Surfaceome of pathogenic yeasts, Candida parapsilosis and Candida tropicalis, revealed with the use of cell surface shaving method and shotgun proteomic approach

In the course of infections caused by pathogenic yeasts from the genus Candida, the fungal cell surface is the first line of contact with the human host. As the surface-exposed proteins are the key players in these interactions, their identification can significantly contribute to discovering the mechanisms of pathogenesis of two emerging pathogens from this genus, C. parapsilosis and C. tropicalis. Therefore, the aim of the present study was to identify the cell wall-attached proteins of these two species with the use of cell surface shaving and a shotgun proteomic approach. Different morphological forms of C. parapsilosis and C. tropicalis cells obtained after growth under various conditions were subjected to this treatment. This allowed to indicate the most abundant cell surface proteins on the basis of the normalized spectral abundance factors. In case of yeast-like forms these were, among others, proteins similar to a chitinase, glyceraldehyde-3-phosphate dehydrogenase and an inducible acid phosphatase for C. parapsilosis, and a constitutive acid phosphatase, pyruvate decarboxylase and glyceraldehyde-3-phosphate dehydrogenase for C. tropicalis. In case of pseudohyphal forms, proteins similar to a cell surface mannoprotein Mp65, chitinase and glycosylphosphatidylinositol-anchored transglycosylase Crh11 were identified at the cell surface of C. parapsilosis. The Rbt1 cell wall protein, a hyphally regulated cell wall protein and proteins from agglutinin-like sequence protein family were found as the most abundant on C. tropicalis pseudohyphae. Apart from the abovementioned proteins, several additional covalently bound and atypical cell wall proteins were also identified. These results extend the current knowledge regarding the molecular basis of virulence of these two non-albicans Candida species

Interaction of human fibronectin with Candida glabrata epithelial adhesin 6 (Epa6)

Adherence of pathogens to extracellular matrix proteins and host cells is one of the essential steps in the microbial colonization of the human organism. The adhesion of C. glabrata, i.e. the second major causative agent of human disseminated candidiases after C. albicans, to the host epithelium mainly engages specific fungal cell wall proteins - epithelial adhesins (Epa) - in particular, Epa1, Epa6 and Epa7. The aim of the present study was to identify the major Epa protein involved in the interactions with the human extracellular matrix protein - fibronectin - and to present the kinetic and thermodynamic characteristics of these interactions. A relatively novel gel-free approach, i.e. the "cell surface shaving" that consists in short treatment of fungal cells with trypsin was employed to identify the C. glabrata surfaceome. Epa6 was purified, and the isolated protein was characterized in terms of its affinity to human fibronectin using a microplate ligand-binding assay and surface plasmon resonance measurements. The dissociation constants for the binding of Epa6 to fibronectin were determined to range between 9.03 × 10-9 M and 7.22 × 10-8 M, depending on the method used (surface plasmon resonance measurements versus the microplate ligand-binding assay, respectively). The identified fungal pathogen-human host protein-protein interactions might become a potential target for novel anticandidal therapeutic approaches

Binding of human plasminogen and high-molecular-mass kininogen by cell surface-exposed proteins of Candida parapsilosis

Pathogenic microbes can recruit to their cell surface human proteins that are components of important proteolytic cascades involved in coagulation, fibrinolysis and innate immune response. Once located at the bacterial or fungal surface, such deployed proteins might be utilized by pathogens to facilitate invasion and dissemination within the host organism by interfering with functionality of these systems or by exploiting specific activity of the bound enzymes. Aim of the study presented here was to characterize this phenomenon in Candida parapsilosis (Ashford) Langeron et Talice - an important causative agent of systemic fungal infections (candidiases and candidemias) in humans. We have investigated the interactions of fungal surface-exposed proteins with plasminogen (HPG) and high-molecular-mass kininogen (HK) - the crucial components of human fibrinolytic system and proinflammatory/procoagulant contact-activated kinin-forming system, respectively. After confirming ability of the fungal surface-exposed proteins to bind HPG and HK, four of them - two agglutinin-like sequence (Als) proteins CPAR2_404780 and CPAR2_404800, a heat shock protein Ssa2 and a moonlighting protein 6-phosphogluconate dehydrogenase 1 - were purified using ion-exchange chromatography, gel filtration and chromatofocusing. Then, their affinities to HPG and HK were characterized with surface plasmon resonance measurements. The determined dissociation constants for the investigated protein-protein complexes were within a 10-7 M order for the HPG binding and in a range of 10-8-10-9 M for the HK binding. Detailed characterization of adsorption of these two important plasma proteins on the fungal cell surface may help to increase our understanding of molecular mechanisms of C. parapsilosis-dependent candidiasis

Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts

Background : Candida parapsilosis and C. tropicalis increasingly compete with C. albicans—the most common fungal pathogen in humans—as causative agents of severe candidiasis in immunocompromised patients. In contrast to C. albicans, the pathogenic mechanisms of these two non-albicans Candida species are poorly understood. Adhesion of Candida yeast to host cells and the extracellular matrix is critical for fungal invasion of hosts. Methods : The fungal proteins involved in interactions with extracellular matrix proteins were isolated from mixtures of β-1,3-glucanase– or β-1,6-glucanase–extractable cell wall-associated proteins by use of affinity chromatography and chemical cross-linking methods, and were further identified by liquid chromatography-coupled tandem mass spectrometry. Results : In the present study, we characterized the binding of three major extracellular matrix proteins—fibronectin, vitronectin and laminin—to C. parapsilosis and C. tropicalis pseudohyphae. The major individual compounds of the fungal cell wall that bound fibronectin, vitronectin and laminin were found to comprise two groups: (1) true cell wall components similar to C. albicans adhesins from the Als, Hwp and Iff/Hyr families; and (2) atypical (cytoplasm-derived) surface-exposed proteins, including malate synthase, glucose-6-phosphate isomerase, 6-phosphogluconate dehydrogenase, enolase, fructose-1,6-bisphosphatase, transketolase, transaldolase and elongation factor 2. Discussion : The adhesive abilities of two investigated non-albicans Candida species toward extracellular matrix proteins were comparable to those of C. albicans suggesting an important role of this particular virulence attribute in the pathogenesis of infections caused by C. tropicalis and C. parapsilosis. Conclusions : Our results reveal new insight into host–pathogen interactions during infections by two important, recently emerging, fungal pathogens

New triazine derivatives as serotonin 5−HT65-HT_6 receptor ligands

Since the number of people with Alzheimer’s disease (AD) continues to rise, new and effective drugs are urgently needed to not only slow down the progression of the disease, but to stop or even prevent its development. Serotonin 5-HT6 receptor (5-HT6R) ligands are still a promising therapeutic target for the treatment of AD. 1,3,5-Triazine derivatives, as novel structures lacking an indole or a sulfone moiety, have proven to be potent ligands for this receptor. In present work, new derivatives of the compound MST4 (4-((2-isopropyl-5-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine), the potent 5-HT6R antagonist (Ki = 11 nM) with promising ADMET and in vivo properties, were designed. The synthesized compounds were tested for their affinity towards 5-HT6R and other receptor (off)targets (serotonin 5-HT2A, 5-HT7 and dopamine D2). Based on the new results, 4-(2-tert-butylphenoxy)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (3) was selected for extended in vitro studies as a potent and selective 5-HT6R ligand (Ki = 13 nM). Its ability to permeate the blood–brain barrier (BBB) and its hepatotoxicity were evaluated. In addition, X-ray crystallography and solubility studies were also performed. The results obtained confirm that 6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine derivatives, especially compound 3, are promising structures for further pharmacological studies as 5-HT6R ligands

Computer-Aided Studies for Novel Arylhydantoin 1,3,5-Triazine Derivatives as 5-HT6 Serotonin Receptor Ligands with Antidepressive-Like, Anxiolytic and Antiobesity Action In Vivo

This study focuses on the design, synthesis, biological evaluation, and computer-aided structure-activity relationship (SAR) analysis for a novel group of aromatic triazine-methylpiperazines, with an hydantoin spacer between 1,3,5-traizine and the aromatic fragment. New compounds were synthesized and their affinities for serotonin 5-HT6, 5-HT1A, 5-HT2A, 5-HT7, and dopamine D2 receptors were evaluated. The induced-fit docking (IFD) procedure was performed to explore the 5-HT6 receptor conformation space employing two lead structures. It resulted in a consistent binding mode with the activity data. For the most active compounds found in each modification line, anti-obesity and anti-depressive-like activity in vivo, as well as “druglikeness” in vitro, were examined. Two 2-naphthyl compounds (18 and 26) were identified as the most active 5-HT6R agents within each lead modification line, respectively. The 5-(2-naphthyl)hydantoin derivative 26, the most active one in the series (5-HT6R: Ki = 87 nM), displayed also significant selectivity towards competitive G-protein coupled receptors (6–197-fold). Docking studies indicated that the hydantoin ring is stabilized by hydrogen bonding, but due to its different orientation, the hydrogen bonds form with S5.44 and N6.55 or Q6.58 for 18 and 26, respectively. Compound 26 exerted anxiolytic-like and antidepressant-like activities. Importantly, it demonstrated anti-obesity properties in animals fed palatable feed, and did not show toxic effects in vitro

Candida albicans shields the periodontal killer Porphyromonas gingivalis from recognition by the host immune system and supports the bacterial infection of gingival tissue

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen—Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host–biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection