Attenuation of the Activity of Caspofungin at High Concentrations against Candida albicans: Possible Role of Cell Wall Integrity and Calcineurin Pathways

ABSTRACT Caspofungin had diminished activity in vitro against Candida albicans at concentrations of 8 to 32 μg/ml. This phenomenon was markedly attenuated in a Δmkc1/Δmkc1 deletion mutant and by the addition of cyclosporine to the wild type. Short exposure to these caspofungin concentrations resulted in MKC1 up-regulation, suggesting roles of cell wall integrity and calcineurin pathways.

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Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans

Genes ◽

10.3390/genes9090447 ◽

2018 ◽

Vol 9 (9) ◽

pp. 447 ◽

Cited By ~ 2

Author(s):

Toni Ciudad ◽

Alberto Bellido ◽

Encarnación Andaluz ◽

Belén Hermosa ◽

Germán Larriba

Keyword(s):

Candida Albicans ◽

Homologous Recombination ◽

Excision Repair ◽

Metabolic Energy ◽

Resting Cells ◽

Wild Type ◽

Base Damage

Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

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Candida albicans cell wall integrity transcription factors regulate polymicrobial biofilm formation with Streptococcus gordonii

PeerJ ◽

10.7717/peerj.7870 ◽

2019 ◽

Vol 7 ◽

pp. e7870 ◽

Cited By ~ 4

Author(s):

Jennifer Chinnici ◽

Lisa Yerke ◽

Charlene Tsou ◽

Sujay Busarajan ◽

Ryan Mancuso ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Transcription Factors ◽

Biofilm Formation ◽

Mixed Cultures ◽

Cell Wall Integrity ◽

Antibiotic Tolerance ◽

Streptococcus Gordonii ◽

Knock Out

Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacterium Streptococcus gordonii is known to attach to the hyphal cell wall of the fungus Candida albicans to form corn-cob like structures in biofilms. However, the role of C. albicans in formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role of C. albicans transcription factors in regulation of polymicrobial biofilms and antibiotic tolerance of S. gordonii. The proteins secreted by C. albicans and S. gordonii in mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance of S. gordonii to ampicillin and erythromycin was determined in mixed cultures and mixed biofilms with C. albicans. Additionally, biofilm formation of S. gordonii with C. albicans knock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms with S. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed that S. gordonii had significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures. C. albicans mutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms of S. gordonii with mutants of C. albicans transcription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates that C. albicans may have an important role in mixed biofilm formation as well as antibiotic tolerance of S. gordonii in polymicrobial biofilms. C. albicans may play a facilitating role than being just an innocent bystander in oral biofilms and infections.

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Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

10.1101/2021.12.31.474627 ◽

2022 ◽

Author(s):

Rocio Garcia-Rodas ◽

Hayet Labbaoui ◽

François Orange ◽

Norma Solis ◽

Oscar Zaragoza ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Mouse Model ◽

Membrane Trafficking ◽

Filamentous Growth ◽

Cell Wall Integrity ◽

Wild Type ◽

Phosphatidylinositol Phosphates ◽

Phosphatidylinositol 4

Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3∆/∆ and ypp1∆/∆ mutant was ~60% and ~40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4∆/∆ mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3∆/∆ had wild-type virulence, the ypp1∆/∆ mutant had attenuated virulence and the stt4∆/∆ mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1∆/∆ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans.

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Role of the yakA gene in morphogenesis and stress response in Penicillium marneffei

Microbiology ◽

10.1099/mic.0.080689-0 ◽

2014 ◽

Vol 160 (9) ◽

pp. 1929-1939 ◽

Cited By ~ 7

Author(s):

Sumanun Suwunnakorn ◽

Chester R. Cooper ◽

Aksarakorn Kummasook ◽

Nongnuch Vanittanakom

Keyword(s):

Cell Wall ◽

Stress Response ◽

Penicillium Marneffei ◽

Cell Wall Integrity ◽

South East Asia ◽

Wild Type ◽

Phase Growth ◽

Dimorphic Fungus ◽

Heat Stress Response

Penicillium marneffei is a thermally dimorphic fungus and a highly significant pathogen of immunocompromised individuals living in or having travelled in south-east Asia. At 25 °C, P. marneffei grows filamentously. Under the appropriate conditions, these filaments (hyphae) produce conidiophores bearing chains of conidia. Yet, when incubated at 37 °C, or upon infecting host tissue, P. marneffei grows as a yeast that divides by binary fission. Previously, an Agrobacterium-mediated transformation system was used to randomly mutagenize P. marneffei, resulting in the isolation of a mutant defective in normal patterns of morphogenesis and conidiogenesis. The interrupted gene was identified as yakA. In the current study, we demonstrate that the yakA mutant produced fewer conidia at 25 °C than the wild-type and a complemented strain. In addition, disruption of the yakA gene resulted in early conidial germination and perturbation of cell wall integrity. The yakA mutant exhibited abnormal chitin distribution while growing at 25 °C, but not at 37 °C. Interestingly, at both temperatures, the yakA mutant possessed increased chitin content, which was accompanied by amplified transcription of two chitin synthase genes, chsB and chsG. Moreover, the expression of yakA was induced during post-exponential-phase growth as well as by heat shock. Thus, yakA is required for normal patterns of development, cell wall integrity, chitin deposition, appropriate chs expression and heat stress response in P. marneffei.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.702779 ◽

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.

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Antifungal Activity Directed Toward the Cell Wall by 2-Cyclohexylidenhydrazo- 4-Phenyl-Thiazole Against Candida albicans

Infectious Disorders - Drug Targets ◽

10.2174/1871526518666180531101605 ◽

2019 ◽

Vol 19 (4) ◽

pp. 428-438 ◽

Cited By ~ 1

Author(s):

Nívea P. de Sá ◽

Ana P. Pôssa ◽

Pilar Perez ◽

Jaqueline M.S. Ferreira ◽

Nayara C. Fonseca ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Antifungal Activity ◽

Mammalian Cells ◽

C Elegans ◽

Buccal Epithelial Cells ◽

Mutant Strains ◽

Low Toxicity ◽

High Concentrations ◽

Mic Value

Background: The increasing incidence of invasive forms of candidiasis and resistance to antifungal therapy leads us to seek new and more effective antifungal compounds. Objective: To investigate the antifungal activity and toxicity as well as to evaluate the potential targets of 2- cyclohexylidenhydrazo-4-phenyl-thiazole (CPT) in Candida albicans. Methods: The antifungal activity of CPT against the survival of C. albicans was investigated in Caenorhabditis elegans. Additionally, we determined the effect of CPT on the inhibition of C. albicans adhesion capacity to buccal epithelial cells (BECs), the toxicity of CPT in mammalian cells, and the potential targets of CPT in C. albicans. Results: CPT exhibited a minimum inhibitory concentration (MIC) value of 0.4-1.9 µg/mL. Furthermore, CPT at high concentrations (>60 x MIC) showed no or low toxicity in HepG2 cells and <1% haemolysis in human erythrocytes. In addition, CPT decreased the adhesion capacity of yeasts to the BECs and prolonged the survival of C. elegans infected with C. albicans. Analysis of CPT-treated cells showed that their cell wall was thinner than that of untreated cells, especially the glucan layer. We found that there was a significantly lower quantity of 1,3-β-D-glucan present in CPT-treated cells than that in untreated cells. Assays performed on several mutant strains showed that the MIC value of CPT was high for its antifungal activity on yeasts with defective 1,3-β-glucan synthase. Conclusion: In conclusion, CPT appears to target the cell wall of C. albicans, exhibits low toxicity in mammalian cells, and prolongs the survival of C. elegans infected with C. albicans.

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Soybean germination limits the role of cell wall integrity in controlling protein physicochemical changes during cooking and improves protein digestibility

Food Research International ◽

10.1016/j.foodres.2021.110254 ◽

2021 ◽

Vol 143 ◽

pp. 110254

Author(s):

Mostafa Zahir ◽

Vincenzo Fogliano ◽

Edoardo Capuano

Keyword(s):

Cell Wall ◽

Protein Digestibility ◽

Cell Wall Integrity ◽

Physicochemical Changes

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Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

Applied Sciences ◽

10.3390/app11156865 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6865

Author(s):

Eun Seon Lee ◽

Joung Hun Park ◽

Seong Dong Wi ◽

Ho Byoung Chae ◽

Seol Ki Paeng ◽

...

Keyword(s):

Cold Stress ◽

Wild Type ◽

Rna Chaperone ◽

E Coli ◽

Cold Sensitive ◽

Thioredoxin H ◽

Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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Suppressive Role of Bam32/DAPP1 in Chemokine-Induced Neutrophil Recruitment

International Journal of Molecular Sciences ◽

10.3390/ijms22041825 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1825

Author(s):

Li Hao ◽

Aaron J. Marshall ◽

Lixin Liu

Keyword(s):

Small Gtpases ◽

Neutrophil Recruitment ◽

Neutrophil Chemotaxis ◽

Wild Type ◽

Adaptor Molecule ◽

Geranylgeranyltransferase I ◽

And Migration ◽

Rap1 Activation

Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32−/− mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32−/− mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32−/− neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32−/− mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.

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