Secretory Protease Research Articles

Fungal-Host Interaction: Curcumin Modulates Proteolytic Enzyme Activity of Candida albicans and Inflammatory Host Response In Vitro.

Current treatments for Candida albicans infection are limited due to the limited number of antifungal drugs available and the increase in antifungal resistance. Curcumin is used as a spice, food preservative, flavoring, and coloring agent that has been shown to have many pharmacological activities. Thus, this study evaluated the modulatory effects of curcumin on major virulence factors associated with the pathogenicity of C. albicans. The minimum inhibitory concentration (MIC) of curcumin against C. albicans (SC5314) was determined. Biofilm formation was quantified and the proteinase and phospholipase secretion was measured. The cytotoxicity was tested in oral fibroblast cells. A cocultured model was used to analyze the gene expression of proinflammatory cytokines (IL-1β, IL-1α, and IL-6) from host cells, as well SAP-1 and PLB-1 by RT-PCR. The MIC was between 6.25 and 12.5 µM, and the activity of proteinase enzyme was significantly decreased in biofilms treated with curcumin. However, proteinase gene expression was not downregulated after curcumin treatment. Furthermore, gene expressions of host inflammatory response, IL-1β and IL-1α, were significantly downregulated after exposure to curcumin. In conclusion, curcumin exhibited antifungal activity against C. albicans and modulated the proteolytic enzyme activities without downregulating the gene expression. In host inflammatory response, curcumin downregulated IL-1β and IL-1α gene expression.

Genome-Wide Screening for Novel Candidate Virulence Related Response Regulator Genes in Xanthomonas oryzae pv. oryzicola.

Two-component regulatory system (TCS), a major type of cellular signal transduction system, is widely used by bacteria to adapt to different conditions and to colonize certain ecological niches in response to environmental stimuli. TCSs are of distinct functional diversity, genetic diversity, and species specificity (pathovar specificity, even strain specificity) across bacterial groups. Although TCSs have been demonstrated to be crucial to the virulence of Xanthomonas, only a few researches have been reported about the studies of TCSs in Xanthomonas oryzae pathovar oryzicola (hereafter Xoc), the pathogen of rice bacterial streak disease. In the genome of Xoc strain GX01, it has been annotated 110 TCSs genes encoding 54 response regulators (RRs), 36 orthodox histidine kinase (HKs) and 20 hybrid histidine kinase (HyHKs). To evaluate the involvement of TCSs in the stress adaptation and virulence of Xoc, we mutated 50 annotated RR genes in Xoc GX01 by homologous vector integration mutagenesis and assessed their phenotypes in given conditions and tested their virulence on host rice. 17 RR genes were identified to be likely involved in virulence of Xoc, of which 10 RR genes are novel virulence genes in Xanthomonas, including three novel virulence genes for bacteria. Of the novel candidate virulence genes, some of which may be involved in the general stress adaptation, exopolysaccharide production, extracellular protease secretion and swarming motility of Xoc. Our results will facilitate further studies on revealing the biological functions of TCS genes in this phytopathogenic bacterium.

Growth and protease secretion of Scedosporium aurantiacum under conditions of hypoxia

One of the micro-environmental stresses that fungal pathogens, such as Scedosporium aurantiacum, colonising human lungs encounter in vivo is hypoxia, or deficiency of oxygen. In this work, we studied the impacts of a hypoxic micro-environment (oxygen levels ≤1%) on the growth of a clinical S. aurantiacum isolate (WM 06.482; CBS 136046) and an environmental strain (S. aurantiacum WM 10.136; CBS 136049) on mucin-containing synthetic cystic fibrosis sputum medium. Additionally, profiles of secreted proteases were compared between the two isolates and protease activity was assessed using class-specific substrates and inhibitors. Overall, both isolates grew slower and produced less biomass under hypoxia compared to normoxic conditions. The pH of the medium decreased to 4.0 over the cultivation time, indicating that S. aurantiacum released acidic compounds into the medium. Accordingly, secreted proteases of the two isolates were dominated by acidic proteases, including aspartic and cysteine proteases, with optimal protease activity at pH 4.0 and 6.0 respectively. The clinical isolate produced higher aspartic and cysteine protease activities. Conversely, all serine proteases, including elastase-like, trypsin-like, chymotrypsin-like and subtilisin-like proteases had higher activities in the environmental isolate. Sequence similarities to 13 secreted proteases were identified by mass spectrometry (MS) by searching against other fungal proteases in the NCBI database. Results from MS analysis were consistent with those from activity assays. The clinical highly-virulent, and environmental low-virulence S. aurantiacum isolates responded differently to hypoxia in terms of the type of proteases secreted, which may reflect their different virulence properties.

Approaches to the mechanism of antifungal activity of Zuccagnia punctata-Larrea nitida bi-herbal combination

BackgroundIn our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA). PurposeTo study the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work. Materials and methodsMinimum Fungicidal Concentration (MFC) and rate of killing of ZpE-LnE were assessed with the microbroth dilution and the time-kill assays respectively. Morphological alterations were observed with both confocal and fluorescence microscopy on the yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the effects on the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors was assessed with previously reported methods. The effect of ZpE-LnE and of ZpE or LnE alone on cell viability was determined on human hepatoma cells line Huh7. ResultsZpE-Ln E was fungicidal killing C. albicans in a shorter time than amphotericin B and produced malformations in S. pombe cells. ZpE-LnE showed to bind to ergosterol but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS. Regarding inhibition of virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic buccal epithelial cells (BECs), did not inhibit the germ tube formation and inhibited the secretion of phospholipases and proteinases but not of haemolysins. ZpE-LnE demonstrated very low toxicity on Huh7 cells, much lower than that each extract alone. ConclusionThe fungicidal properties of ZpE-LnE against C. albicans, its dual mechanism of action targeting the fungal membrane's ergosterol as well as the cell wall, its capacity of inhibiting several important virulence factors added to its low toxicity, make ZpE-LnE a good candidate for the development of a new antifungal bi-Herbal Medicinal Product.

Improving of hydrolases biosythesis by solid-state fermentation of Penicillium camemberti on rapeseed cake

The study show usefulness of rapeseed cake, rich in fats and proteins byproduct generated after oil production, which may be used as a microbial medium for lipase and protease biosynthesis. Of 26 different filamentous fungi screened by solid-state fermentation, Penicillium camemberti AM83 was found to abundantly produce lipase and protease. Various process parameters were then optimized to maximize lipase and protease secretion, including carbon and nitrogen source, C/N ratio, metal ions, temperature, moisture content, initial pH, and inoculum size. Lipase production increased approximately 11.2-fold in solid-state cultures on rapeseed cake supplemented with lactose and calcium chloride, alkalinized to pH 8, hydrated to 80%, and inoculated with 1.2 × 106 spores/mL. Similarly, protease production increased approximately 8.4-fold in optimized cultures inoculated with 3.2 × 108 spores/mL, and grown on rapeseed cake with lactose and ammonium sulfate at pH 9 and moisture content 60%. The results highlight the potential economic value of solid-state fermentation on rapeseed cake to produce industrial hydrolases.

N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding.

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

Incidence of leaf spot disease on cotton caused by Curvularia verruculosa and role of its hydrolytic enzymes in pathogenesis

The finding described in this study is the first report of leaf spot disease of cotton caused by Curvularia verruculosa surveyed in the state of Maharashtra (India). The isolated phytopathogenic fungal strain was identified using morphological characteristics and molecular identification of ITS gene sequence (MF784436) and D1D2 region of LSU gene (KY978073). The ability of fungal strain to secrete hydrolytic enzymes viz., pectinase, xylanase, protease, cellulase and lipase was detected. The secretion profile of hydrolytic enzymes by C. verruculosa was also examined in planta and in vitro. The secretion of cellulase, xylanase and protease was found to be inducible on cotton-stalk powder containing media; while secretion of pectinase and lipase was constitutive in glucose containing medium. The hydrolytic enzymes secretion during etiological progression of disease was detected on cotton leaves at regular interval of 24h up to 10days. A significant correlation (P<0.05) was observed between hydrolytic enzymes secretion and disease severity index. The increased level of hydrolytic enzymes in infected plant sample indicates their role in disease progression. The newly documented fungal phytopathogen Curvularia verruculosa was deposited at National Fungal Culture Collection of India, Pune with accession number of NFCCI-4119.

Withania somnifera as a Potential Anxiolytic and Anti-inflammatory Candidate Against Systemic Lipopolysaccharide-Induced Neuroinflammation.

Reactive gliosis, microgliosis, and subsequent secretion of various inflammatory mediators like cytokines, proteases, reactive oxygen, and nitrogen species are the suggested key players associated with systemic inflammation-driven neuroinflammation and cognitive impairments in various neurological disorders. Conventionally, non-steroidal anti-inflammatory drugs are prescribed to suppress inflammation but due to their adverse effects, their usage is not well accepted. Natural products are emerging better therapeutic agents due to their affordability and inherent pleiotropic biological activities. In Ayurveda, Ashwagandha (Withania somnifera) is well known for its immunomodulatory properties. The current study is an extension of our previous report on in vitro model system and was aimed to investigate anti-neuroinflammatory potential of water extract from the Ashwagandha leaves (ASH-WEX) against systemic LPS-induced neuroinflammation and associated behavioral impairments using in vivo rat model system. Oral feeding of ASH-WEX for 8weeks significantly ameliorated the anxiety-like behavior as evident from Elevated plus maze test. Suppression of reactive gliosis, inflammatory cytokines production like TNF-α, IL-1β, IL-6, and expression of nitro-oxidative stress enzymes like iNOS, COX2, NOX2 etc were observed in ASH-WEX-treated animals. NFκB, P38, and JNK MAPKs pathways analysis showed their involvement in inflammation suppression which was further confirmed by inhibitor studies. The current study provides first ever preclinical evidence and scientific validation that ASH-WEX exhibits the anti-neuroinflammatory potential against systemic LPS-induced neuroinflammation and ameliorates associated behavioral abnormalities. Aqueous extract from Ashwagandha leaves and its active phytochemicals may prove to be promising candidates to prevent neuroinflammation associated with various neuropathologies.

Modulation of Receptor Protein Tyrosine Phosphatase Sigma Increases Chondroitin Sulfate Proteoglycan Degradation through Cathepsin B Secretion to Enhance Axon Outgrowth.

Severed axon tips reform growth cones following spinal cord injury that fail to regenerate, in part, because they become embedded within an inhibitory extracellular matrix. Chondroitin sulfate proteoglycans (CSPGs) are the major axon inhibitory matrix component that is increased within the lesion scar and in perineuronal nets around deafferented neurons. We have recently developed a novel peptide modulator (intracellular sigma peptide) of the cognate receptor of CSPGs, protein tyrosine phosphatase σ (RPTPσ), which has been shown to markedly improve sensorimotor function, micturition, and coordinated locomotor behavior in spinal cord contused rats. However, the mechanism(s) underlying how modulation of RPTPσ mediates axon outgrowth through inhibitory CSPGs remain unclear. Here, we describe how intracellular sigma peptide modulation of RPTPσ induces enhanced protease Cathepsin B activity. Using DRG neurons from female Sprague Dawley rats cultured on an aggrecan/laminin spot assay and a combination of biochemical techniques, we provide evidence suggesting that modulation of RPTPσ regulates secretion of proteases that, in turn, relieves CSPG inhibition through its digestion to allow axon migration though proteoglycan barriers. Understanding the mechanisms underlying RPTPσ modulation elucidates how axon regeneration is impaired by proteoglycans but can then be facilitated following injury.SIGNIFICANCE STATEMENT Following spinal cord injury, chondroitin sulfate proteoglycans (CSPGs) upregulate and potently inhibit axon regeneration and functional recovery. Protein tyrosine phosphatase σ (RPTPσ) has been identified as a critical cognate receptor of CSPGs. We have previously characterized a synthetic peptide (intracellular sigma peptide) that targets the regulatory intracellular domain of the receptor to allow axons to regenerate despite the presence of CSPGs. Here, we have found that one important mechanism by which peptide modulation of the receptor enhances axon outgrowth is through secretion of a protease, Cathepsin B, which enables digestion of CSPGs. This work links protease secretion to the CSPG receptor RPTPσ for the first time with implications for understanding the molecular mechanisms underlying neural regeneration and plasticity.

The Role of Macrophage/B-Cell Interactions in the Pathophysiology of B-Cell Lymphomas.

Macrophages (MPs) are heterogeneous, multifunctional, myeloid-derived leukocytes that are part of the innate immune system, playing wide-ranging critical roles in basic biological activities, including maintenance of tissue homeostasis involving clearance of microbial pathogens. Tumor-associated MPs (TAMs) are MPs with defined specific M2 phenotypes now known to play central roles in the pathophysiology of a wide spectrum of malignant neoplasms. Also, TAMs are often intrinsic cellular components of the essential tumor microenvironment (TME). In concert with lymphoid-lineage B and T cells at various developmental stages, TAMs can mediate enhanced tumor progression, often leading to poor clinical prognosis, at least partly through secretion of chemokines, cytokines, and various active proteases shown to stimulate tumor growth, angiogenesis, metastasis, and immunosuppression. Researchers recently showed that TAMs express certain key checkpoint-associated proteins [e.g., programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1)] that appear to be involved in T-cell activation and that these proteins are targets of other specific checkpoint-blocking immunotherapies (anti-PD-1/PD-L1) currently part of new therapeutic paradigms for chemotherapy-resistant neoplasms. Although much is known about the wide spectrum and flexibility of MPs under many normal and neoplastic conditions, relatively little is known about the increasingly important interactions between MPs and B-lymphoid cells, particularly in the TME in patients with aggressive B-cell non-Hodgkin lymphoma (NHL-B). Normal and neoplastic lymphoid and myeloid cell/MP lineages appear to share many primitive cellular characteristics as well as transcriptional factor interactions in human and animal ontogenic studies. Such cells are capable of ectopic transcription factor-induced lineage reprogramming or transdifferentiation from early myeloid/monocytic lineages to later induce B-cell lymphomagenesis in experimental in vivo murine systems. Close cellular interactions between endogenous clonal neoplastic B cells and related aberrant myeloid precursor cells/MPs appear to be important interactive components of aggressive NHL-B that we discuss herein in the larger context of the putative role of B-cell/MP cellular lineage interactions involved in NHL-B pathophysiology during ensuing lymphoma development.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry.KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth.Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

Astrocyte-T cell signaling via Junctional Adhesion Molecule-A (JAM-A) controls cytokine and protease secretion, T cell infiltration of the CNS and clinical disability during autoimmune attack (P5.035)

Astrocyte-T cell signaling via Junctional Adhesion Molecule-A (JAM-A) controls cytokine and protease secretion, T cell infiltration of the CNS and clinical disability during autoimmune attack (P5.035)

Structural characterization and in vivo pro-tumor properties of a highly conserved matrikine.

Elastin-derived peptides (EDPs) exert protumor activities by increasing tumor growth, migration and invasion. A number of studies have highlighted the potential of VGVAPG consensus sequence-derived elastin-like polypeptides whose physicochemical properties and biocompatibility are particularly suitable for in vivo applications, such as drug delivery and tissue engineering. However, among the EDPs, the influence of elastin-derived nonapeptides (xGxPGxGxG consensus sequence) remains unknown. Here, we show that the AGVPGLGVG elastin peptide (AG-9) present in domain-26 of tropoelastin is more conserved than the VGVAPG elastin peptide (VG-6) from domain-24 in mammals. The results demonstrate that the structural features of AG-9 and VG-6 peptides are similar. CD, NMR and FTIR spectroscopies show that AG-9 and VG-6 present the same conformation, which includes a mixture of random coils and β-turn structures. On the other hand, the supraorganization differs between peptides, as demonstrated by AFM. The VG-6 peptide gathers in spots, whereas the AG-9 peptide aggregates into short amyloid-like fibrils. An in vivo study showed that AG-9 peptides promote tumor progression to a greater extent than do VG-6 peptides. These results were confirmed by in vitro studies such as 2D and 3D proliferation assays, migration assays, adhesion assays, proteinase secretion studies and pseudotube formation assays to investigate angiogenesis. Our findings suggest the possibility that the AG-9 peptide present in patient sera may dramatically influence cancer progression and could be used in the design of new, innovative antitumor therapies.

Sodium thiosulphate induced immobilized bacterial disintegration of sludge: An energy efficient and cost effective platform for sludge management and biomethanation

The present study aimed to gain better insights into profitable biomethanation through sodium thiosulphate induced immobilized protease secreting bacterial disintegration (STS-IPBD) of sludge. STS disperse the flocs at 0.08 g/g SS of dosage and assists the subsequent bacterial disintegration. Immobilization of bacteria increases the hydrolytic activity of cells towards effective liquefaction of sludge. A higher liquefaction of 22% was accomplished for STS-IPBD when compared to immobilized protease secreting bacterial disintegration (IPBD alone). The kinetic parameters of Line Weaver Burk plot analysis revealed a maximal specific growth rate (µmax) of 0.320 h−1 for immobilized cells when compared to suspended free cells showing the benefit of immobilization. Floc dispersion and immobilization of bacteria imparts a major role in biomethanation as the methane generation (0.32 gCOD/g COD) was higher in STS-IPBD sample. The cost analysis showed that STS – IPBD was a feasible process with net profit of 2.6 USD/Ton of sludge.

The First Whole-Cell Proteome- and Lysine-Acetylome-Based Comparison between Trichophyton rubrum Conidial and Mycelial Stages.

Trichophyton rubrum is the most common fungal pathogen in the world, which has been studied as an important dermatophyte model organism. Despite the prevalence of T. rubrum, the available antifungal therapies are not sufficiently efficient. In this study, we performed the first comparison between the two major growth stages of T. rubrum: conidial and mycelial stages, based on their whole-cell proteomes and lysine acetylomes. In total, 4343 proteins were identified in both stages, and 1879 proteins were identified as differentially expressed between the two stages. The results showed that secretory proteases were more abundant in conidia, while aerobic metabolism and protein synthesis were significantly activated in the mycelial stage. In addition, 386 acetylated sites on 285 proteins and 5414 acetylated sites on 2335 proteins were identified in conidia and mycelia, respectively. The acetylation modifications were highly involved in metabolism and protein synthesis in both stages but differentially involved in Kyoto Encyclopedia of Genes and Genomes pathways and in epigenetic regulation between the two stages. Furthermore, inhibition of acetyltransferases or deacetylases significantly inhibited fungal growth and induced apoptosis. These results will enhance our understanding of the biological and physiological characteristics of T. rubrum and facilitate the development of improved therapies targeting these medically important pathogenic fungi.

Production of Proteinase with Plasmin-Like and Prekallikrein Activating Activity by the Micromycete Aspergillus terreus

The effect of nitrogen sources in the fermentation medium and the cultivation conditions on the production of proteinases with plasmin-like and prekallikrein activation activity by micromycete Aspergillus terreus 2 was investigated. The highest secretion of proteinases was achieved when the micromycete was cultivated on a medium containing both amine- and mineral nitrogen sources at an initial pH of 5.5 and at 28°С. It was established that the extracellular micromycete proteinases are equally capable of hydrolyzing fibrin and fibrinogen.

Anticandidal activity of bioinspired ZnO NPs: effect on growth, cell morphology and key virulence attributes of Candida species

The pathogenicity of Candida species in human is dependent on a variety of virulence factor such as adhesion factors, germ tube and hyphal formation, secretion of hydrolytic phospholipases and proteinases and drug resistance biofilm. ZnO NPs have been synthesized by using leaf extract of Crinum latifolium and were characterized by UV–Vis spectrophotometer, FTIR, SEM, EDX and TEM. In this study for the first time, potent inhibitory effects of ZnO NPs on principal virulence factors of Candida albicans and non-albicans such as germ tube formation, secretion of hydrolytic phospholipases and proteinases and biofilm formation has been investigated. ZnO NPs remarkably reduced the germ tube formation of C. albicans at 1 (86.4%), 0.5 (75.0%), 0.25 (61.4%), 0.125 (34.1%) and 0.062 mg/ml (11.4%). ZnO NPs significantly lowered the phospholipase and proteinase secretion by 58.8 and 95.2% at 0.25 mg/ml, respectively. CSLM results showed that ZnO NPs suppressed biofilm formation up to 85% at 0.25 mg/ml. SEM and TEM micrograph showed that ZnO NPs penetrated inside the cell and causes extensive damaged in cell wall and cell membrane. Inhibition of Candida growth and various virulent factors by ZnO NPs provides an insight towards their therapeutic application for the treatment of Candida-associated infections.

A common evolutionary pathway for maintaining quorum sensing in Pseudomonas aeruginosa.

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the "tragedy of the commons". Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided "tragedy of the commons". The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.

Hepatitis C virus regulates proprotein convertase subtilisin/kexin type 9 promoter activity

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine protease mainly expressed in liver. Although PCSK9 has been shown to inhibit hepatitis C virus (HCV) entry and replication, whether HCV regulates PCSK9 transcription has not been well studied. PCSK9 promoter activity is modulated by numerous transcription factors including sterol-regulatory element binding protein (SREBP)-1a, -1c, −2, hepatocyte nuclear factor-1 (HNF-1), and forkhead box O3 (FoxO3). Since they are differently regulated by HCV, we studied the effects of these transcription factors on PCSK9 promoter activity in the context of HCV infection and replication. We demonstrated that PCSK9 promoter activity was up-regulated after HCV infection and in HCV genomic replicon cells. We also studied the effects of HCV proteins on the PCSK9 promoter activity. While HCV structural proteins core, E1, and E2 had no effect, NS2, NS3, NS3-4A, NS5A and NS5B enhanced, and p7 and NS4B decreased PCSK9 promoter activity. Furthermore, we showed that transcription factors SREBP-1c, HNF-1α and specificity protein 1 increased PCSK9 promoter activity in HCV replicon cells, whereas SREBP-1a, HNF-1β and FoxO3 had an inhibitory effect. These results demonstrated the molecular mechanisms of how HCV modulates PCSK9 promoter activity and advanced our understanding on the mutual interactions between HCV and PCSK9.

УЧАСТИЕ ТУЧНЫХ КЛЕТОК В АДАПТАЦИИ ЖЕЛУДКА МОНГОЛЬСКИХ ПЕСЧАНОК К ГРАВИТАЦИОННОМУ ФАКТОРУ

The aim was to study the morphofunctional state of the mast cells (MC) population in the stomach of Mongolian gerbils after a 12-day space flight. Material and methods. The experiment was performed on 35 Mongolian gerbils, 12 of which were in the 12-day space flight onboard the “Foton-M” No. 3 spacecraft, 11 animals made up a group of synchronous experiments, simulating some conditions of orbital flight, 12 – a vivarium group. The object of the study – the MC of the stomach fundus. Histochemical (revealing the chloroacetyl esterase activity, staining with toluidine blue) and immunomorphological (identification of tryptase, MC chymase and growth factor of MC and stem cells receptor – CD117) staining techniques were used. Results. A decrease in the volume of the gastric MC population was observed, which was combined with a change in the secretion and expression of the growth factor receptor of mast cells and stem cells after orbital flight. The increase in the number of chloroacetyl esterase-positive MC, in the intensity of migration of mature MC precursors in the gastric membrane was found. The decrease in tryptase-containing MC in the population, indicating the active secretion of protease under the influence of real space flight factors, was accompanied by an increase of chymase expression. The results of the synchronous experiment on modeling the orbital flight conditions in the model of ground equipment were not characterized by such pronounced changes. Conclusions. MC are a multifunctional element of the stomach anti-gravity system and a promising target for the study of adaptive processes in tissue microenvironment in zero gravity. MC take an active part in the formation of gravisensitivity of the stomach tissue structures of Mongolian gerbils and determine the development of adaptive processes under the influence of weightlessness at the local and systemic levels.