scholarly journals The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungusCryptococcus neoformans.

2018 ◽  
Author(s):  
Mélissa Caza ◽  
Guanggan Hu ◽  
Eric David Neilson ◽  
Minsu Cho ◽  
Won Hee Jung ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mitochondrial Function ◽  
Iron Uptake ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Uptake System ◽  
Poor Growth ◽  
Enhanced Sensitivity ◽  
Electron Transport Complexes ◽  
Sm Protein

ABSTRACTThe battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus,Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, avps45deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and avps45mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, thevps45mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of thevps45mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

scholarly journals The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans

2018 ◽  
Vol 14 (8) ◽  
pp. e1007220 ◽  
Author(s):  
Mélissa Caza ◽  
Guanggan Hu ◽  
Erik David Nielson ◽  
Minsu Cho ◽  
Won Hee Jung ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mitochondrial Function ◽  
Iron Uptake ◽  
Pathogenic Fungus ◽  
Sm Protein

scholarly journals Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Adriana Oliveira Manfiolli ◽  
Filipe Silva Siqueira ◽  
Thaila Fernanda dos Reis ◽  
Patrick Van Dijck ◽  
Sanne Schrevens ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Cross Talk ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Mitogen Activated Protein Kinase ◽  
Melanin Production ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Salvage Pathways

ABSTRACT The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade. IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

scholarly journals Role of Ferroxidases in Iron Uptake and Virulence of Cryptococcus neoformans

2009 ◽  
Vol 8 (10) ◽  
pp. 1511-1520 ◽  
Author(s):  
Won Hee Jung ◽  
Guanggan Hu ◽  
Wayne Kuo ◽  
James W. Kronstad
Keyword(s):  
Cryptococcus Neoformans ◽  
Membrane Trafficking ◽  
Iron Uptake ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Antifungal Drugs ◽  
Iron Limitation ◽  
Ergosterol Biosynthesis ◽  
Uptake System ◽  
Intracellular Iron

ABSTRACT Iron acquisition is a critical aspect of the virulence of many pathogenic microbes, and iron limitation is an important defense mechanism for mammalian hosts. We are examining mechanisms of iron regulation and acquisition in the fungal pathogen Cryptococcus neoformans, and here, we characterize the roles of the ferroxidases Cfo1 and Cfo2. Cfo1 is required for the reductive iron uptake system that mediates the utilization of transferrin, an important iron source for C. neoformans during infection. The virulence of a cfo1 mutant was attenuated in a mouse model of cryptococcosis, and the mutant also displayed increased sensitivities to the antifungal drugs fluconazole and amphotericin B. Wild-type levels of drug sensitivity were restored by the addition of exogenous heme, which suggested that reduced levels of intracellular iron may curtail heme levels and interfere with ergosterol biosynthesis. We constructed green fluorescent protein (GFP) fusion proteins and found elevated expression of Cfo1-GFP upon iron limitation, as well as localization of the fusion to the plasma membrane. Trafficking to this location was disrupted by a defect in the catalytic subunit of cyclic AMP-dependent protein kinase. This result is consistent with findings from studies indicating an influence of the kinase on the expression of protein-trafficking functions in C. neoformans.

scholarly journals Molecular Analysis of the Candida albicans Homolog of Saccharomyces cerevisiae MNN9, Required for Glycosylation of Cell Wall Mannoproteins

1999 ◽  
Vol 181 (24) ◽  
pp. 7439-7448 ◽  
Author(s):  
Susan B. Southard ◽  
Charles A. Specht ◽  
Chitra Mishra ◽  
Joan Chen-Weiner ◽  
Phillips W. Robbins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Gene Family ◽  
Molecular Analysis ◽  
Transmembrane Domain ◽  
Antifungal Drugs ◽  
Northern Analysis ◽  
Fungal Cell ◽  
Poor Growth

ABSTRACT The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the Candida albicans homolog ofSaccharomyces cerevisiae MNN9, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption. CaMNN9 has significant homology with S. cerevisiae MNN9, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in Candida. CaMNN9 resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of CaMNN9 leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with β-1,3-glucanase. Like all members of the S. cerevisiae MNN9 gene family theCamnn9Δ strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the Camnn9Δ strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of MNN9 gene family members CaVAN1and CaANP1 in the Camnn9Δ strain showed that transcription of those genes is not affected in the absence ofCaMNN9 transcription. Our results suggest that, while the role MNN9 plays in glycosylation in bothCandida and Saccharomyces is conserved, loss ofMNN9 function in C. albicans leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target.

scholarly journals The Novel J-Domain Protein Mrj1 Is Required for Mitochondrial Respiration and Virulence in Cryptococcus neoformans

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Linda C. Horianopoulos ◽  
Guanggan Hu ◽  
Mélissa Caza ◽  
Kerstin Schmitt ◽  
Peter Overby ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Mitochondrial Function ◽  
Mitochondrial Respiration ◽  
Antifungal Drugs ◽  
Complex Iii ◽  
Content Type ◽  
Mitochondrial Functions ◽  
J Domain ◽  
Domain Protein

ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans must adapt to the mammalian environment to establish an infection. Proteins facilitating adaptation to novel environments, such as chaperones, may be required for virulence. In this study, we identified a novel mitochondrial co-chaperone, Mrj1 (mitochondrial respiration J-domain protein 1), necessary for virulence in C. neoformans. The mrj1Δ and J-domain-inactivated mutants had general growth defects at both routine laboratory and human body temperatures and were deficient in the major virulence factor of capsule elaboration. The latter phenotype was associated with cell wall changes and increased capsular polysaccharide shedding. Accordingly, the mrj1Δ mutant was avirulent in a murine model of cryptococcosis. Mrj1 has a mitochondrial localization and co-immunoprecipitated with Qcr2, a core component of complex III of the electron transport chain. The mrj1 mutants were deficient in mitochondrial functions, including growth on alternative carbon sources, growth without iron, and mitochondrial polarization. They were also insensitive to complex III inhibitors and hypersensitive to an alternative oxidase (AOX) inhibitor, suggesting that Mrj1 functions in respiration. In support of this conclusion, mrj1 mutants also had elevated basal oxygen consumption rates which were completely abolished by the addition of the AOX inhibitor, confirming that Mrj1 is required for mitochondrial respiration through complexes III and IV. Furthermore, inhibition of complex III phenocopied the capsule and cell wall defects of the mrj1 mutants. Taken together, these results indicate that Mrj1 is required for normal mitochondrial respiration, a key aspect of adaptation to the host environment and virulence. IMPORTANCE Cryptococcus neoformans is the causative agent of cryptococcal meningitis, a disease responsible for ∼15% of all HIV-related deaths. Unfortunately, development of antifungal drugs is challenging because potential targets are conserved between humans and C. neoformans. In this context, we characterized a unique J-domain protein, Mrj1, which lacks orthologs in humans. We showed that Mrj1 was required for normal mitochondrial respiration and that mutants lacking Mrj1 were deficient in growth, capsule elaboration, and virulence. Furthermore, we were able to phenocopy the defects in growth and capsule elaboration by inhibiting respiration. This result suggests that the role of Mrj1 in mitochondrial function was responsible for the observed virulence defects and reinforces the importance of mitochondria to fungal pathogenesis. Mitochondria are difficult to target, as their function is also key to human cells; however, Mrj1 presents an opportunity to target a unique fungal protein required for mitochondrial function and virulence in C. neoformans.

scholarly journals cspAInfluences Biofilm Formation and Drug Resistance in Pathogenic FungusAspergillus fumigatus

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Zhongqi Fan ◽  
Zhe Li ◽  
Zongge Xu ◽  
Hongyan Li ◽  
Lixiang Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Wall ◽  
Biofilm Formation ◽  
Pathogenic Fungus ◽  
Antifungal Drugs ◽  
Lung Epithelial Cells ◽  
Targeted Gene Disruption ◽  
Lung Epithelial ◽  
Conidial Surface ◽  
Biofilm Development

The microbial cell wall plays a crucial role in biofilm formation and drug resistance.cspAencodes a repeat-rich glycophosphatidylinositol-anchored cell wall protein in the pathogenic fungusAspergillus fumigatus. To determine whethercspAhas a significant impact on biofilm development and sensitivity to antifungal drugs inA. fumigatus, a ΔcspAmutant was constructed by targeted gene disruption, and we then reconstituted the mutant to wild type by homologous recombination of a functionalcspAgene. Deletion ofcspAresulted in a rougher conidial surface, reduced biofilm formation, decreased resistance to antifungal agents, and increased internalization by A549 human lung epithelial cells, suggesting thatcspAnot only participates in maintaining the integrity of the cell wall, but also affects biofilm establishment, drug response, and invasiveness ofA. fumigatus.

The Regulatory Function of the Molecular Chaperone Hsp90 in the Cell Wall Integrity of Pathogenic Fungi

2018 ◽  
Vol 16 (1) ◽  
pp. 44-53
Author(s):  
Marina Campos Rocha ◽  
Camilla Alves Santos ◽  
Iran Malavazi
Keyword(s):  
Cell Wall ◽  
Antifungal Therapy ◽  
Molecular Chaperone ◽  
Regulatory Protein ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Hsp90 Inhibition

Different signaling cascades including the Cell Wall Integrity (CWI), the High Osmolarity Glycerol (HOG) and the Ca2+/calcineurin pathways control the cell wall biosynthesis and remodeling in fungi. Pathogenic fungi, such as Aspergillus fumigatus and Candida albicans, greatly rely on these signaling circuits to cope with different sources of stress, including the cell wall stress evoked by antifungal drugs and the host’s response during infection. Hsp90 has been proposed as an important regulatory protein and an attractive target for antifungal therapy since it stabilizes major effector proteins that act in the CWI, HOG and Ca2+/calcineurin pathways. Data from the human pathogen C. albicans have provided solid evidence that loss-of-function of Hsp90 impairs the evolution of resistance to azoles and echinocandin drugs. In A. fumigatus, Hsp90 is also required for cell wall integrity maintenance, reinforcing a coordinated function of the CWI pathway and this essential molecular chaperone. In this review, we focus on the current information about how Hsp90 impacts the aforementioned signaling pathways and consequently the homeostasis and maintenance of the cell wall, highlighting this cellular event as a key mechanism underlying antifungal therapy based on Hsp90 inhibition.

scholarly journals Expression Patterns in Reductive Iron Assimilation and Functional Consequences during Phagocytosis of Lichtheimia corymbifera, an Emerging Cause of Mucormycosis

2021 ◽  
Vol 7 (4) ◽  
pp. 272
Author(s):  
Felicia Adelina Stanford ◽  
Nina Matthias ◽  
Zoltán Cseresnyés ◽  
Marc Thilo Figge ◽  
Mohamed I. Abdelwahab Hassan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Uptake ◽  
Intracellular Transport ◽  
Pathogenic Fungus ◽  
Expression Patterns ◽  
Iron Depletion ◽  
Iron Assimilation ◽  
Human Pathogenic Fungus ◽  
Hyphal Formation ◽  
Yeast Mutants

Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.

Sign in / Sign up

Export Citation Format

Share Document