scholarly journals Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

2011 ◽  
Vol 80 (1) ◽  
pp. 429-440 ◽  
Author(s):  
Xianping Li ◽  
Meihua Gao ◽  
Xuelin Han ◽  
Sha Tao ◽  
Dongyu Zheng ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Aspergillus Fumigatus ◽  
Phospholipase D ◽  
Morphological Characteristics ◽  
Pathogenic Fungi ◽  
Lung Epithelial Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Important Virulence Factor ◽  
The Impact

ABSTRACTAspergillus fumigatusis the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge of the impact of phospholipases onA. fumigatusvirulence is rather limited. In this study, disruption of thepldgene encoding phospholipase D (PLD), an important member of the phospholipase protein family inA. fumigatus, was confirmed to significantly decrease both intracellular and extracellular PLD activity ofA. fumigatus. Thepldgene disruption did not alter conidial morphological characteristics, germination, growth, and biofilm formation but significantly suppressed the internalization ofA. fumigatusinto A549 epithelial cells without affecting conidial adhesion to epithelial cells. Importantly, the suppressed internalization was fully rescued in the presence of 100 μM phosphatidic acid, the PLD product. Indeed, complementation ofpldrestored the PLD activity and internalization capacity ofA. fumigatus. Phagocytosis ofA. fumigatusconidia by J774 macrophages was not affected by the absence of thepldgene. Pretreatment of conidia with 1-butanol and a specific PLD inhibitor decreased the internalization ofA. fumigatusinto A549 epithelial cells but had no effect on phagocytosis by J774 macrophages. Finally, loss of thepldgene attenuated the virulence ofA. fumigatusin mice immunosuppressed with hydrocortisone acetate but not with cyclophosphamide. These data suggest that PLD ofA. fumigatusregulates its internalization into lung epithelial cells and may represent an important virulence factor forA. fumigatusinfection.

scholarly journals Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice

2021 ◽  
Vol 22 (11) ◽  
pp. 6146
Author(s):  
Dominik H. W. Leitz ◽  
Julia Duerr ◽  
Surafel Mulugeta ◽  
Ayça Seyhan Agircan ◽  
Stefan Zimmermann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Fibrosis ◽  
Growth Failure ◽  
Lung Epithelial Cells ◽  
Na Channel ◽  
Preclinical Development ◽  
Neonatal Mice ◽  
Mucin Expression ◽  
Lung Epithelial ◽  
The Impact

Recent studies found that expression of Nedd4‑2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4‑2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFβ signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4‑2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4‑2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4‑2fl/fl/CCSP‑rtTA2S‑M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP‑C trafficking. We found that the congenital deletion of Nedd4‑2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4‑2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4‑2 may support studies of the pathogenesis and preclinical development of therapies for chILD.

scholarly journals Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

2014 ◽  
Vol 80 (20) ◽  
pp. 6465-6472 ◽  
Author(s):  
Sarah L. Robinson ◽  
Daniel G. Panaccione
Keyword(s):  
Aspergillus Fumigatus ◽  
Candidate Genes ◽  
Mass Spectra ◽  
Ergot Alkaloids ◽  
Lysergic Acid ◽  
Precursor Feeding ◽  
Gene Encoding ◽  
Content Type ◽  
Multiple Reactions ◽  
Important Pathway

ABSTRACTDifferent lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungusEpichloë festucaevar.lolii×Epichloë typhinaisolate Lp1 (henceforth referred to asEpichloësp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate geneseasHandcloAwere expressed in a mutant strain of the moldAspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with theEpichloësp. Lp1 allele ofeasA, which encodes an enzyme that catalyzed the synthesis of agroclavine from anA. fumigatusintermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressingeasAandcloAfromEpichloësp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result ofEpichloësp. Lp1easAexpression in the absence ofcloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.

scholarly journals Preexposure to Isavuconazole Increases the Virulence of Mucorales but Not Aspergillus fumigatus in a Drosophila melanogaster Infection Model

2018 ◽  
Vol 63 (2) ◽  
pp. e01896-18 ◽  
Author(s):  
Sebastian Wurster ◽  
Russell E. Lewis ◽  
Nathaniel D. Albert ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Drosophila Melanogaster ◽  
Aspergillus Fumigatus ◽  
Clinical Isolates ◽  
Infection Model ◽  
Content Type ◽  
The Impact

ABSTRACT Breakthrough mucormycosis in patients receiving isavuconazole prophylaxis or therapy has been reported. We compared the impact of isavuconazole and voriconazole exposure on the virulence of clinical isolates of Aspergillus fumigatus and different Mucorales species in a Drosophila melanogaster infection model. In contrast to A. fumigatus, a hypervirulent phenotype was found in all tested Mucorales upon preexposure to either voriconazole or isavuconazole. These findings may contribute to the explanation of breakthrough mucormycosis in isavuconazole-treated patients.

scholarly journals Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus

mBio ◽  
2021 ◽  
Author(s):  
José P. Guirao-Abad ◽  
Martin Weichert ◽  
Ginés Luengo-Gil ◽  
Sarah Sze Wah Wong ◽  
Vishukumar Aimanianda ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Resistance ◽  
Secretory Pathway ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Gene Encoding ◽  
Content Type ◽  
Downstream Target ◽  
P Type ◽  
Er Homeostasis

The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus .

scholarly journals A Fungus-Specific Ras Homolog Contributes to the Hyphal Growth and Virulence of Aspergillus fumigatus

2005 ◽  
Vol 4 (12) ◽  
pp. 1982-1989 ◽  
Author(s):  
Jarrod R. Fortwendel ◽  
Wei Zhao ◽  
Ruchi Bhabhra ◽  
Steven Park ◽  
David S. Perlin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Growth Control ◽  
Biomass Accumulation ◽  
Hyphal Growth ◽  
Pathogenic Fungi ◽  
Total Biomass ◽  
Gene Encoding ◽  
Directional Growth ◽  
Solid Media ◽  
Hyphal Morphology

ABSTRACT The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The ΔrasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited ∼65% survival compared to ∼10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

scholarly journals Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606Twith Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

2012 ◽  
Vol 80 (3) ◽  
pp. 1015-1024 ◽  
Author(s):  
Jennifer A. Gaddy ◽  
Brock A. Arivett ◽  
Michael J. McConnell ◽  
Rafael López-Rojas ◽  
Jerónimo Pachón ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Iron Acquisition ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Content Type ◽  
Alveolar Epithelial

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606Ttype strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays usingGalleria mellonellalarvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606Tcells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with theseex vivoandin vivoapproaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606Tto establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606Tstrain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin.

scholarly journals Chemotherapy with Phage Lysins Reduces Pneumococcal Colonization of the Respiratory Tract

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Bruno Corsini ◽  
Roberto Díez-Martínez ◽  
Leire Aguinagalde ◽  
Fernando González-Camacho ◽  
Esther García-Fernández ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Bacterial Load ◽  
Antimicrobial Activities ◽  
Lung Epithelial Cells ◽  
Lytic Enzymes ◽  
Nasopharyngeal Colonization ◽  
Content Type ◽  
Human Epithelial Cells ◽  
Pneumococcal Colonization

ABSTRACT Bacteriophage-borne lytic enzymes, also named lysins or enzybiotics, are efficient agents for the killing of bacterial pathogens. The colonization of the respiratory tract by Streptococcus pneumoniae is a prerequisite for the establishment of the infection process. Hence, we have evaluated the antibacterial activities of three different lysins against pneumococcal colonization using human nasopharyngeal and lung epithelial cells as well as a mouse model of nasopharyngeal colonization. The lysins tested were the wild-type Cpl-1, the engineered Cpl-7S, and the chimera Cpl-711. Moreover, we included amoxicillin as a comparator antibiotic. Human epithelial cells were infected with three different multidrug-resistant clinical isolates of S. pneumoniae followed by a single dose of the corresponding lysin. The antimicrobial activities of these lysins were also evaluated using a mouse nasopharyngeal carriage model. The exposure of the infected epithelial cells to Cpl-7S did not result in the killing of any of the pneumococcal strains investigated. However, the treatment with Cpl-1 or Cpl-711 increased the killing of S. pneumoniae organisms adhered to both types of human epithelial cells, with Cpl-711 being more effective than Cpl-1, at subinhibitory concentrations. In addition, a treatment with amoxicillin had no effect on reducing the carrier state, whereas mice treated by the intranasal route with Cpl-711 showed significantly reduced nasopharyngeal colonization, with no detection of bacterial load in 20 to 40% of the mice. This study indicates that Cpl-1 and Cpl-711 lysins might be promising antimicrobial candidates for therapy against pneumococcal colonization.

scholarly journals Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Helen R. Clark ◽  
Allison B. Powell ◽  
Kelsey A. Simmons ◽  
Tariq Ayubi ◽  
Shiv D. Kale
Keyword(s):  
Epithelial Cells ◽  
Aspergillus Fumigatus ◽  
Clinical Isolate ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Initial Contact ◽  
Airway Epithelial ◽  
Class Iii ◽  
Content Type ◽  
Therapeutic Development

ABSTRACT Aspergillus fumigatus is a ubiquitous mold that produces small airborne conidia capable of traversing deep into the respiratory system. Recognition, processing, and clearance of A. fumigatus conidia by bronchial airway epithelial cells are thought to be relevant to host defense and immune signaling. Using z-stack confocal microscopy, we observed that only 10 to 20% of adherent conidia from the AF293 clinical isolate are internalized by BEAS-2B cells 6 h postchallenge and not prior. Similar percentages of internalization were observed for the CEA10 clinical isolate. A large subset of both AF293 and CEA10 conidia are rendered metabolically inactive without internalization at 3 h postchallenge by BEAS-2B cells. A significantly larger percentage of CEA10 conidia are metabolically active at 9 and 12 h postchallenge in comparison to the AF293 isolate, demonstrating heterogeneity among clinical isolates. We identified 7 host markers (caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1) that consistently localized around internalized conidia 9 h postchallenge. Transient gene silencing of RAB5C, PIK3C3, and flotillin-2 resulted in a larger population of metabolically active conidia. Our findings emphasize the abundance of both host phosphatidylinositol 3-phosphate (PI3P) and PI4P around internalized conidia, as well as the importance of class III PI3P kinase for conidial processing. Therapeutic development focused on RAB5C-, PIK3C3-, and flotillin-2-mediated pathways may provide novel opportunities to modulate conidial processing and internalization. Determination of how contacted, external conidia are processed by airway epithelial cells may also provide a novel avenue to generate host-targeted therapeutics. IMPORTANCE Conidia from the fungus Aspergillus fumigatus are notorious for their ability to stay airborne. This characteristic is believed to allow conidia to penetrate into the cleanest environments. Several hundred conidia are thought to be inhaled each day by a given individual and then expelled by mucociliary clearance. Given that airway epithelial cells make up a significant portion of the pulmonary-air interface, we set out to determine the percentage of conidia that are actually internalized after initial contact with airway epithelial cells. We determined this through an in vitro assay using an immortalized bronchial airway epithelial cell line known as BEAS-2B. Our results suggest a small fraction of conidia are internalized by BEAS-2B cells, while the majority stay adherent to the surface of cells or are washed away during sample processing. Internalization of conidia was observed at 6 h postchallenge and not prior. Our data also indicate conidia are rendered metabolically inactive within 3 h of challenge, suggesting BEAS-2B cells process a large number of conidia without internalization in this early time frame. We have also identified several host endocytosis markers that localize around internalized conidia as well as contribute to the processing of conidia. Understanding how these host endocytosis markers affect the processing of internal and/or external conidia may provide a novel avenue for therapeutic development.

scholarly journals Linocin and OmpW Are Involved in Attachment of the Cystic Fibrosis-Associated Pathogen Burkholderia cepacia Complex to Lung Epithelial Cells and Protect Mice against Infection

2016 ◽  
Vol 84 (5) ◽  
pp. 1424-1437 ◽  
Author(s):  
Siobhán McClean ◽  
Marc E. Healy ◽  
Cassandra Collins ◽  
Stephen Carberry ◽  
Luke O'Shaughnessy ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Burkholderia Cepacia ◽  
Therapeutic Option ◽  
Lung Epithelial Cells ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Content Type ◽  
Lung Epithelial ◽  
Proteomics Approach

Members of theBurkholderia cepaciacomplex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species:Burkholderia cenocepacia, the most virulent, andB. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species.Escherichia colistrains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responsesin vivo. Mice immunized with either recombinant linocin or OmpW were protected fromB. cenocepaciaandB. multivoranschallenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.

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