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1.
Sci Rep ; 12(1): 17942, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289280

RESUMO

New Schiff base ligand, derived from antiviral valacyclovir, and its novel Cr(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II) complexes have been synthesized. By using a variety of analytical and spectroscopic techniques, the type of bonding between the ligand and the metal ions in the recently formed complexes was clarified. The Schiff base ligand act as a bidentate and coordinated with the metal ions through the azomethine-N and the phenolic-O centers, in a mono-deprotonated form. Except for the Zn(II) complex, which displayed a tetrahedral geometry, all complexes displayed octahedral geometry. The TGA findings supported that the stability and decomposition properties of the metal complexes were entirely distinct from one another. The thermogram showed decomposition of all investigated metal complexes above 200 °C in three, four or five steps, and indicated the high thermal stability of these complexes. According to XRD patterns, the particles of these complexes were located at the nanoscale. Moreover, for all the samples analyzed, the TEM images showed uniform and homogeneous surface morphology. The biological activity revealing the high efficiencies of the screened complexes as antibacterial and antitumor agents. The antimicrobial activity of the ligand and its complexes was examined against a variety of pathogenic bacteria and fungi including Escherichia coli, Staphylococcus aureus and Candida albicans. The data obtained revealed that the metal ion in the complexes enhanced the antimicrobial activity compared to the free ligand. The high efficiencies toward S. aureus, E. coli, and C. albicans appeared by Cu(II) complex 23, Ni(II) complex 20, and Ni(II) complex 19, respectively. The antitumor activity of the ligand and its complexes was tested against Hepatocellular carcinoma cell line (HepG-2 cells), the residue 28 which produced after heating the Cu(II) complex 25 at 200 °C for 1 h, exhibited strong inhibition of HepG-2 cell growth. The results of the DNA cleavage investigation demonstrated the ability of investigated Cu(II) complex to degrade DNA. The docking findings showed strong interactions of both the ligand and its examined Cu(II) complex, revealing their ability to cleavage DNA and their potent inhibitory effects on tumor cells. The electrical conductivity study confirmed that the ligand and its investigated complexes had semiconducting properties.


Assuntos
Antineoplásicos , Complexos de Coordenação , Bases de Schiff/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Ligantes , Staphylococcus aureus , Escherichia coli/metabolismo , Valaciclovir/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Metais/farmacologia , Candida albicans/metabolismo , Zinco/química , DNA/metabolismo , Íons/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Testes de Sensibilidade Microbiana , Espectrofotometria Infravermelho
2.
J Comput Chem ; 43(32): 2121-2130, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36190786

RESUMO

Novel agents to treat invasive fungal infections are urgently needed because the small number of established targets in pathogenic fungi makes the existing drug repertoire particularly vulnerable to the emergence of resistant strains. Recently, we reported that Candida albicans Bdf1, a bromodomain and extra-terminal domain (BET) bromodomain with paired acetyl-lysine (AcK) binding sites (BD1 and BD2) is essential for fungal cell growth and that an imidazopyridine (1) binds to BD2 with selectivity versus both BD1 and human BET bromodomains. Bromodomain binding pockets contain a conserved array of structural waters. Molecular dynamics simulations now reveal that one water molecule is less tightly bound to BD2 than to BD1, explaining the site selectivity of 1. This insight is useful in the performance of ligand docking studies to guide design of more effective Bdf1 inhibitors, as illustrated by the design of 10 new imidazopyridine BD2 ligands 1a-j, for which experimental binding and site selectivity data are presented.


Assuntos
Candida albicans , Fatores de Transcrição , Humanos , Candida albicans/metabolismo , Ligantes , Fatores de Transcrição/metabolismo , Sítios de Ligação
3.
Proc Natl Acad Sci U S A ; 119(41): e2209699119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36191236

RESUMO

Fungi and bacteria often engage in complex interactions, such as the formation of multicellular biofilms within the human body. Knowledge about how interkingdom biofilms initiate and coalesce into higher-level communities and which functions the different species carry out during biofilm formation remain limited. We found native-state assemblages of Candida albicans (fungi) and Streptococcus mutans (bacteria) with highly structured arrangement in saliva from diseased patients with childhood tooth decay. Further analyses revealed that bacterial clusters are attached within a network of fungal yeasts, hyphae, and exopolysaccharides, which bind to surfaces as a preassembled cell group. The interkingdom assemblages exhibit emergent functions, including enhanced surface colonization and growth rate, stronger tolerance to antimicrobials, and improved shear resistance, compared to either species alone. Notably, we discovered that the interkingdom assemblages display a unique form of migratory spatial mobility that enables fast spreading of biofilms across surfaces and causes enhanced, more extensive tooth decay. Using mutants, selective inactivation of species, and selective matrix removal, we demonstrate that the enhanced stress resistance and surface mobility arise from the exopolymeric matrix and require the presence of both species in the assemblage. The mobility is directed by fungal filamentation as hyphae extend and contact the surface, lifting the assemblage with a "forward-leaping motion." Bacterial cell clusters can "hitchhike" on this mobile unit while continuously growing, to spread across the surface three-dimensionally and merge with other assemblages, promoting community expansion. Together, our results reveal an interkingdom assemblage in human saliva that behaves like a supraorganism, with disease-causing emergent functionalities that cannot be achieved without coassembly.


Assuntos
Biofilmes , Saliva , Streptococcus mutans , Candida albicans/metabolismo , Criança , Doença , Humanos , Hifas/fisiologia , Dinâmica Populacional , Saliva/microbiologia
4.
Elife ; 112022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36200752

RESUMO

Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here, we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1 and Frp2 redistribute to the plasma membrane in the presence of hemin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from FREs to enable extracellular heme uptake.


Hosts and disease-causing fungi are often locked into a battle over resources. The host will attempt to withhold molecules that the fungus needs to survive, while the pathogen will try to find alternative routes to obtain them. Candida albicans, for example, can go after the atoms of iron embedded in the proteins of the organism it infects. To do so it releases molecules known as hemophores, which scavenge the iron-containing heme molecule that equips oxygen-carrying proteins in the blood. Once captured, the heme is carried across the wall that protects C. albicans from the environment and brought to the membrane of the cell. It is then taken in and trafficked inside vesicles to its destination. However, the identity of the molecular actors which help to bridge the internal and external segments of the heme journey remain unclear. Previous studies have shown that the hemophore Pga7 is involved, but this protein is attached to the outside of the cell membrane, where it cannot directly interact with the import machinery. Roy et al. set out to discover this missing link. Examining the genomes of fungal species related to C. albicans highlighted two membrane proteins, Frp1 and Frp2, which could participate in heme uptake. Protein sequence comparison revealed that Frp1 and Frp2 were closely related to ferric reductases, a group of membrane enzymes which can chemically alter extracellular iron prior to uptake. Deleting the genes for Frp1 and Frp2 rendered C. albicans cells incapable of taking in heme. Conversely, a fungal species which cannot normally uptake heme could efficiently internalise these complexes when artificially equipped with Frp1 and Pga7, suggesting that the two proteins work closely together. Finally, protein structure comparisons highlighted that an extracellular domain present in ferric reductases but absent in Frp1 and Frp2 is, in fact, related to Pga7 and other hemophores. This implies that the iron and heme uptake systems may share a common evolutionary origin. Overall, the work by Roy et al. reveals a new family of proteins which allow disease-causing fungi to steal iron from their hosts. This knowledge may be useful to design better anti-fungal treatments.


Assuntos
Candida albicans , FMN Redutase , Animais , FMN Redutase/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
5.
Appl Microbiol Biotechnol ; 106(21): 7251-7263, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36195704

RESUMO

The cross-kingdom interactions between Candida albicans and Streptococcus mutans have played important roles in early childhood caries (ECC). However, the key pathways of C. albicans promoting the cariogenicity of S. mutans are still unclear. Here, we found that C. albicans CHK1 gene was highly upregulated in their dual-species biofilms. C. albicans chk1Δ/Δ significantly reduced the synergistical growth promotion, biofilm formation, and exopolysaccharides (EPS) production of S. mutans, the key cariogenic agent, compared to C. albicans wild type (WT) and CHK1 complementary strains. C. albicans WT upregulated the expressions of S. mutans EPS biosynthesis genes gtfB, gtfC, and gtfD, and their regulatory genes vicR and vicK, but chk1Δ/Δ had no effects. Both C. albicans WT and chk1Δ/Δ failed to promote the biofilm formation and EPS production of S. mutans ΔvicK and antisense-vicR strains, indicating that C. albicans CHK1 upregulated S. mutans vicR and vicK to increase the EPS biosynthesis gene expression, then enhanced the EPS production and biofilm formation to promote the cariogenicity. In rat caries model, the coinfection with chk1Δ/Δ and S. mutans decreased the colonization of S. mutans and developed less caries especially the severe caries compared to that from the combinations of S. mutans with C. albicans WT, indicating the essential role of C. albicans CHK1 gene in the development of dental caries. Our study for the first time demonstrated the key roles of C. albicans CHK1 gene in dental caries and suggested that it may be a practical target to reduce or treat ECC. KEY POINTS: • C. albicans CHK1 gene is important for its interaction with S. mutans. • CHK1 regulates S. mutans two-component system to promote its cariogenicity. • CHK1 gene regulates the cariogenicity of S. mutans in rat dental caries.


Assuntos
Cárie Dentária , Streptococcus mutans , Animais , Pré-Escolar , Humanos , Ratos , Biofilmes , Candida albicans/metabolismo , Suscetibilidade à Cárie Dentária , Streptococcus mutans/genética , Proteínas Fúngicas/genética , Quinase 1 do Ponto de Checagem/genética
6.
Pathog Dis ; 80(1)2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36302135

RESUMO

Extracellular vesicles (EVs) are nano-sized-particles that play an important role in cellular cross-talk. The aim of this study was to understand the proteomic cargo of EVs, released by Retinal Pigment Epithelial (RPE) cells challenged with Candida albicans (C-CA) and Aspergillus flavus (C-AF). EVs were isolated from culture supernatant of retinal cells infected with fungal pathogens and characterized by dynamic light scattering, SEM, and western blot. EV proteome was then evaluated by mass spectrometry (LC-MS/MS). Isolated EVs were approximately 120-150 nm and higher in number in infected group compared to control. Proteomic profiling of EVs from infected cells, showed a total of 419 and 254 differentially expressed proteins, of which 218 were upregulated in C-CA group and 81 proteins were upregulated in C-AF group. Gene ontology revealed majority of proteins associated with transport, cell migration, and in activation of innate immune response. Proteins identified were annexins, calpain, and Sorcin proteins. Additionally, KEGG analysis unveiled involvement of MAPK, HIF-1, and PI3K-AKT signalling pathways. Proteomic results indicate that EVs cargo derived from fungal-infected retinal cells can activate immune signalling pathways and might contribute to the pathogenesis of endophthalmitis, indicating the potential use of EVs as theranostic marker for management of fungal infections.


Assuntos
Endoftalmite , Vesículas Extracelulares , Humanos , Candida albicans/metabolismo , Proteômica/métodos , Aspergillus flavus , Cromatografia Líquida , Fosfatidilinositol 3-Quinases/metabolismo , Espectrometria de Massas em Tandem , Vesículas Extracelulares/química , Endoftalmite/metabolismo
7.
Genetics ; 222(4)2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36226807

RESUMO

Candida albicans is an opportunistic fungal pathogen that causes superficial infections in immunocompetent individuals, as well as life-threatening systemic disease in immunocompromised patients. A key virulence trait of this pathogen is its ability to transition between yeast and filamentous morphologies. A functional genomic screen to identify novel regulators of filamentation previously revealed VPS53 as being important for morphogenesis. Vps53 belongs to the Golgi-associated retrograde protein (GARP) complex, which mediates retrograde trafficking from the endosome to the trans-Golgi network. Here, we explored the role of the entire GARP complex in regulating morphogenesis. Deletion of any of the four genes encoding GARP complex subunits severely impaired filamentation in response to diverse filament-inducing cues, including upon internalization by macrophages. Genetic pathway analysis revealed that while hyperactivation of protein kinase A (PKA) signaling is insufficient to drive filamentation in GARP complex mutants, these strains are capable of filamentation upon overexpression of transcriptional activators or upon deletion of transcriptional repressors of hyphal morphogenesis. Finally, compromise of the GARP complex induced lipotoxicity, and pharmacological inhibition of sphingolipid biosynthesis phenocopied genetic compromise of the GARP complex by impairing filamentation. Together, this work identifies the GARP complex as an important mediator of filamentation in response to multiple inducing cues, maps genetic circuitry important for filamentation upon compromise of GARP function, and supports a model whereby GARP deficiency impairs lipid homeostasis, which is important for supporting filamentous growth in C. albicans.


Assuntos
Candida albicans , Proteínas Fúngicas , Humanos , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas , Morfogênese/genética , Fatores de Transcrição/genética , Regulação Fúngica da Expressão Gênica
8.
Front Cell Infect Microbiol ; 12: 944611, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36237434

RESUMO

Candida albicans is a pathogenic yeast that causes candidiasis in immunocompromised patients. The overuse of antifungal drugs has led to the development of resistance to such drugs by this fungus, which is a major challenge in antifungal chemotherapy. One approach to this problem involves the utilization of new natural products as an alternative source of antifungals. Curcumin, one such natural product, has been widely studied as a drug candidate and is reported to exhibit antifungal activity against C. albicans. Although studies of the mechanism of curcumin against human cancer cells have shown that it inhibits heat shock protein 90 (Hsp90), little is known about its function against C. albicans. In this paper, using a doxycycline-mediated HSP90 strain and an HSP90-overexpressing strain of C. albicans, we demonstrated that the curcumin triggered a decrease in Hsp90 by affecting it at the post-transcriptional level. This also led to the downregulation of HOG1 and CDR1, resulting in a reduction of the stress response and efflux pump activity of C. albicans. However, the inhibition of HSP90 by curcumin was not due to the inhibition of transcription factors HSF1 or AHR1. We also found that curcumin can not only decrease the transcriptional expression of CDR1, but also inhibit the efflux pump activity of Cdr1. Hence, we conclude that disruption of HSP90 by curcumin could impair cell growth, stress responses and efflux pump activity of C. albicans.


Assuntos
Produtos Biológicos , Curcumina , Antifúngicos/farmacologia , Candida albicans/metabolismo , Curcumina/farmacologia , Doxiciclina , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90 , Humanos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Microb Pathog ; 172: 105765, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36087690

RESUMO

An accumulating trend of research demonstrates that diabetic patients are susceptible to skin infections with Candida albicans, but the mechanism still remains unclear. The intense oxidative stress (OS) responses were occurred in the lesion of diabetic mice footpads after C. albicans infection. Localised skin infections would lead to more severe complications while the severity of the condition worsens or the inadequate treatment. Notably, in this study, through the investigation of murine diabetic footpad C. albicans infection model and molecular biotechnology, including histopathological staining, immunofluorescence (IF) staining, quantitative real-time PCR (qPCR), western blot (WB), flow cytometry (FCM), sandwich enzyme-linked immunosorbent assay (ELISA) assays, we found that intense OS responses in the footpad tissue not only mediated the activation of NF-κB protein complex, but also triggered downstream pyroptosis and apoptosis through NLRP3 inflammasome, which is one of the potential reasons for the severe condition of infectious skin injuries in diabetic mice. Caspase-1, a classical signal pathway protein in pyroptosis, could promote pore formation on cell membranes and the release of the cytokine after NLRP3 inflammasome activation. With intense immune-inflammatory responses, the organism also stimulates immune organs such as the spleen and lymph nodes to produce negative feedback regulation and generate CD4+CD25+Foxp3+ Treg cells to rectify the process. Therefore, combined with the results of this work, it is possible to design and screen relevant drugs for NLRP3 inflammasomes as core targets to keep the OS response at a low level in the footpad tissues.


Assuntos
Diabetes Mellitus Experimental , Pé Diabético , Animais , Camundongos , Piroptose/fisiologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Candida albicans/metabolismo , NF-kappa B/metabolismo , Diabetes Mellitus Experimental/complicações , Caspase 1/metabolismo , Estresse Oxidativo/fisiologia , Citocinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo
10.
PLoS Genet ; 18(9): e1010390, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36084128

RESUMO

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph.


Assuntos
Heme , Proteínas de Saccharomyces cerevisiae , Candida albicans/genética , Candida albicans/metabolismo , Heme/genética , Heme/metabolismo , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/metabolismo , Hemina/metabolismo , Hemina/farmacologia , Homeostase/genética , Ferro/metabolismo , Peroxidases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
PLoS Genet ; 18(9): e1010405, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36121853

RESUMO

In order to successfully induce disease, the fungal pathogen Candida albicans regulates exposure of antigens like the cell wall polysaccharide ß(1,3)-glucan to the host immune system. C. albicans covers (masks) ß(1,3)-glucan with a layer of mannosylated glycoproteins, which aids in immune system evasion by acting as a barrier to recognition by host pattern recognition receptors. Consequently, enhanced ß(1,3)-glucan exposure (unmasking) makes fungal cells more visible to host immune cells and facilitates more robust fungal clearance. However, an understanding of how C. albicans regulates its exposure levels of ß(1,3)-glucan is needed to leverage this phenotype. Signal transduction pathways and their corresponding effector genes mediating these changes are only beginning to be defined. Here, we report that the phosphatase calcineurin mediates unmasking of ß(1,3)-glucan in response to inputs from the Cek1 MAPK pathway and in response to caspofungin exposure. In contrast, calcineurin reduces ß-glucan exposure in response to high levels of extracellular calcium. Thus, depending on the input, calcineurin acts as a switchboard to regulate ß(1,3)-glucan exposure levels. By leveraging these differential ß(1,3)-glucan exposure phenotypes, we identified two novel effector genes in the calcineurin regulon, FGR41 and C1_11990W_A, that encode putative cell wall proteins and mediate masking/unmasking. Loss of either effector caused unmasking and attenuated virulence during systemic infection in mice. Furthermore, immunosuppression restored the colonization decrease seen in mice infected with the fgr41Δ/Δ mutant to wild-type levels, demonstrating a reliance on the host immune system for virulence attenuation. Thus, calcineurin and its downstream regulon are general regulators of unmasking.


Assuntos
Candida albicans , Proteínas Fúngicas/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , beta-Glucanas , Animais , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Caspofungina/farmacologia , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Glucanos/metabolismo , Camundongos , beta-Glucanas/metabolismo
12.
Redox Biol ; 56: 102461, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36087429

RESUMO

Fungal infections cause serious health problems, especially in patients with an immune-deficiency. Histone deacetylase 11 (HDAC11) mediates various immune functions, yet little is known about its role in regulating host immune responses to fungal infection. Here we report that HDAC11 negatively controls antifungal immunity in macrophages and dendritic cells. Deleting Hdac11 protects mice from morbidity and markedly improves their survival rate upon systemic infection with Candida albicans (C. albicans). Moreover, HDAC11 deficiency results in increased production of NO and reactive oxygen species, which enhances fungal killing. Mechanistically, loss of HDAC11 increases histone 3 and 4 acetylation at the Nos2 promoter and leads to enhanced Nos2 transcription and corresponding iNOS levels in macrophages. In addition, STAT3, a transcriptional repressor of Nos2, physically interacts with HDAC11, serving as a scaffold protein supporting the HDAC11 association with the Nos2 promoter. Notably, treatment with the HDAC11 inhibitor, FT895, exhibits antifungal therapeutic effects in both mouse and human cells challenged with C. albicans. These data support that HDAC11 may be a therapeutic target for fungal infection.


Assuntos
Antifúngicos , Histonas , Animais , Antifúngicos/farmacologia , Candida albicans/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
13.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077373

RESUMO

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.


Assuntos
Candida albicans , Ergosterol , Proteínas de Membrana Transportadoras/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Esfingolipídeos/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/citologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Ceramidas/metabolismo , Farmacorresistência Fúngica , Ergosterol/metabolismo , Proteínas Fúngicas/metabolismo , Inositol/farmacologia , Proteínas de Membrana Transportadoras/análise , Testes de Sensibilidade Microbiana
14.
Cell Rep ; 40(12): 111374, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36130496

RESUMO

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.


Assuntos
Candida albicans , Micotoxinas , Candida albicans/metabolismo , Morte Celular , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Hifas/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Micotoxinas/metabolismo , Micotoxinas/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
15.
Microbiol Res ; 263: 127132, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35940106

RESUMO

Intra-abdominal candidiasis (IAC) occurs due to the direct inoculation of Candida into the sterile peritoneal cavity or leakage of the gastrointestinal tract. An important difference between the two forms of the disease is the presence of fecal material, which is exclusive to the latter condition. However, the influence of fecal material on the prognosis of IAC is still poorly understood. Furthermore, methodologies that use the quantification of fungal load by culture methods have low sensitivity, as they do not adequately show the precocity of the infectious process. Here, we developed a new method to evaluate the aspects of the pathophysiology of IAC, mainly the influence of fecal material on the prognosis of infection, by using C. albicans radiolabeled with technetium-99 m (99 mTc). C. albicans was successfully radiolabeled with 99 mTc (18.5 MBq) using dihydrate stannous chloride (100 µM) as a reducing agent. This binding was stable for 72 h. Viability, yeast-to-hyphae transition, morphology, and antifungal susceptibility were not altered by radiolabeling C. albicans with 99 mTc. The biomass and the fungal load of 99 mTc-C. albicans biofilms were reduced compared to the C. albicans non-radiolabeled after 72 h and 48 h of incubation, respectively. In the IAC model, the fungal load in the biodistribution of 99 mTc-C. albicans and culture assays was higher in animals receiving fungal inoculum without fecal material, suggesting that the presence of this component reduces the invasiveness of the pathogen.


Assuntos
Candida albicans , Candidíase , Animais , Antifúngicos/metabolismo , Candida albicans/metabolismo , Candidíase/diagnóstico por imagem , Candidíase/tratamento farmacológico , Modelos Animais de Doenças , Camundongos , Tecnécio , Distribuição Tecidual
16.
Microbiol Res ; 263: 127146, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35940108

RESUMO

The sensing of environmental conditions such as nutrient availability and the ability to adapt and respond to changing conditions are crucial for the survival of living organisms. Evidence from several organisms have revealed that some metabolic enzymes act as sensors of nutrient status and regulate the expression of sets of genes required for nutrients utilization and condition specific environmental adaptation. Thus metabolic enzymes regulate the signaling pathway by acting as transcriptional regulators and providing required metabolites. The commensal yeast, Candida albicans has recently emerged as a model system for understanding the N-acetylglucosamine (GlcNAc) signaling pathway in eukaryotes. GlcNAc kinase (Hxk1), the first enzyme of the catabolic cascade, has been shown to perform several functions such as regulation of gene expression and regulation of the metabolic status of the cell thereby resulting in a change in cell morphology (yeast-hyphal transition, white-opaque switching), metabolic gene expression, synthesis of metabolic precursors, induction of glycolytic flux rate and biofilm formation. Here, in this review we have discussed various roles of Hxk1that have not been reported in other organisms previously. The enzyme exhibits dynamic changes in subcellular localization consistent with its expanded functions inside the cell. Thus Hxk1 in C. albicans orchestrates several dynamic cellular processes and this signaling system can act as a paradigm to understand the cell fate and metabolic specialization in other eukaryotes too. Still, the molecular cues involved in Hxk1 mediating functions are yet to be unveiled; the relationship between Hxk1 sensing and its signaling effects is also not understood yet.


Assuntos
Candida albicans , Regulação Fúngica da Expressão Gênica , Acetilglucosamina/genética , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
17.
PLoS One ; 17(8): e0273663, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36040970

RESUMO

Candidalysin, a peptide toxin produced specifically from hyphae of Candida albicans, plays a crucial role in C. albicans pathogenesis in the oral cavity and vagina. Synthetic peptides have been widely used in previous studies to investigate the bioactivity of candidalysin. Although the solubility of the peptide, which is expected to have a hydrophobic property, has not been well characterized, candidalysin solutions are usually prepared in water. In this study, we prepared the synthetic peptide candidalysin in water (CLw) or in dimethyl sulfoxide (CLd) and compared their cytotoxicity and interleukin (IL)-1ß-producing activity to determine whether the activity of the peptide would be affected. In addition, we evaluated whether the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway or other pathways were involved in their activities. Unexpectedly, we found that CLw was not completely solubilized and contained abundant insoluble microparticles. CLw was active at comparably high concentrations (≥ 10 µM). In contrast, CLd is completely solubilized and sufficiently active at low concentrations, that is, 1 µM or less. CLw showed weak cytotoxicity and NLRP3-dependent and cathepsin B-dependent IL-1ß-producing activity, whereas CLd showed strong cytotoxicity and cathepsin B-dependent IL-1ß-producing activity. Fractionation of CLw revealed that NLRP3-dependent activity was caused by insoluble microparticles. Furthermore, nanoparticle tracking of CLd revealed that the peptide was present as nanoparticles with a size of 96 nm. CLw contained a small amount of such nanoparticles. Thus, the bioactivities of the synthetic peptide candidalysin, especially the IL-1ß-producing activity, are affected by the solubility of the peptide depending on the solvent employed. The NLRP3-dependent activity of the synthetic peptide is caused by insoluble microparticles and may not be the intrinsic activity of candidalysin.


Assuntos
Catepsina B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Candida albicans/metabolismo , Catepsina B/metabolismo , Proteínas Fúngicas , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Solubilidade , Água/metabolismo
18.
mBio ; 13(5): e0193722, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-35993746

RESUMO

Candida albicans is among the most significant human fungal pathogens. However, the vast majority of C. albicans studies have focused on a single clinical isolate and its marked derivatives. We investigated natural variation among clinical C. albicans isolates in gene regulatory control of biofilm formation, a process crucial to virulence. The transcription factor Efg1 is required for biofilm-associated gene expression and biofilm formation. Previously, we found extensive variation in Efg1-responsive gene expression among 5 diverse clinical isolates. However, chromatin immunoprecipitation sequencing analysis showed that Efg1 binding to genomic loci was uniform among the isolates. Functional dissection of strain differences identified three transcription factors, Brg1, Tec1, and Wor1, for which small changes in expression levels reshaped the Efg1 regulatory network. Brg1 and Tec1 are known biofilm activators, and their role in Efg1 network variation may be expected. However, Wor1 is a known repressor of EFG1 expression and an inhibitor of biofilm formation. In contrast, we found that a modest increase in WOR1 RNA levels, reflecting the expression differences between C. albicans strains, could augment biofilm formation and expression of biofilm-related genes. The analysis of natural variation here reveals a novel function for a well-characterized gene and illustrates that strain diversity offers a unique resource for elucidation of network interactions. IMPORTANCE Clinical isolates of all pathogens vary in the strength of traits linked to disease. In this study, we focused on variation in a pathogenicity trait of the fungal pathogen Candida albicans, biofilm formation. This trait is under the control of the cell type regulator Efg1. Expression of Efg1 is known from previous studies to be repressed by a second cell type regulator, Wor1. However, we found that natural variation in biofilm formation and biofilm-related gene expression was driven by collaboration between Efg1 and Wor1. Our findings show that analysis of natural isolates can reveal unexpected features of gene function, even for well-studied genes.


Assuntos
Candida albicans , Proteínas Fúngicas , Biofilmes , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Regulação Fúngica da Expressão Gênica , RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
J Biol Chem ; 298(10): 102419, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36037968

RESUMO

Candida albicans (C. albicans) is a dimorphic commensal human fungal pathogen that can cause severe oropharyngeal candidiasis (oral thrush) in susceptible hosts. During invasive infection, C. albicans hyphae invade oral epithelial cells (OECs) and secrete candidalysin, a pore-forming cytolytic peptide that is required for C. albicans pathogenesis at mucosal surfaces. Candidalysin is produced in the hyphal invasion pocket and triggers cell damage responses in OECs. Candidalysin also activates multiple MAPK-based signaling events that collectively drive the production of downstream inflammatory mediators that coordinate downstream innate and adaptive immune responses. The activities of candidalysin are dependent on signaling through the epidermal growth factor receptor (EGFR). Here, we interrogated known EGFR-MAPK signaling intermediates for their roles mediating the OEC response to C. albicans infection. Using RNA silencing and pharmacological inhibition, we identified five key adaptors, including growth factor receptor-bound protein 2 (Grb2), Grb2-associated binding protein 1 (Gab1), Src homology and collagen (Shc), SH2-containing protein tyrosine phosphatase-2 (Shp2), and casitas B-lineage lymphoma (c-Cbl). We determined that all of these signaling effectors were inducibly phosphorylated in response to C. albicans. These phosphorylation events occurred in a candidalysin-dependent manner and additionally required EGFR phosphorylation, matrix metalloproteinases (MMPs), and cellular calcium flux to activate a complete OEC response to fungal infection. Of these, Gab1, Grb2, and Shp2 were the dominant drivers of ERK1/2 activation and the subsequent production of downstream innate-acting cytokines. Together, these results identify the key adaptor proteins that drive the EGFR signaling mechanisms that underlie oral epithelial responses to C. albicans.


Assuntos
Candida albicans , Candidíase Bucal , Receptores ErbB , Proteínas Fúngicas , Mucosa Bucal , Humanos , Candida albicans/metabolismo , Candida albicans/patogenicidade , Citocinas/metabolismo , Receptores ErbB/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Candidíase Bucal/metabolismo , Candidíase Bucal/microbiologia , Mucosa Bucal/metabolismo , Mucosa Bucal/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia
20.
PLoS One ; 17(8): e0272844, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35947573

RESUMO

Green methods have become vital for sustainable development of the scientific and commercial sphere; however, they can bring new challenges, including the need for detailed characterization and elucidation of efficacy of their products. In this study, green method of silver nanoparticles (AgNPs) production was employed using an extract from grapevine canes. The aim of the study was to contribute to the knowledge about biosynthesized AgNPs by focusing on elucidation of their antifungal efficiency based on their size and/or hypothesized synergy with bioactive substances from Vitis vinifera cane extract. The antifungal activity of AgNPs capped and stabilized with bioactive compounds was tested against the opportunistic pathogenic yeast Candida albicans. Two dispersions of nanoparticles with different morphology (characterized by SEM-in-STEM, DLS, UV-Vis, XRD, and AAS) were prepared by modification of reaction conditions suitable for economical production and their long-term stability monitored for six months was confirmed. The aims of the study included the comparison of the antifungal effect against suspension cells and biofilm of small monodisperse AgNPs with narrow size distribution and large polydisperse AgNPs. The hypothesis of synergistic interaction of biologically active molecules from V. vinifera extracts and AgNPs against both cell forms were tested. The interactions of all AgNPs dispersions with the cell surface and changes in cell morphology were imaged using SEM. All variants of AgNPs dispersions were found to be active against suspension and biofilm cells of C. albicans; nevertheless, surprisingly, larger polydisperse AgNPs were found to be more effective. Synergistic action of nanoparticles with biologically active extract compounds was proven for biofilm cells (MBIC80 20 mg/L of polydisperse AgNPs in extract), while isolated nanoparticles suspended in water were more active against suspension cells (MIC 20 mg/L of polydisperse AgNPs dispersed in water). Our results bring new insight into the economical production of AgNPs with defined characteristics, which were proven to target a specific mode of growth of significant pathogen C. albicans.


Assuntos
Nanopartículas Metálicas , Prata , Antibacterianos/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Biofilmes , Candida albicans/metabolismo , Testes de Sensibilidade Microbiana , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Prata/metabolismo , Prata/farmacologia , Água/metabolismo
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