

Extracellular Matrix ◽

Epithelial Cells ◽

Group B Streptococcus ◽

Surface Protein ◽

Chronically Ill ◽

Host Cells ◽

Upstream Open Reading Frame ◽

Content Type ◽

Group B

ABSTRACTStreptococcus agalactiae(group BStreptococcus[GBS]) is a leading cause of neonatal sepsis and meningitis, peripartum infections in women, and invasive infections in chronically ill or elderly individuals. GBS can be isolated from the gastrointestinal or genital tracts of up to 30% of healthy adults, and infection is thought to arise from invasion from a colonized mucosal site. Accordingly, bacterial surface components that mediate attachment of GBS to host cells or the extracellular matrix represent key factors in the colonization and infection of the human host. We identified a conserved GBS gene of unknown function that was predicted to encode a cell wall-anchored surface protein. Deletion of the gene and a cotranscribed upstream open reading frame (ORF) in GBS strain 515 reduced bacterial adherence to VK2 vaginal epithelial cellsin vitroand reduced GBS binding to fibronectin-coated microtiter wells. Expression of the gene product inLactococcus lactisconferred the ability to adhere to VK2 cells, to fibronectin and laminin, and to fibronectin-coated ME-180 cervical epithelial cells. Expression of the recombinant protein inL. lactisalso markedly increased biofilm formation. The adherence function of the protein, namedbacterialsurfaceadhesin of GBS (BsaB), depended both on a central BID1 domain found in bacterial intimin-like proteins and on the C-terminal portion of the BsaB protein. Expression of BsaB in GBS, like that of several other adhesins, was regulated by the CsrRS two-component system. We conclude that BsaB represents a newly identified adhesin that participates in GBS attachment to epithelial cells and the extracellular matrix.

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Evaluation of the Role of Staphylococci in the Pathomechanism of Conjunctivitis

10.21203/rs.3.rs-191872/v1 ◽

2021 ◽

Author(s):

Ewa Jasińska ◽

Agnieszka Bogut ◽

Agnieszka Magryś ◽

Alina Olender

Keyword(s):

Antibiotic Resistance ◽

Epithelial Cells ◽

Biofilm Formation ◽

Methicillin Resistance ◽

Microtiter Plate ◽

Host Cells ◽

Diffusion Method ◽

Microtiter Plate Assay ◽

Low Prevalence

Abstract Purpose: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.Methods: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtiter plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.Results: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.Conclusions: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

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The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822012000600016 ◽

2012 ◽

Vol 45 (6) ◽

pp. 739-744 ◽

Cited By ~ 2

Author(s):

Francisco Laurindo da Silva ◽

Raphael Sanzio Pimenta ◽

Juliana Fonseca Moreira da Silva ◽

Déborah Aparecida Negrão Corrêa ◽

Ary Corrêa Junior

Keyword(s):

Epithelial Cell ◽

Paracoccidioides Brasiliensis ◽

Yeast Cells ◽

Epithelial Cell Line ◽

Con A ◽

Culture Methods ◽

Disease Treatment ◽

Adhesion Behavior ◽

And Control

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

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The ultrastructure of Candida albicans infections

Canadian Journal of Microbiology ◽

10.1139/m81-181 ◽

1981 ◽

Vol 27 (11) ◽

pp. 1156-1164 ◽

Cited By ~ 36

Author(s):

Thomas J. Marrie ◽

J. William Costerton

Keyword(s):

Candida Albicans ◽

Oral Candidiasis ◽

Ruthenium Red ◽

Host Cells ◽

Yeast Cells ◽

Positive Matrix ◽

Urine Sediment ◽

Bacteria Adhesion

Scrapings of Candida albicans plaques from the tongue and buccal mucosa of patients with oral candidiasis were examined electron microscopy. In addition, urine sediment from patients with infection of their catheterized urinary tracts was similar examined. Three types of C. albicans – oral epithelial cell interactions were noted: a loose adherence apparently mediated by ruthenium red positive matrix, a "tight" adherence where no space could be seen between the host and yeast cell, and invasions host cells by yeast hyphal elements. Adhesion of Candida blastospores to hyphal elements and adhesion of bacteria to Candida cells was also frequently observed.Urine sediments from patients with mixed bacteria–yeast infections demonstrated adhesion of the bacteria to the yeast cells. This phenomenon was also demonstrated in in vitro experiments and fibrous ruthenium red material invariably occupied the zo*** of adhesion.Phagocytosis of yeast by polymorphonuclear leukocytes was found in urinary, but not in oral, candidiasis. Our in vivo and vitro observations indicate that a ruthenium red positive matrix covers the surfaces involved in the yeast to yeast, yeast to ho and yeast to bacteria adhesion.

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Protein Coding and Long Noncoding RNA (lncRNA) Transcriptional Landscape in SARS-CoV-2 Infected Bronchial Epithelial Cells Highlight a Role for Interferon and Inflammatory Response

Genes ◽

10.3390/genes11070760 ◽

2020 ◽

Vol 11 (7) ◽

pp. 760 ◽

Cited By ~ 10

Author(s):

Radhakrishnan Vishnubalaji ◽

Hibah Shaath ◽

Nehad M. Alajez

Keyword(s):

Epithelial Cells ◽

Inflammatory Response ◽

Noncoding Rna ◽

Cell Model ◽

Bronchial Epithelial Cells ◽

Host Cells ◽

Protein Coding ◽

Bronchial Epithelial ◽

Upstream Regulator

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.

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Increased Virulence of a Fibronectin-Binding Protein Mutant of Staphylococcus aureus in a Rat Model of Pneumonia

Infection and Immunity ◽

10.1128/iai.70.7.3865-3873.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3865-3873 ◽

Cited By ~ 42

Author(s):

Mary C. McElroy ◽

David J. Cain ◽

Christine Tyrrell ◽

Timothy J. Foster ◽

Christopher Haslett

Keyword(s):

Staphylococcus Aureus ◽

Epithelial Cells ◽

Rat Model ◽

Binding Proteins ◽

Binding Protein ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Fibronectin Binding Protein ◽

Fibronectin Binding

ABSTRACT Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia.

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Efficacy of Plant-Derived Antimicrobials in Controlling Enterohemorrhagic Escherichia coli Virulence In Vitro

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-104 ◽

2016 ◽

Vol 79 (11) ◽

pp. 1965-1970 ◽

Cited By ~ 11

Author(s):

SANGEETHA ANANDA BASKARAN ◽

ANUP KOLLANOOR-JOHNY ◽

MEERA SURENDRAN NAIR ◽

KUMAR VENKITANARAYANAN

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Antimicrobial Agents ◽

Intestinal Epithelial Cells ◽

Enterohemorrhagic Escherichia Coli ◽

Host Cells ◽

Intestinal Epithelial ◽

Virulence Gene Expression ◽

Human Intestinal Epithelial Cells

ABSTRACTEscherichia coli O157:H7 is a major foodborne pathogen that can cause serious human illness characterized by hemorrhagic diarrhea and kidney failure. The pathology of enterohemorrhagic E. coli O157:H7 (EHEC) infection is primarily mediated by verotoxins, which bind to the globotriaosylceramide receptor on host cells. Antibiotics are contraindicated for treating EHEC infection because they lead to increased verotoxin release, thereby increasing the risk of renal failure and death in patients. Thus, alternative strategies are needed for controlling EHEC infections in humans. This study investigated the effect of subinhibitory concentrations of five plant-derived antimicrobial agents (PDAs) that are generally considered as safe, i.e., trans-cinnamaldehyde, eugenol, carvacrol, thymol, and β-resorcylic acid, on EHEC motility, adhesion to human intestinal epithelial cells, verotoxin production, and virulence gene expression. All tested PDAs reduced EHEC motility and attachment to human intestinal epithelial cells (P < 0.05) and decreased verotoxin synthesis by EHEC. The reverse transcription real-time PCR data revealed that PDAs decreased the expression of critical virulence genes in EHEC (P < 0.05). The results collectively suggest that these PDAs could be used to reduce EHEC virulence, but follow-up studies in animal models are necessary to validate these findings.

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