Association of the hypha‐related protein Pra1 and zinc transporter Zrt1 with biofilm formation by the pathogenic yeast Candida albicans

Bloodstream infection by the pathogenic fungus Candida albicans is a major health problem. Candidemia is often associated with medical devices, which can act as substrates for biofilm development. Biofilm‐related infections are relatively difficult to treat because of their resistance to antimicrobial agents. It is therefore important to explore the mechanisms of biofilm formation. Dimorphism is a major contributor to biofilm formation in C. albicans. To determine whether the hypha‐related proteins Pra1 (pH‐regulated antigen) and Zrt1 (zinc transporter) are responsible for biofilm formation, the ability of pra1 and zrt1 deletion mutants to form biofilms was investigated. Biofilm formation by both deletion mutants was less than that of the wild‐type strain. Because Pra1 and Zrt1 are also related to the zinc homeostasis system, the effects of adding zinc on biofilm formation were also examined. Biofilm formation was increased in the presence of zinc. These data suggest that Pra1 and Zrt1 regulate biofilm formation through zinc homeostasis.

     

Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment.     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Biofilm formation of Candida albicans on the surface of a soft denture‐lining material

Background: Soft denture lining‐materials are more susceptible to microbial adhesion than hard denture base acrylic resin. Poor oral hygiene and Candida albicans infection are common among elderly denture wearers as these patients usually have difficulty in keeping them clean. Purpose: To evaluate the influence of the oral hygiene methods on the formation of a biofilm over a soft denture‐lining material. Material and methods: Twenty volunteers were randomly separated into two groups: G1 and G2. Ten volunteers performed daily hygiene of the prostheses with a soft toothbrush and toothpaste. The G2 performed a treatment identical to G1 but also immersed the prostheses in sodium hypochlorite 0.5% for 20 min, once a week. Quantification of the mean score values of biofilm formation at different times were statistically analysed using analysis of variance and Tukey’s test (α = 0.05). Results: G1 (0.65 ± 0.52) showed the lowest mean score values of biofilm formation. There was statistical difference between G1 and G2. The highest mean score values were found at 6 weeks (1.3 ± 1.08) and were statistically different from other times. Conclusion: The oral hygiene methods had a significant effect in the formation of the biofilm over a soft denture‐lining material.     

In vitro assessment of the growth and plasma membrane H+‐ATPase inhibitory activity of ebselen and structurally related selenium‐ and sulfur‐containing compounds in Candida albicans

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H⁺‐ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)‐resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC₅₀ ～ 18 μM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC₅₀ ～ 14 μM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU‐resistant fungi.     

Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation

Plasma membrane (PM) blebs are dynamic actin-rich cell protrusions that occur, e.g., during cytokinesis, amoeboid cell motility and cell attachment. Using a targeted siRNA screen against 21 actin nucleation factors, we identify a novel and essential role of the human diaphanous formin DIAPH3 in PM blebbing during cell adhesion. Suppression of DIAPH3 inhibited blebbing to promote rapid cell spreading involving β1-integrin. Multiple isoforms of DIAPH3 were detected on the mRNA and protein level of which isoforms 3 and 7 were the largest and most abundant isoforms that however did not induce formation of actin-rich protrusions. Rather, PM blebbing specifically involved the low abundance isoform 1 of DIAPH3 and activation of isoform 7 by deletion of the diaphanous-autoregulatory domain caused the formation of filopodia. Dimerization and actin assembly activity were essential for induction of specific cell protrusions by DIAPH3 isoforms 1 and 7. Our data suggest that the N-terminal region comprising the GTPase-binding domain determined the subcellular localization of the formin as well as its protrusion activity between blebs and filopodia. We propose that isoform-selective actin assembly by DIAPH3 exerts specific and differentially regulated functions during cell adhesion and motility.     

Distinct roles for β‐arrestin2 and arrestin‐domain‐containing proteins in β2 adrenergic receptor trafficking

β‐arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven‐transmembrane receptor trafficking and signalling. Other proteins with predicted ‘arrestin‐like’ structural domains but lacking sequence homology have been indicated to function like β‐arrestin in receptor regulation. We demonstrate that β‐arrestin2 is the primary adaptor that rapidly binds agonist‐activated β₂ adrenergic receptors (β₂ARs) and promotes clathrin‐dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin‐dependent lysosomal degradation of the receptor. The arrestin‐domain‐containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized β₂AR–Nedd4 complexes on endosomes and do not affect the adaptor roles of β‐arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4–β₂AR complexes to a subpopulation of early endosomes.     

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly‐γ‐glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self‐assembles as mono‐disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)‐solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET_A) in their active conformations. Furthermore, ET_A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.     

Substrate relay in an Hsp70‐cochaperone cascade safeguards tail‐anchored membrane protein targeting

Membrane proteins are aggregation‐prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post‐translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail‐anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo. Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how “substrate funneling” through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.     

The carboxylic acid transporters Jen1 and Jen2 affect the architecture and fluconazole susceptibility of Candida albicans biofilm in the presence of lactate

Candida albicans has the ability to adapt to different host niches, often glucose-limited but rich in alternative carbon sources. In these glucose-poor microenvironments, this pathogen expresses JEN1 and JEN2 genes, encoding carboxylate transporters, which are important in the early stages of infection. This work investigated how host microenvironments, in particular acidic containing lactic acid, affect C. albicans biofilm formation and antifungal drug resistance. Multiple components of the extracellular matrix were also analysed, including their impact on antifungal drug resistance, and the involvement of both Jen1 and Jen2 in this process. The results show that growth on lactate affects biofilm formation, morphology and susceptibility to fluconazole and that both Jen1 and Jen2 might play a role in these processes. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity.     

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca²⁺‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l⁻¹, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca²⁺‐ATPase (PMCA) activity, Ca²⁺/CaM activated EGM Ca²⁺ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca²⁺/CaM to activate EGM Ca²⁺‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.     

Distinct haplotype structure at the innate immune receptor Toll‐like receptor 2 across bank vole populations and lineages in Europe

Parasite‐mediated selection may contribute to the maintenance of genetic variation at host immune genes over long time scales. To date, the best evidence for the long‐term maintenance of immunogenetic variation in natural populations comes from studies on the major histocompatibility complex (MHC) genes, whereas evidence for such processes from other immune genes remains scarce. In the present study, we show that, despite pronounced population differentiation and the occurrence of numerous private alleles within populations, the innate immune gene Toll‐like receptor 2 (TLR2) displays a distinct haplotype structure in 21 bank vole (Myodes glareolus) populations across Europe. Haplotypes from all populations grouped in four clearly differentiated clusters, with the three main clusters co‐occurring in at least three previously described mitochondrial lineages. This pattern indicates that the distinct TLR2 haplotype structure may precede the split of the mitochondrial lineages 0.19–0.56 Mya and suggests that haplotype clusters at this innate immune receptor are maintained over prolonged time in wild bank vole populations.     

Pleiotropic effects of the histone deacetylase Hos2 linked to H4‐K16 deacetylation,H3‐K56 acetylation,and H2A‐S129 phosphorylation in Beauveria bassiana

Histone acetyltransferases and deacetylases maintain dynamics of lysine acetylation/deacetylation on histones and nonhistone substrates involved in gene regulation and cellular events. Hos2 is a Class I histone deacetylases that deacetylates unique histone H4‐K16 site in yeasts. Here, we report that orthologous Hos2 deacetylates H4‐K16 and is also involved in the acetylation of histone H3‐K56 and the phosphorylation of histone H2A‐S129 and cyclin‐dependent kinase 1 CDK1‐Y15 in Beauveria bassiana, a filamentous fungal insect pathogen. These site‐specific modifications are evidenced with hyperacetylated H4‐K16, hypoacetylated H3‐K56, and both hypophosphorylated H2A‐S129 and CDK1‐Y15 in absence of hos2. Consequently, the Δhos2 mutant suffered increased sensitivities to DNA‐damaging and oxidative stresses, disturbed cell cycle, impeded cytokinesis, increased cell size or length, reduced conidiation capacity, altered conidial properties, and attenuated virulence. These phenotypic changes correlated well with dramatic repression of many genes that are essential for DNA damage repair, G₁/S transition and DNA synthesis, hyphal septation, and asexual development. The uncovered ability for Hos2 to directly deacetylate H4‐K16 and to indirectly modify H3‐K56, H2A‐S129, and CDK1‐Y15 provides novel insight into more subtle regulatory role for Hos2 in genomic stability and diverse cellular events in the fungal insect pathogen than those revealed previously in nonentomophathogenic fungi.     

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml⁻¹. In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml⁻¹. The results obtained from real-time PCR and analysis of the ∆∆C_q values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.     

Syngas fermentation of Clostridium carboxidivoran P7 in a hollow fiber membrane biofilm reactor: Evaluating the mass transfer coefficient and ethanol production performance

Gasification followed by syngas fermentation is a unique hybrid process for converting lignocellulosic biomass into fuels and chemicals. Current syngas fermentation faces several challenges with low gas–liquid mass transfer being one of the major bottlenecks. The aim of this work is to evaluate the performance of hollow fiber membrane biofilm reactor (HFM-BR) as a reactor configuration for syngas fermentation. The volumetric mass transfer coefficient (K_La) of the HFM-BR was determined at abiotic conditions within a wide range of gas velocity/flowrate passing through the hollow fiber lumen and liquid velocity/flowrate passing through the membrane module shell. The K_La values of the HFM-BR were higher than most reactor configurations such as stir tank reactors and bubble columns. A continuous syngas fermentation of Clostridium carboxidivorans P7 was implemented in the HFM-BR system at different operational conditions, including the syngas flow rate, liquid recirculation between the module and reservoir, and the dilution rate. It was found that the syngas fermentation performance such as syngas utilization efficiency, ethanol concentration and productivity, and ratio of ethanol to acetic acid depended not only on the mass transfer efficiency but also the characteristics of biofilm attached on the membrane module (biofouling or abrading of the biofilm). The HFM-BR results in a highest ethanol concentration of 23.93 g/L with an ethanol to acetic acid ratio of 4.79. Collectively, the research shows the HFM-BR is an efficient reactor system for syngas fermentation with high mass transfer.