scholarly journals O-Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) Is Crucial for Cell Wall Integrity and Conidium Morphology, Especially at an Elevated Temperature

2007 ◽  
Vol 6 (12) ◽  
pp. 2260-2268 ◽  
Author(s):  
Hui Zhou ◽  
Hongyan Hu ◽  
Lijuan Zhang ◽  
Ruoyu Li ◽  
Haomiao Ouyang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Elevated Temperature ◽  
Aspergillus Fumigatus ◽  
Cell Wall Integrity ◽  
Secretory Proteins ◽  
Experimental Conditions ◽  
Human Fungal Pathogen ◽  
Conidium Formation

ABSTRACT Protein O-mannosyltransferases initiate O mannosylation of secretory proteins, which are of fundamental importance in eukaryotes. In this study, the PMT gene family of the human fungal pathogen Aspergillus fumigatus was identified and characterized. Unlike the case in Saccharomyces cerevisiae, where the PMT family is highly redundant, only one member of each PMT subfamily, namely, Afpmt1, Afpmt2, and Afpmt4, is present in A. fumigatus. Mutants with a deletion of Afpmt1 are viable. In vitro and in vivo activity assays confirmed that the protein encoded by Afpmt1 acts as an O-mannosyltransferase (AfPmt1p). Characterization of the ΔAfpmt1 mutant showed that a lack of AfPmt1p results in sensitivity to elevated temperature and defects in growth and cell wall integrity, thereby affecting cell morphology, conidium formation, and germination. In a mouse model, Afpmt1 was not required for the virulence of A. fumigatus under the experimental conditions used.

scholarly journals Monoclonal Immunoglobulin G1 Directed against Aspergillus fumigatus Cell Wall Glycoprotein Protects against Experimental Murine Aspergillosis

2005 ◽  
Vol 12 (9) ◽  
pp. 1063-1068 ◽  
Author(s):  
Ashok K. Chaturvedi ◽  
A. Kavishwar ◽  
G. B. Shiva Keshava ◽  
P. K. Shukla
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Protective Efficacy ◽  
Wide Spectrum ◽  
Kidney Tissue ◽  
Survival Times ◽  
Fungal Cell ◽  
New Strategy

ABSTRACT Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log10 units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 × 105 CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.

scholarly journals Inhibition of classical and alternative modes of respiration in C. albicans leads to cell wall remodelling and increased macrophage recognition

2018 ◽  
Author(s):  
Lucian Duvenage ◽  
Louise A. Walker ◽  
Aleksandra Bojarczuk ◽  
Simon A. Johnston ◽  
Donna M. MacCallum ◽  
...  
Keyword(s):  
Cell Wall ◽  
Electron Transport ◽  
Fungal Pathogen ◽  
Fungal Infections ◽  
Oxidase Inhibitor ◽  
Nitric Oxide Donor ◽  
Mammalian Host ◽  
Human Fungal Pathogen

AbstractThe human fungal pathogen C. albicans requires respiratory function for normal growth, morphogenesis and virulence. As such the mitochondria represent an enticing target for the development of new antifungal strategies. This possibility is further bolstered by the presence of fungal specific characteristics. However, respiration in C. albicans, as is the case in many fungal organisms, is facilitated by redundant electron transport mechanisms that makes direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity in mammals to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevent respiration, lead to a loss in viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings also demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals.Author SummaryCurrent approaches to tackling fungal infections are limited and new targets must be identified to protect against the emergence of resistant strains. We investigate the potential of targeting mitochondria, organelles required for energy production, growth and virulence, in the yeast human fungal pathogen Candida albicans. Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However release of C. albicans cells from mitochondrial inhibition appears to activate a stress response that increases traits associated with virulence. Our results make it clear that mitochondria are a valid target for the development of anti-fungal strategies but that we must determine the mechanisms by which they regulate stress signalling and virulence ahead of successful therapeutic advance.

scholarly journals Yapsins Are a Family of Aspartyl Proteases Required for Cell Wall Integrity in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (8) ◽  
pp. 1364-1374 ◽  
Author(s):  
Damian J. Krysan ◽  
Elizabeth L. Ting ◽  
Claudia Abeijon ◽  
Lee Kroos ◽  
Robert S. Fuller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Specific Activity ◽  
Wall Stress ◽  
Cell Wall Integrity ◽  
Dependent Manner ◽  
Glucan Synthesis ◽  
Aspartyl Proteases

ABSTRACT The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Δ2Δ mutant and the quintuple yapsin mutant (5ypsΔ) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5ypsΔ and yps1Δ2Δ mutants have decreased amounts of 1,3- and 1,6-β-glucan. Although there is decreased incorporation of both 1,3- and 1,6-β-glucan in the 5ypsΔ mutant in vivo, in vitro specific activity of both 1,3- and 1,6-β-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsΔ. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Δ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

scholarly journals Critical Assessment of Cell Wall Integrity Factors Contributing to in vivo Echinocandin Tolerance and Resistance in Candida glabrata

2021 ◽  
Vol 12 ◽  
Author(s):  
Rocio Garcia-Rubio ◽  
Rosa Y. Hernandez ◽  
Alissa Clear ◽  
Kelley R. Healey ◽  
Erika Shor ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Glabrata ◽  
Fungal Infections ◽  
Critical Role ◽  
Cell Wall Integrity ◽  
Fungal Cell ◽  
Fungal Host

Fungal infections are on the rise, and emergence of drug-resistant Candida strains refractory to treatment is particularly alarming. Resistance to azole class antifungals, which have been extensively used worldwide for several decades, is so high in several prevalent fungal pathogens, that another drug class, the echinocandins, is now recommended as a first line antifungal treatment. However, resistance to echinocandins is also prominent, particularly in certain species, such as Candida glabrata. The echinocandins target 1,3-β-glucan synthase (GS), the enzyme responsible for producing 1,3-β-glucans, a major component of the fungal cell wall. Although echinocandins are considered fungicidal, C. glabrata exhibits echinocandin tolerance both in vitro and in vivo, where a subset of the cells survives and facilitates the emergence of echinocandin-resistant mutants, which are responsible for clinical failure. Despite this critical role of echinocandin tolerance, its mechanisms are still not well understood. Additionally, most studies of tolerance are conducted in vitro and are thus not able to recapitulate the fungal-host interaction. In this study, we focused on the role of cell wall integrity factors in echinocandin tolerance in C. glabrata. We identified three genes involved in the maintenance of cell wall integrity – YPS1, YPK2, and SLT2 – that promote echinocandin tolerance both in vitro and in a mouse model of gastrointestinal (GI) colonization. In particular, we show that mice colonized with strains carrying deletions of these genes were more effectively sterilized by daily caspofungin treatment relative to mice colonized with the wild-type parental strain. Furthermore, consistent with a role of tolerant cells serving as a reservoir for generating resistant mutations, a reduction in tolerance was associated with a reduction in the emergence of resistant strains. Finally, reduced susceptibility in these strains was due both to the well described FKS-dependent mechanisms and as yet unknown, FKS-independent mechanisms. Together, these results shed light on the importance of cell wall integrity maintenance in echinocandin tolerance and emergence of resistance and lay the foundation for future studies of the factors described herein.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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