Candida Albicans Protein Research Articles

Molecular Docking and Zone Inhibition Analysis of Fractionated Ethanol Extract of Zingiber officinale var. rubrum Against Candida albicans as Oral Antifungal

Ginger is a herb that can be used as an alternative medicine because it has anti-bacterial, antioxidant, anti-inflammatory, analgesic and antifungal properties. This study aims to identify the potency of red ginger ethanol extract against Candida albicans protein by using in silico and in vitro approaches. Several ginger-derived compounds were obtained from PubChem, while the target protein 4J14 was obtained from the Protein Data Bank (PDB) database. Prediction of Activity Spectra for Substances (PASS) Online was used to predict each compound’s biological function. Before molecular docking analysis, the 3D ligand site web server determined the binding site coordinates. PyRx was then used to perform molecular docking analysis on ginger compounds and the target protein 4J14, with fluconazole and posaconazole serving as controls. Additional analysis was used to identify amino acid residues in the complexes. The Chimera software was employed to describe protein compound complexes. The inhibitory zones of red ginger and fluconazole on fungal growth were determined in vitro. The molecular docking results showed that gamma-sitosterol had a more negative binding affinity (−9.6 kcal/mol) than fluconazole (−8.7 kcal/mol). Moreover, it affected the biological response of the target protein 4J14 or cytochrome 450, which is an essential protein in the fungal infection process. In vitro tests proved that a red ginger extract concentration of 15 mg/mL had antifungal potential. In silico and in vitro studies revealed that red ginger extract has the potential to be an antifungal agent. HIGHLIGHTS Since there have been numerous studies on the content of red ginger compounds with essential oils that have been shown to have antifungal potential, the fractionation approach was used to extract active compounds in red ginger that are devoid of essential oils in order to determine the antifungal potential The biological response of cytochrome 450, also known as target protein 4J14, is impacted by gamma-sitosterol. This protein is crucial for the mechanisms involved in fungal infection An extract from red ginger exhibited the same antifungal potential as fluconazole, according to in vitro experiments GRAPHICAL ABSTRACT

Optimizing Spectronaut Search Parameters to Improve Data Quality with Minimal Proteome Coverage Reductions in DIA Analyses of Heterogeneous Samples.

Data-independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimized to minimize the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings, we analyzed Mus musculus macrophages and compared their proteome to macrophages spiked withCandida albicans. This experimental system enabled the identification of "false positives" as Candida albicans peptides and proteins should not be present in the Mus musculus-only samples. We show that adjusting the search parameters reduced "false positive" identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimized parameters incurred a moderate cost, only reducing the overall number of "true positive" identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analyzing heterogeneous populations of cell types or tissues.

In vitro antimicrobial, anticancer evaluation, and in silico studies of mannopyranoside analogs against bacterial and fungal proteins: Acylation leads to improved antimicrobial activity

Carbohydrate analogs are an important, well-established class of clinically useful medicinal agents that exhibit potent antimicrobial activity. Thus, we explored the various therapeutic potential of methyl α-D-mannopyranoside (MαDM) analogs, including their ability to synthesize and assess their antibacterial, antifungal, and anticancer properties; additionally, molecular docking, molecular dynamics simulation, and ADMET analysis were performed. The structure of the synthesized MαDM analogs was ascertained by spectroscopic techniques and physicochemical and elemental analysis. In vitro antimicrobial activity was assessed and revealed significant inhibitory effects, particularly against gram-negative bacteria along with the prediction of activity spectra for substances (PASS). Concurrently, MαDM analogs showed good results against antifungal pathogens and exhibited promising anticancer effects in vitro, demonstrating dose-dependent cytotoxicity against Ehrlich ascites carcinoma (EAC) cancer cells while sparing normal cells from compound 5, with an IC50 of 4511.65 µg/mL according to the MTT colorimetric assay. A structure–activity relationship (SAR) study revealed that hexose combined with the acyl chains of decanoyl (C-10) and benzenesulfonyl (C6H5SO2-) had synergistic effects on the bacteria and fungi that were examined. Molecular docking was performed against the Escherichia coli (6KZV) and Candida albicans (1EAG) proteins to acquire insights into the molecular interactions underlying the observed biological activities. The docking results were further supported by 100 ns molecular dynamics simulations, which provided a dynamic view of the stability and flexibility of complexes involving MαDM and its targets. In addition, ADMET analysis was used to evaluate the toxicological and pharmacokinetic profiles. Owing to their promising drug-like properties, these MαDM analogs exhibit potential as prospective therapeutic candidates for future development.

Impact of polar (DMSO, ethanol, water) solvation on geometry, spectroscopy (FT-IR, UV, NMR), quantum chemical parameters, and the antifungal activities of benzothiazole derivative by molecular docking approach

Due to their ubiquity and the rise of drug-resistant forms, Candida albicans infections pose a serious threat to world health. Exploring new molecular possibilities is essential in order to create newer antifungal medicines to address this challenge. Herein, the use of density functional theory at the B3LYP-D3BJ/aug-cc-pVDZ method along with the in silico molecular docking was utilized to examine the effects of polar (DMSO, ethanol, water) solvation on the reactivity, spectral (NMR, UV, FT-IR) investigation, and the antifungal potential of a bis[ethyl2-(4-hydroxy-3-{(E)-[(1,3-benzothiazol-2-yl)inimo]methyl} phenyl)-4-methyl-1,3-thiazole-5-carbo -xylate (BTZ). The study finds that polar solvents exert a notable influence on BTZ's reactivity, with the highest energy gap observed in the gas phase with a value of 3.4939 eV while in the solvents; the values are 3.4477, 3.4477, and 3.4422 eV for DMSO, ethanol, and water, respectively. This observation implies that BTZ may exhibit varying degrees of reactivity under different solvents. To evaluate BTZ's suitability as a potential antifungal agent, absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies were conducted which reveals that BTZ adheres to Lipinski's rule of five, demonstrating its drug-like potential. Molecular docking simulations against Candida albicans proteins (1ZAP and 6ZDU) show promising binding affinities, with BTZ exhibiting a strong interaction with 1ZAP (-5.4 kcal/mol). The findings of this research contribute valuable insights into the reactivity and potential antifungal activity of BTZ, providing a promising candidate for further exploration in the quest for effective treatments against Candida infections.

Antifungal activity of compounds from Gordonia sp. WA8-44 isolated from the gut of Periplaneta americana and molecular docking studies

Invasive fungal infections are on the rise, leading to a continuous demand for antifungal antibiotics. Rare actinomycetes have been shown to contain a variety of interesting compounds worth exploring. In this study, 15 strains of rare actinobacterium Gordonia were isolated from the gut of Periplaneta americana and screened for their anti-fungal activity against four human pathogenic fungi. Strain WA8-44 was found to exhibit significant anti-fungal activity and was selected for bioactive compound production, separation, purification, and characterization. Three anti-fungal compounds, Collismycin A, Actinomycin D, and Actinomycin X2, were isolated from the fermentation broth of Gordonia strain WA8-44. Of these, Collismycin A was isolated and purified from the secondary metabolites of Gordonia for the first time, and its anti-filamentous fungi activity was firstly identified in this study. Molecular docking was carried out to determine their hypothetical binding affinities against nine target proteins of Candida albicans. Chitin Synthase 2 was found to be the most preferred antimicrobial protein target for Collismycin A, while 1,3-Beta-Glucanase was the most preferred anti-fungal protein target for Actinomycin D and Actinomycin X2. ADMET prediction revealed that Collismycin A has favorable oral bioavailability and little toxicity, making it a potential candidate for development as an orally active medication.

Functional genomic analysis of Candida albicans protein kinases reveals modulators of morphogenesis in diverse environments

Candida albicans is a leading cause of mycotic infection. The ability to transition between yeast and filamentous forms is critical to C.albicans virulence and complex signaling pathways regulate this process. Here, we screened a C.albicans protein kinase mutant library in six environmental conditions to identify regulators of morphogenesis. We identified the uncharacterized gene orf19.3751 as a negative regulator of filamentation and follow-up investigations implicated a role for orf19.3751 in cell cycle regulation. We also uncovered a dual role for the kinases Ire1 and protein kinase A (Tpk1 and Tpk2) in C.albicans morphogenesis, specifically as negative regulators of wrinkly colony formation on solid medium but positive regulators of filamentation in liquid medium. Further analyses suggested Ire1 modulates morphogenesis in both media states in part through the transcription factor Hac1 and in part through independent mechanisms. Overall, this work provides insights into the signaling governing morphogenesis in C.albicans.

Site-Selectivity of the Reaction of 3-Amino-4-Cyano-5-Phenyl-1H-Pyrrole-2-Carboxylic Acid Amide with α-Halocarbonyl Compounds. Antimicrobial Activity and Docking Study for COVID-19 of the Products

In this context, we are interested to study the site-selectivity of the reaction of 3-amino-4-cyano-5-phenyl-1H-pyrrole-2-carboxylic acid amide 1 with different types of hydrazonoyl halides as well as the α-haloketones. The products of these reactions were confirmed from their spectral data and conformational studies to be pyrrolo[1,2-d][1,2,4]triazine and pyrrolo[1,2-a]pyrazine derivatives. We used docking studies to test the ability of the new synthesized pyrrolo[1,2-d][1,2,4]triazine and pyrrolo[1,2-a]pyrazine derivatives to dock and inhibit the microbes and COVID-19 proteins. The results of both docking studies revealed a strong fit of almost the tested derivatives into the active sites of peroxiredoxin 5 (Pdb: 2WFC; the overexpressed proteins in Candida albicans) and COVID-19 main protease (Pdb: 6LU7; severe acute respiratory syndrome coronavirus 2). Moreover, the antimicrobial activity of the products was investigated against two selected Fungi species and two G+ and two G− bacteria. The results of antimicrobial activity indicated that almost all derivatives showed no activity against fungi species, while all of them have moderate activity against bacteria strains (G+ and G− bacteria).

B-Cell Epitope Mapping from Eight Antigens of Candida albicans to Design a Novel Diagnostic Kit: An Immunoinformatics Approach

Invasive candidiasis is an emerging fungal infection and a leading cause of morbidity in health care facilities. Despite advances in antifungal therapy, increased antifungal drug resistance in Candida albicans has enhanced patient fatality. The most common method for Candida albicans diagnosing is blood culture, which has low sensitivity. Therefore, there is an urgent need to establish a valid diagnostic method. Our study aimed to use the bioinformatics approach to design a diagnostic kit for detecting Candida albicans with high sensitivity and specificity. Eight antigenic proteins of Candida albicans (HYR1, HWP1, ECE1, ALS, EAP1, SAP1, BGL2, and MET6) were selected. Next, a construct containing different immunodominant B-cell epitopes was derived from the antigens and connected using a suitable linker. Different properties of the final construct, such as physicochemical properties, were evaluated. Moreover, the designed construct underwent 3D modeling, reverse translation, and codon optimization. The results confirmed that the designed construct could identify Candida albicans with high sensitivity and specificity in serum samples of patients with invasive candidiasis. However, experimental studies are needed for final confirmation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10989-022-10413-1.

Suppressor genetics reveals novel inter-domain crosstalk within the multidrug transporter Mdr1 protein

The Multidrug resistance-1 protein (Mdr1p) of Candida albicans is a crucial drug/H+ antiporter within the Major Facilitator Superfamily of proteins involved in the efflux of a broad spectrum of structurally diverse xenobiotic compounds. As a member of the DHA1 subfamily, Mdr1p consists of 12 transmembrane helices (TMHs), divided equally into two Transmembrane Domains (TMDs). How the pseudo-symmetrically positioned TMHs, and the TMDs they compose, communicate with each other remains poorly characterized. In that direction, the recovery of spontaneous chromosomal mutants that negatively affect the primary mutant phenotype can provide essential inter-domain communication insights. For this purpose, in the current study we have performed a suppressor screen for a critically transport deficient mutant G230A, located within TMH-4 of Mdr1p, predicted towards the intracellular space. The recovered suppressor (P528H), that restores the transport capacity of this initially drug susceptible mutant, map to TMH-12, very close to the extracellular space. Since the mutant and suppressor sites occupy the N-domain and C-domain, respectively, and locate at a pseudo-symmetrical position, these results hint to a novel pattern of crosstalk. Additionally, the recovered suppressor mutation restores wild type phenotypes for all tested xenobiotic substrates except cycloheximide, thus implying substrate selectivity. Furthermore, the molecular modeling and docking results suggest a novel compensatory mechanism which is independent of drug binding. Altogether, the present study is a first attempt to gain insights into the transport mechanism of drug/H+ antiporter using the suppressor genetics approach.

Proteinous Components of Neutrophil Extracellular Traps Are Arrested by the Cell Wall Proteins of Candida albicans during Fungal Infection, and Can Be Used in the Host Invasion

One of defense mechanisms of the human immune system to counteract infection by the opportunistic fungal pathogen Candida albicans is the recruitment of neutrophils to the site of invasion, and the subsequent production of neutrophil extracellular traps (NETs) that efficiently capture and kill the invader cells. In the current study, we demonstrate that within these structures composed of chromatin and proteins, the latter play a pivotal role in the entrapment of the fungal pathogen. The proteinous components of NETs, such as the granular enzymes elastase, myeloperoxidase and lactotransferrin, as well as histones and cathelicidin-derived peptide LL-37, are involved in contact with the surface of C. albicans cells. The fungal partners in these interactions are a typical adhesin of the agglutinin-like sequence protein family Als3, and several atypical surface-exposed proteins of cytoplasmic origin, including enolase, triosephosphate isomerase and phosphoglycerate mutase. Importantly, the adhesion of both the elastase itself and the mixture of proteins originating from NETs on the C. albicans cell surface considerably increased the pathogen potency of human epithelial cell destruction compared with fungal cells without human proteins attached. Such an implementation of adsorbed NET-derived proteins by invading C. albicans cells might alter the effectiveness of the fungal pathogen entrapment and affect the further host colonization.

Safety, immunogenicity, and efficacy of NDV-3A against Staphylococcus aureus colonization: A phase 2 vaccine trial among US Army Infantry trainees

BackgroundMilitary trainees are at increased risk for Staphylococcus aureus colonization and infection. Disease prevention strategies are needed, but a S. aureus vaccine does not currently exist. MethodsWe enrolled US Army Infantry trainees (Fort Benning, GA) in a phase 2, randomized, double-blind, placebo-controlled trial of NDV-3A, a vaccine containing a recombinant adhesin/invasion protein of Candida albicans that has structural similarity to the S. aureus protein clumping factor A. Study participants received one intramuscular dose of NDV-3A or placebo (adjuvant alone) within 72 h of arrival on base. Longitudinal nasal and oral (throat) swabs were collected throughout the 14-week Infantry training cycle. Safety, immunogenicity, and efficacy of NDV-3A against S. aureus nasal / oral acquisition were the endpoints. ResultsThe NDV-3A candidate had minimal reactogenicity and elicited robust antigen-specific B- and T-cell responses. During the 56-day post-vaccination period, there was no difference in the incidence of S. aureus nasal acquisition between those who were randomized to receive NDV-3A vs. placebo (25.6% vs. 29.1%; vaccine efficacy [VE]: 12.1%; p = 0.31). In time-to-event analysis, there was no difference between study groups with respect to the S. aureus colonization-free interval (VE: 13%; p = 0.29). Similarly, the efficacy of NDV-3A against S. aureus oral acquisition was poor (VE: 2.4%; p = 0.52). ConclusionsA single dose of NDV-3A did not prevent nasal nor oral acquisition of S. aureus in a population of military trainees at high risk for colonization.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

ABSTRACTFluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.

Determination of Candida albicans proteins concentration by enzyme-linked immunosorbent assay method at subcutaneous introduction in candidiasis therapy

Therapeutic effect of C. albicans proteins in concentrations 1, 2, 3, 4 and 5 mg/ml has been examined in white mice. Animals were infected i.p. with suspension of C. albicans strain CCM 335–867 in the amount of 20 million cells per 1 ml volume. After 5 days and repeatedly after 14 days mice were injected s.c. in the upper part of right hind paw with proteins of Candida cells of volume 0.2 ml. 14 days after each injection, the determination of the animal body protective functions has been carried out by the titer of specific antibodies during immunoassay. According to the data obtained, it has been found that in the s.c. administration, after the first and second injection with C. albicans protein of concentration 1, 2, 3, 4 and 5 mg/ml antibody titers increased 2 times, indicating that there is no stimulation of immune protection.

Ras activates the first step of GPI anchor biosynthesis in Candida albicans

Glycosylphosphatidylinositol (GPI) anchor biosynthesis is a multi‐step pathway found in all eukaryotes. GPI anchored proteins are involved in a variety of functions like adhesion, host cell attachment, invasion and biofilm formation in Candida albicans, a human pathogenic fungus. The first and committed step of the pathway i.e., transfer of GlcNAc from UDP‐GlcNAc to phosphatidylinositol (PI) to form GlcNAc‐PI, involves an enzyme complex (GPI‐GnT) of six subunits viz. CaGpi1, CaGpi2, CaGpi3, CaGpi15, CaGpi19 and CaEri1. A cross‐talk exists between GPI anchor biosynthesis, Ras signaling and ergosterol biosynthesis in Candida albicans, in which CaGPI2 controls hyphal morphogenesis through CaRAS1 while CaGPI19 seems to regulate azole drug response via co‐regulation of CaERG11. Interestingly, both CaGPI2 and CaGPI19 also negatively regulate each other. Further investigations on how Ras signaling cross‐talks with GPI biosynthesis in C. albicans suggest that Ras activates GPI‐GnT activity, which runs counter to what is reported in Saccharomyces cerevisiae. Out of the two Ras proteins of Candida albicans, CaRas1 alone activates the GPI‐GnT activity. Different forms of CaRas1 too can activate the GPI‐GnT in vivo. This evidence is further strengthened by the fact that CaRas1 physically interacts with CaGpi2, as evidenced by FRET. Furthermore, CaGpi2 appears to be important for virulence since a heterozygous mutant of CaGPI2 is unable to kill macrophage cells and the mutant is more susceptible for macrophage‐mediated killing. Current work is focused on identifying the domains of interaction between CaGpi2 and CaRas1. Understanding this interaction will enable us to target any or both of these molecules to control the virulence of this pathogen.Support or Funding InformationSSK received funding from Department of Science and Technology (DST) India Grant SB/SO/BB‐011/2014 and an umbrella grant to the School of Life Sciences from the DST under Promotion of University research and Scientific Excellence Grant DST PURSE No. SR/PURSE/PHASE 2/11 (C), and Department of Biotechnology (DBT) India Grant BT/PR3749/BRB/10/986/2011. SCS received Junior and Senior Research Fellowships from University Grants Commission (UGC), India.

Bioinformatic analysis and functional characterization of the CFEM proteins in maize anthracnose fungus Colletotrichum graminicola

Fungal secreted proteins that contain the Common in Fungal Extracellular Membrane (CFEM) domain are important for pathogenicity. The hemibiotrophic fungus Colletotrichum graminicola causes the serious anthracnose disease of maize. In this study, we identified 24 CgCFEM proteins in the genome of C. graminicola. Phylogenic analysis revealed that these 24 proteins (CgCFEM1—24) can be divided into 2 clades based on the presence of the trans-membrane domain. Sequence alignment analysis indicated that the amino acids of the CFEM domain are highly conserved and contain 8 spaced cysteines, with the exception that CgCFEM1 and CgCFEM24 lack 1 and 2 cysteines, respectively. Ten CgCFEM proteins with a signal peptide and without the trans-membrane domain were considered as candidate effectors and, thus were selected for structural prediction and functional analyses. The CFEM domain in the candidate effectors can form a helical-basket structure homologous to the Csa2 protein in Candida albicans, which is responsible for haem acquisition and pathogenicity. Subcellular localization analysis revealed that these effectors accumulate in the cell membrane, nucleus, and cytosolic bodies. Additionally, 5 effectors, CgCFEM6, 7, 8, 9 and 15, can suppress the BAX-induced programmed cell death in Nicotiana benthamiana with or without the signal peptide. These results demonstrate that these 10 CgCFEM candidate effectors with different structures and subcellular localizations in host cells may play important roles during the pathogenic processes on maize plants.

Multiple Drug Targeting Potential of Novel Ligands Against Virulent Proteins of Candida albicans

Systemic candidiasis is one of the most common nosocomial systemic infections causing high mortality and morbidity. Although infections caused by other Candida species are increasing the majority of candidiasis is commonly caused by Candida albicans which accounts for 40–60% of the reported cases. Current antifungal treatment regime is feeble due to persistent multiple drug resistance. Moreover, antifungal agents are mostly synthetic drugs that cause toxic side effects on immune-compromised patients. So, not only finding novel drug candidates from natural sources is necessary but to identify such compounds that have multiple targeting potential is essential. Hence, in this study, we propose to screen and identify bioactive natural compounds based on flavonoids, terpenes, and alkaloids for their potential antifungal activity. This in silico study was carried out by targeting 16 major virulent protein targets of C. albicans with 91 ligands. We have reported few ligands having multi-targeting potential and further analyzed theirs in silico pharmacokinetic profile. In silico ADMET analysis and protein–protein interaction can not only help in refining better drug candidates but also shortlist ideal protein for drug targeting.

Network analysis of hyphae forming proteins in Candida albicans identifies important proteins responsible for pathovirulence in the organism

Candida albicans causes two types of major infections in humans: superficial infections, such as skin and mucosal infection, and life-threatening systemic infections, like airway and catheter-related blood stream infections. It is a polymorphic fungus with two distinct forms (yeast and hyphal) and the morphological plasticity is strongly associated with many disease causing proteins. In this study, 137 hyphae associated proteins from Candida albicans (C. albicans) were collected from different sources to create a Protein-Protein Interaction (PPI) network. Out of these, we identified 18 hub proteins (Hog1, Hsp90, Cyr1, Cdc28, Pkc1, Cla4, Cdc42, Tpk1, Act1, Pbs2, Bem1, Tpk2, Ras1, Cdc24, Rim101, Cdc11, Cdc10 and Cln3) that were the most important ones in hyphae development. Ontology and functional enrichment analysis of these proteins could categorize these hyphae associated proteins into groups like signal transduction, kinase activity, biofilm formation, filamentous growth, MAPK signaling etc. Functional annotation analysis of these proteins showed that the protein kinase activity to be essential for hyphae formation in Candida. Additionally, most of the proteins from the network were predicted to be localized on cell surface or periphery, suggesting them as the main protagonists in inducing infections within the host. The complex hyphae formation phenomenon of C. albicans is an attractive target for exploitation to develop new antifungals and anti-virulence strategies to combat C. albicans infections. We further tried to characterize few of the most crucial proteins, especially the kinases by their sequence and structural prospects. Therefore, through this article an attempt to understand the hyphae forming protein network analysis has been made to unravel and elucidate the complex pathogenesis processes with the principal aim of systems biological research involving novel Bioinformatics strategies to combat fungal infections.

Alcohol dehydrogenase of Candida albicans triggers differentiation of THP-1 cells into macrophages

Candida albicans proteins located on the cell wall and in the cytoplasm have gained great attention because they are not only involved in cellular metabolism and the maintenance of integrity but also interact with host immune systems. Previous research has reported that enolase from C. albicans exhibits high immunogenicity and effectively protects mice against disseminated candidiasis. In this study, alcohol dehydrogenase (ADH) of C. albicans was cloned and purified for the first time, and this study focused on evaluating its effects on the differentiation of the human monocytic cell line THP-1. The morphological features of THP-1 cells exposed to ADH were similar to those of phorbol-12-myristate acetate-differentiated (PMA-differentiated) macrophages. Functionally, ADH enhanced the adhesion, phagocytosis, and killing capacities of THP-1 cells. A flow cytometric assay demonstrated that ADH-induced THP-1 cells significantly increased CD86 and CD11b expression. The production of IL-1β, IL-6, and TNF-α by cells increased in the presence of ADH. As expected, after pretreatment with a MEK inhibitor (U0126), ADH-induced THP-1 cells exhibited unaltered morphological features, eliminated ERK1/2 phosphorylation, prevented CD86/CD11b upregulation and inhibited pro-inflammatory cytokine increase. Collectively, these results suggest that ADH enables THP-1 cells to differentiate into macrophages via the ERK pathway, and it may play an important role in the immune response against fungal invasion.

Dissection of the regulatory role for the N-terminal domain in Candida albicans protein phosphatase Z1.

The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1–16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1–16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.

In Silico Inhibition of Essential Candida albicans Proteins by Arenicin, a Marine Antifungal Peptide

Fungal infections increased substantially in the last years, becoming a relevant public health problem. Many of these infections account for high rates of morbidity and mortality. The emergence of resistant fungal clinical isolates have also motivate studies to find new antifungal therapies. Candida albicans is an oportunistic pathogen and affects a great number of immunocompromised patients worldwide. The marine ecosystem has been considered a rich source of bioactive metabolites due to the complexity and originality of its structures. Proteins and peptides from marine organisms have been shown to have antiviral, anti-inflammatory, antimalarial, anticancer, antimicrobial and antifungal properties. Arenicins are antimicrobial peptides isolated from the marine lugworm Arenicola marina with 21 amino acid residues in a β-hairpin structure. Dihydrofolate reductase, exo-b-(1,3)-glucanase and sterol 14α-demethylase are essential C. albincas enzymes that take part in DNA, cell wall and membrane metabolism, respectively. The present study evaluates the interaction of arenicin with important enzymes of C. albicans related to cell wall, ergosterol and DNA metabolism in order to elucidate possible molecular targets. We showed through an in silico approach, that a single compound from a marine worm (A. marina), can bind to three C. albicans essential proteins. The interaction occurs in regions inside the active site or at least near, with amino acid residues evaluated as hot spots. Arenicin is a new promising antifugal drug. The next step is to investigate protein-protein interactions performed by DHFR, EBG and CYP51 and assess whether arenicin is able to disrupt essential interaction or not.