Anti-EFG1 2'-OMethylRNA oligomer inhibits Candida albicans filamentation and attenuates the candidiasis in Galleria mellonella.
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ABSTRACT:
SUBMITTER: Araujo D
PROVIDER: S-EPMC8728520 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Candida albicans is an important cause of morbidity in hospitalized and immunosuppressed patients. Virulence factors of C. albicans include: filamentation, proteinases, adherence proteins and biofilm formation. The objective of this work was to use Galleria mellonella as a model to study the roles of C. albicans filamentation in virulence. We focused our study to five genes BCR1, FLO8, KEM1, SUV3 and TEC1 that have been shown to play a role in filamentation. Filaments are necessary for biofilm formation and evading interaction with macrophages in mammalian infections. Among the five mutant strain tested, we found that only the flo8/flo8 mutant strain did not form filaments within G. mellonella. This strain also exhibited reduced virulence in the larvae. Another strain that exhibited reduced pathogenicity in the G. mellonella model was tec1/tec1 but by contrast, the tec1/tec1 strain retained the ability to form filaments. Overexpression of TEC1 in the flo8/flo8 mutant restored filamentation but did not restore virulence in the larvae as well as in a mouse model of C. albicans infection. The filamentation phenotype did not affect the ability of hemocytes, the immune cells of G. mellonella, to associate with the various mutant strains of C. albicans. The capacities of the tec1/tec1 mutant and the flo8/flo8 TDH3-TEC1 strains to form filaments with impaired virulence suggest that filamentation alone is not sufficient to kill G. mellonella and suggest other virulence factors may be associated with genes that regulate filamentation.
| S-EPMC2883670 | biostudies-other
Project description:Antisense oligomers and their analogs have been successfully utilized to silence gene expression for the treatment of many human diseases; however, the control of yeast's virulence determinants has never been exploited before. In this sense, this work is based on the key hypothesis that if a pathogen's genetic sequence is a determinant of virulence, it will be possible to synthesize a nucleic acid mimic based on antisense therapy (AST) that will bind to the mRNA produced, blocking its translation into protein and, consequently, reducing the pathogen virulence phenotype. EFG1 is an important determinant of virulence that is involved in the regulation of the Candida albicans switch from yeast to filamentous form. Thus, our main goal was to design and synthesize an antisense oligonucleotide (ASO) targeting the EFG1 mRNA and to validate its in vitro applicability. The results show that the anti-EFG1 2'-OMethylRNA (2'OMe) oligomer was able to significantly reduce the levels of EFG1 gene expression and of Efg1p protein translation (both approximately 60%), as well as effectively prevent filamentation of C. albicans cells (by 80%). Moreover, it was verified that anti-EFG1 2'OMe keeps the efficacy in different simulated human body fluids. Undeniably, this work provides potentially valuable information for future research into the management of Candida infections, regarding the development of a credible and alternative method to control C. albicans infections, based on AST methodology.
| S-EPMC6838528 | biostudies-literature
Project description:The incidence of resistant Candida isolates has increased continuously in recent decades, especially Candida albicans. To overcome this resistance, research on antifungal sensitizers has attracted considerable attention. Linezolid was found to inhibit the growth of Pythium insidiosum and synergize with amphotericin B against Cryptococcus neoformans. The objective of this study was to determine the interactions of linezolid and azoles against C. albicans in vitro and in vivo. In vitro, linezolid combined with azoles induced synergistic effects not only against some susceptible C. albicans isolates, but also against all tested resistant C. albicans isolates. For all resistant isolates, exposure to the combination of linezolid with azoles induced a significant decrease in the minimum inhibitory concentrations (MIC) of azoles, from >512 to 0.5-1 μg/mL for fluconazole, from >16 to 0.25-1 μg/mL for itraconazole, and from >16 to 0.03-0.25 μg/mL for voriconazole. Additionally, linezolid synergized with fluconazole against biofilms that were preformed for ≤ 12 h from both susceptible and resistant C. albicans, and the sessile MIC of fluconazole decreased from >1024 to 1-4 μg/mL. In vivo, linezolid plus azoles prolonged the survival rate of infected Galleria mellonella larvae twofold compared with the azole monotherapy group, significantly decreased the fungal burden of the infected larvae, and reduced the damage of resistant C. albicans to the larval tissue. These findings will contribute to antifungal agent discovery and new approaches for the treatment of candidiasis caused by C. albicans.
| S-EPMC6365414 | biostudies-literature
Project description:This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterized the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 10⁶ yeast cells reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) h post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 h. A range of proteins secreted during infection of G. mellonella by C. albicans were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 10⁶ yeast cells/mL), p < 0.05) as compared to control (0.99 ± 0.01 × 10⁶ yeast cells/mL). C. albicans responded to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins. The results presented here indicate that C. albicans can overcome the fungicidal activity of hemolymph by altering protein synthesis and cellular respiration, and commence invasion and dissemination throughout the host.
| S-EPMC6463115 | biostudies-literature
Project description:Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.
| S-EPMC4249548 | biostudies-literature
Project description:Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether clinical isolates of Lactobacillus spp. are able to provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 107 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these insects compared to the control group (P = 0.0001). After that, we investigated the immune mechanisms involved in the protection against C. albicans infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte quantity (2.38 x 106 cells/mL) in relation to the control group (1.29 x 106 cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the G. mellonella hemolymph. Further, we found that L. paracasei 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in G. mellonella hemolymph. In conclusion, L. paracasei 28.4 can modulate the immune system of G. mellonella and protect against candidiasis.
| S-EPMC5340386 | biostudies-literature
Project description:The aim of the present study was to identify Candida albicans transcription factors (TFs) involved in virulence. Although mice are considered the gold-standard model to study fungal virulence, mini-host infection models have been increasingly used. Here, barcoded TF mutants were first screened in mice by pools of strains and fungal burdens (FBs) quantified in kidneys. Mutants of unannotated genes which generated a kidney FB significantly different from that of wild-type were selected and individually examined in Galleria mellonella. In addition, mutants that could not be detected in mice were also tested in G. mellonella. Only 25% of these mutants displayed matching phenotypes in both hosts, highlighting a significant discrepancy between the two models. To address the basis of this difference (pool or host effects), a set of 19 mutants tested in G. mellonella were also injected individually into mice. Matching FB phenotypes were observed in 50% of the cases, highlighting the bias due to host effects. In contrast, 33.4% concordance was observed between pool and single strain infections in mice, thereby highlighting the bias introduced by the "pool effect." After filtering the results obtained from the two infection models, mutants for MBF1 and ZCF6 were selected. Independent marker-free mutants were subsequently tested in both hosts to validate previous results. The MBF1 mutant showed impaired infection in both models, while the ZCF6 mutant was only significant in mice infections. The two mutants showed no obvious in vitro phenotypes compared with the wild-type, indicating that these genes might be specifically involved in in vivo adapt.
| S-EPMC4419840 | biostudies-literature
Project description:Galleria mellonella larvae were administered an inoculum of Candida albicans and the response to infection over 24 hours was monitored. The yeast cell density in infected larvae declined initially but replication commenced six hours post-infection. The hemocyte density decreased from 5.2 × 106/ml to 2.5 × 106/ml at 2 hours but increased to 4.2 × 106 at 6 hours and decreased subsequently. Administration of β - glucan to larvae also caused a fluctuation in hemocyte density (5.1 ± 0.22 × 106/ml (0 hour) to 6.25 ± 0.25 × 106/ml (6 hour) (p < 0.05) to 5 ± 2.7 × 106 (24 hour)) and the population showed an increase in the density of small, granular cells at 24 hours (p < 0.05). Hemocytes from larvae inoculated with β - glucan for 6 or 24 hours showed faster killing of C. albicans cells (53 ± 4.1% (p < 0.01), 64 ± 3.7%, (p < 0.01), respectively) than hemocytes from control larvae (24 ± 11%) at 60 min. Proteomic analysis indicated increased abundance of immune related proteins cecropin-A (5 fold) and prophenoloxidase-activating proteinase-1 (5 fold) 6 hours post infection but by 24 hours there was elevated abundance of muscle (tropomyosin 2 (141 fold), calponin (66 fold), troponin I (62 fold)) and proteins indicative of cellular stress (glutathione-S-transferase-like protein (114 fold)), fungal dissemination (muscle protein 20-like protein (174 fold)) and tissue breakdown (mitochondrial cytochrome c (10 fold)). Proteins decreased in abundance at 24 hour included β - 1,3 - glucan recognition protein precursor (29 fold) and prophenoloxidase subunit 2 (25 fold).
| S-EPMC5955201 | biostudies-literature
Project description:Efg1p is a key transcriptional regulator in Candida albicans which controls various aspects of morphogenesis and metabolism in this organism. Efg1p contains a central basic helix-loop-helix (bHLH) domain, flanked by sequences highly conserved in fungal APSES proteins, as well as polyglutamine stretches at the N- and C-terminal ends. A systematic deletion approach to specify functional domains of Efg1p revealed that the APSES domain is essential for morphogenesis of the normal yeast and true hyphal cell forms and that bHLH flanking sequences are needed for Efg1p stability. Additional C-terminal sequences were required for hyphal formation on some inducing media, and most Efg1p sequences were needed for chlamydospore morphogenesis. Overexpression of EFG1 led to pseudohypha formation only if a functional APSES domain was present, while a switch from the opaque to the white cell type in addition depended on the presence of certain N- and C-terminal segments. Yeast two-hybrid experiments revealed that binding of Efg1p to its antagonist Czf1p required two regions outside of the APSES domain, which did not coincide with Efg1p sequences needed for its transcriptional repressor activity. Binding of the Flo8 transcription factor to Efg1p did not require the APSES domain but appeared to occur at two or more redundant domains. In contrast, DNA binding of Efg1p to an MluI cell cycle box (MCB) element solely required the APSES domain. Overall, these results suggest that functional domains of Efg1p are spread throughout most of its sequences, including the central APSES domain involved in DNA binding, as well as flanking regions required for various protein interactions and regulatory activities.
| S-EPMC2394972 | biostudies-literature
Project description:This study assessed the development of disseminated candidiasis within Galleria mellonella larvae and characterised the proteomic responses of Candida albicans to larval hemolymph. Infection of larvae with an inoculum of 1 × 106 yeast cell reduced larval viability 24 (53.33 ± 3.33%), 48 (33.33 ± 3.33%) and 72 (6.66 ± 3.33%) hour post infection. C. albicans infection quickly disseminated from the site of inoculation and the presence of yeast and hyphal forms were found in nodules extracted from infected larvae at 6 and 24 hours. A range of proteins secreted during infection of G. mellonella were detected in larval hemolymph and these were enriched for biological processes such as interaction with host and pathogenesis. The candicidal activity of hemolymph after immediate incubation of yeast cells resulted in a decrease in yeast cell viability (0.23 ± 0.03 × 106, p < 0.05) as compared to control (0.99 ± 0.01 × 106). extracellular (in vivo) proteome of C. albicans in larval hemolymph were assessed. C. albicans responds to incubation in hemolymph ex vivo by the induction of an oxidative stress response, a decrease in proteins associated with protein synthesis and an increase in glycolytic proteins.
2019-01-17 | PXD011997 | Pride