Species-specific differences in C-5 sterol desaturase function influence the outcome of azole antifungal exposure.

2021 ◽  
Author(s):  
Arturo Luna-Tapia ◽  
Josie E. Parker ◽  
Steven L. Kelly ◽  
Glen E. Palmer
Keyword(s):  
Fungal Pathogens ◽  
Desaturase Activity ◽  
Hyphal Growth ◽  
Candida Auris ◽  
Membrane Function ◽  
Rhizopus Delemar ◽  
Growth Defects ◽  
Low Levels ◽  
Species Specific ◽  
Residual Growth

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol-diol that disrupts membrane function. In Candida albicans , sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3 . Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensity of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans ( CaERG3 ), Candida glabrata ( CgERG3 ), Candida auris ( CaurERG3 ), Cryptococcus neoformans ( CnERG3 ), Aspergillus fumigatus ( AfERG3A-C ) and Rhizopus delemar ( RdERG3A/B ) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored sterol C-5 desaturase activity, and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant - confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the minimal inhibitory concentration (MIC) and residual growth observed at supra-MIC concentrations. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced varying levels of 14α-methylergosta-8,24(28)-dien-3β,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C5-sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Species Specific Immunoassays to Measure Blood Platelet and Coagulation Activation in the Rat

1996 ◽  
Vol 76 (06) ◽  
pp. 1090-1095 ◽  
Author(s):  
C Ravanat ◽  
M Freund ◽  
S Schuhler ◽  
P Grunert ◽  
L Meyer ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Plasma Concentrations ◽  
Simultaneous Detection ◽  
Blood Platelet ◽  
Coagulation Activation ◽  
Platelet Factor ◽  
Prethrombotic State ◽  
Low Levels ◽  
Species Specific ◽  
Higher Sensitivity

SummaryThe purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thromboplastin (90 to 2400 μl/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.

scholarly journals The cAMP-PKA Pathway Regulates Growth, Sexual and Asexual Differentiation, and Pathogenesis in Fusarium graminearum

2014 ◽  
Vol 27 (6) ◽  
pp. 557-566 ◽  
Author(s):  
Shuai Hu ◽  
Xiaoying Zhou ◽  
Xiaoying Gu ◽  
Shulin Cao ◽  
Chengfang Wang ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Vegetative Growth ◽  
Double Mutant ◽  
Elevated Temperatures ◽  
Hyphal Growth ◽  
Growth Defects ◽  
Plant Infection ◽  
Dependent Protein ◽  
Pka Pathway

Like many other filamentous ascomycetes, Fusarium graminearum contains two genes named CPK1 and CPK2 that encode the catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase A (PKA). To determine the role of cAMP signaling in pathogenesis and development in F. graminearum, we functionally characterized these two genes. In addition, we generated and characterized the cpk1 cpk2 double and fac1 adenylate cyclase gene deletion mutants. The cpk1 mutant was significantly reduced in vegetative growth, conidiation, and deoxynivalenol production but it had increased tolerance to elevated temperatures. It was defective in the production of penetration branches on plant surfaces, colonization of wheat rachises, and spreading in flowering wheat heads. Deletion of CPK1 had no effect on perithecium development but the cpk1 mutant was defective in ascospore maturation and releasing. In contrast, the cpk2 mutant had no detectable phenotypes, suggesting that CPK2 contributes minimally to PKA activities in F. graminearum. Nevertheless, the cpk1 cpk2 double mutant had more severe defects in vegetative growth and rarely produced morphologically abnormal conidia. The double mutant, unlike the cpk1 or cpk2 mutant, was nonpathogenic and failed to form perithecia on self-mating plates. Therefore, CPK1 and CPK2 must have overlapping functions in vegetative growth, differentiation, and plant infection in F. graminearum. The fac1 mutant was also nonpathogenic and had growth defects similar to those of the cpk1 cpk2 mutant. However, deletion of FAC1 had no effect on conidium morphology. These results indicated that CPK1 is the major PKA catalytic subunit gene and that the cAMP-PKA pathway plays critical roles in hyphal growth, conidiation, ascosporogenesis, and plant infection in F. graminearum.

scholarly journals Droplet-based microfluidics platform for antifungal analysis against filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sehrish Iftikhar ◽  
Aurélie Vigne ◽  
Julia Elisa Sepulveda-Diaz
Keyword(s):  
Fungal Pathogens ◽  
Antimicrobial Agents ◽  
Pearson Correlation ◽  
Hyphal Growth ◽  
Bulk Analysis ◽  
Single Spore ◽  
Microfluidic Platform ◽  
Viability Assay ◽  
Wide Range ◽  
Potential Applications

AbstractFungicides are extensively used in agriculture to control fungal pathogens which are responsible for significant economic impact on plant yield and quality. The conventional antifungal screening techniques, such as water agar and 96-well plates, are based on laborious protocols and bulk analysis, restricting the analysis at the single spore level and are time consuming. In this study, we present a droplet-based microfluidic platform that enables antifungal analysis of single spores of filamentous fungus Alternaria alternata. A droplet-based viability assay was developed, allowing the germination and hyphal growth of single A. alternata spores within droplets. The viability was demonstrated over a period of 24 h and the antifungal screening was achieved using Kunshi/Tezuma as antifungal agent. The efficacy results of the droplet-based antifungal analysis were compared and validated with the results obtained from conventional protocols. The percentage inhibitions assessed by the droplet-based platform were equivalent with those obtained by the other two methods, and the Pearson correlation analysis showed high correlation between the three assays. Taken together, this droplet-based microfluidic platform provides a wide range of potential applications for the analysis of fungicide resistance development as well as combinatorial screening of other antimicrobial agents and even antagonistic fungi.

scholarly journals Synthesis and Metabolism of Thyroid Hormones Is Preferentially Maintained in Selenium-Deficient Transgenic Mice

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1306-1313 ◽  
Author(s):  
Lutz Schomburg ◽  
Cornelia Riese ◽  
Marten Michaelis ◽  
Emine Griebert ◽  
Marc O. Klein ◽  
...  
Keyword(s):  
Thyroid Gland ◽  
Thyroid Hormones ◽  
Knockout Mice ◽  
Serum Levels ◽  
Mrna Levels ◽  
Normal Range ◽  
Growth Defects ◽  
Low Levels

The thyroid gland is rich in selenium (Se) and expresses a variety of selenoproteins that are involved in antioxidative defense and metabolism of thyroid hormones (TH). Se deficiency impairs regular synthesis of selenoproteins and adequate TH metabolism. We recently generated mice that lack the plasma Se carrier, selenoprotein P (SePP). SePP-knockout mice display decreased serum Se levels and manifest growth defects and neurological abnormalities partly reminiscent of thyroid gland dysfunction or profound hypothyroidism. Thus, we probed the TH axis in developing and adult SePP-knockout mice. Surprisingly, expression of Se-dependent 5′-deiodinase type 1 was only slightly altered in liver, kidney, or thyroid at postnatal d 60, and 5′-deiodinase type 2 activity in brain was normal in SePP-knockout mice. Thyroid gland morphology, thyroid glutathione peroxidase activity, thyroid Se concentration, and serum levels of TSH, T4, or T3 were within normal range. Pituitary TSHβ transcripts and hepatic 5′-deiodinase type 1 mRNA levels were unchanged, indicating regular T3 bioactivity in thyrotropes and hepatocytes. Cerebellar granule cell migration as a sensitive indicator of local T3 action during development was undisturbed. Collectively, these findings demonstrate that low levels of serum Se or SePP in the absence of other challenges do not necessarily interfere with regular functioning of the TH axis. 5′-deiodinase isozymes are preferentially supplied, and Se-dependent enzymes in the thyroid are even less-dependent on serum levels of Se or SePP than in brain. This indicates a top priority of the thyroid gland and its selenoenzymes with respect to the hierarchical Se supply within the organism.

scholarly journals Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal PathogenRhizopus delemar

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Poppy C. S. Sephton-Clark ◽  
Jose F. Muñoz ◽  
Elizabeth R. Ballou ◽  
Christina A. Cuomo ◽  
Kerstin Voelz
Keyword(s):  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Developmental Process ◽  
Fungal Spores ◽  
Hyphal Growth ◽  
Large Shift ◽  
Rhizopus Delemar ◽  
Content Type ◽  
Transcriptional Changes ◽  
Dormant Spore

ABSTRACTRhizopus delemaris an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores ofRhizopus delemarcause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transformingRhizopus delemarfrom a single-cellular to multicellular organism.IMPORTANCEGermination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.

Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 141-149 ◽  
Author(s):  
S. Mortillo ◽  
P.M. Wassarman
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Transmission Electron ◽  
Acrosomal Membrane ◽  
Membrane Compartments ◽  
Sperm Membrane ◽  
Differential Binding ◽  
Inner Acrosomal Membrane ◽  
Low Levels ◽  
Species Specific

Egg zona pellucida glycoproteins mZP3 and mZP2 serve as primary and secondary sperm receptors, respectively, during initial stages of fertilization in mice [Wassarman (1988) A. Rev. Biochem. 57, 415–442]. These receptors interact with complementary egg-binding proteins (EBPs) located on the sperm surface to support species-specific gamete adhesion. Results of whole-mount autoradiographic experiments suggest that purified egg mZP3 and mZP2 bind preferentially to acrosome-intact (AI) and acrosome-reacted (AR) sperm heads, respectively [Bleil and Wassarman (1986) J. Cell Biol. 102, 1363–1371]. Here, we used purified egg mZP2, egg mZP3 and fetuin, which were coupled directly to colloidal gold (‘gold-probes’), to examine binding of these glycoproteins to membrane compartments of AI and AR sperm by transmission electron microscopy. mZP3 gold-probes were found associated primarily with plasma membrane overlying the acrosomal and post-acrosomal regions of AI sperm heads. They were also found associated with plasma membrane overlying the post-acrosomal region of AR sperm heads. mZP2 gold-probes were found associated primarily with inner acrosomal membrane of AR sperm heads, although some gold was associated with outer acrosomal membrane of AI sperm that had holes in plasma membrane overlying the acrosome. Fetuin gold-probes, used to assess background levels of binding, were bound at relatively low levels to plasma membrane and inner acrosomal membrane of AI and AR sperm, respectively. None of the gold-probes exhibited significant binding to sperm tails, or to red blood cells and residual bodies present in sperm preparations. These results provide further evidence that mZP2 and mZP3 bind preferentially to heads of AR and AI sperm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Zoonotic Microsporidia in Wild Lagomorphs in Southern Spain

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2218
Author(s):  
Anabel Martínez-Padilla ◽  
Javier Caballero-Gómez ◽  
Ángela Magnet ◽  
Félix Gómez-Guillamón ◽  
Fernando Izquierdo ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Enterocytozoon Bieneusi ◽  
Southern Spain ◽  
Health Concern ◽  
Wild Rabbit ◽  
Specific Pcr ◽  
Encephalitozoon Intestinalis ◽  
Wild Lagomorphs ◽  
Species Specific ◽  
Iberian Hare

Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0–1.7%) but not in hares (0.0%; 95% CI: 0.0–4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0–0.8%) and three Iberian hares (3.8%; 95% CI: 0.0–8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0–0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors’ knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.

scholarly journals Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+Homeostasis in Aspergillus nidulans

2016 ◽  
Vol 82 (11) ◽  
pp. 3420-3430 ◽  
Author(s):  
Sha Wang ◽  
Xiao Liu ◽  
Hui Qian ◽  
Shizhu Zhang ◽  
Ling Lu
Keyword(s):  
Salt Stress ◽  
Calcium Channel ◽  
Aspergillus Nidulans ◽  
Transient Receptor Potential ◽  
Feedback Inhibition ◽  
Calcium Influx ◽  
Hyphal Growth ◽  
Salt Stress Response ◽  
Content Type ◽  
Growth Defects

ABSTRACTThe eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion ofcchAsuppresses the hyphal growth defects caused by the loss of calcineurin under salt stress inAspergillus nidulans. Additionally, the hypersensitivity of the ΔcnaAstrain to extracellular calcium and cell-wall-damaging agents can be suppressed bycchAdeletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca2+concentration ([Ca2+]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of eithercnaAorcchAsignificantly decreased the [Ca2+]c, but a deficiency in bothcnaAandcchAswitches the [Ca2+]cto the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction ofcchAin the ΔcnaAstrain dramatically restored the expression ofenaA(a major determinant for sodium detoxification), which was abolished in the ΔcnaAstrain under salt stress. These results suggest that double deficiencies ofcnaAandcchAcould bypass the requirement of calcineurin to induceenaAexpression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca2+channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCEThe feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One7:e46564, 2012,http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion ofcchAcould suppress the hyphal growth defects caused by the loss of calcineurin under salt stress inAspergillus nidulans. In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca2+]c. Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca2+channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca2+homeostasis in response to different environments.

scholarly journals The Maize Lethal Leaf Spot 1 Mutant Has Elevated Resistance to Fungal Infection at the Leaf Epidermis

1998 ◽  
Vol 11 (11) ◽  
pp. 1110-1118 ◽  
Author(s):  
Carl Simmons ◽  
Sabine Hantke ◽  
Susan Grant ◽  
Gurmukh S. Johal ◽  
Steven P. Briggs
Keyword(s):  
Leaf Spot ◽  
Fungal Pathogens ◽  
Hyphal Growth ◽  
Leaf Epidermis ◽  
Puccinia Sorghi ◽  
Leaf Maturity ◽  
Pathogenesis Related ◽  
Lesion Number ◽  
Related Proteins ◽  
Germination And Growth

The maize lethal leaf spot 1 (lls1) mutant exhibits enhanced resistance to fungal pathogens. The lls1 resistance to Cochliobolus heterostrophus has two components: (i) lesion number is reduced 40% relative to wild type; and (ii) the lesions that do form often do not contain viable fungus. This lesion sterility is dependent upon leaf maturity and light, whereas reduced lesion number is not. The lls1 lesions express pathogenesis-related proteins at high levels, so lesion sterility likely results from activation of defense systems and necrosis. Reduced lesion number is correlated with a reduction of C. heterostrophus spore germination, hyphal growth, and haustoria formation on the leaf epidermis. The rust pathogen Puccinia sorghi has reduced pustule formation on lls1, and its germination and growth are also slowed on the epidermis. However, after entering the mesophyll through stomata, P. sorghi can form pustules on lls1, and even green islands within necrotic lls1 lesions. In situ mRNA hybridization shows that Lls1 is predominantly expressed in the leaf epidermis, coincident with the site of resistance in the mutant.

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