Aspartic Proteinase Activity Research Articles

Identification of the enzyme activity of human Demodex aspartic protease and its function to hydrolyse host macromolecules and skin cell proteins

Aspartic protease (ASP), a common proteolytic enzyme, plays an important role in the pathogenesis of numerous parasites. However, its role in Demodex remains unclear. Herein, we studied the expression, purification, enzymatic activity detection, and hydrolysis function of human Demodex ASP. The findings showed that recombinant ASP (rASP) possessed aspartic protease activity, which reached optimum levels at pH 2.5–3.0 and 35 °C. Furthermore, the activity of Demodex folliculorum rASP (Df. ASP) was considerably higher than that of Demodex brevis rASP (Db.rASP). Df.rASP also exhibited a more potent hydrolytic ability than Db.rASP. Df.rASP hydrolysed IgG, IgM, and fibronectin, whereas Db.rASP only slightly hydrolysed IgG. Mass spectrometry analysis revealed that Df.rASP exerted hydrolytic effects on 38 HaCaT proteins, more than the 23 proteins hydrolysed by Db.rASP. Sequence alignment and structure modelling of the substrate binding cleft identified three distinct amino acids between Df. ASP and Db. ASP, which should be the molecular basis for their difference in enzymatic activity and hydrolytic function. These results imply that Df.rASP may play a more critical role in the pathogenesis of human Demodex, and molecular data will provide a scientific basis for future analyses of their molecular pathogenesis.

Research progress on the regulatory cell death of osteoblasts in periodontitis

Periodontitis is a chronic inflammatory disease characterized by progressive destruction of alveolar bone. The most critical mechanism underlying alveolar bone destruction is the imbalance of bone homeostasis, where osteoblast-mediated bone matrix synthesis plays an important role in regulating bone homeostasis. Regulated cell death is instrumental in both the inflammatory microenvironment and the regulation of bone homeostasis. Chronic inflammation, oxidative stress, and other factors can be directly involved in mitochondrial and death receptor-mediated signaling pathways, modulating B-cell lymphoma 2 family proteins and cysteine aspartic acid specific protease (caspase) activity, thereby affecting osteoblast apoptosis and alveolar bone homeostasis. Chronic inflammation and cellular damage induce osteoblast necroptosis via the RIPK1/RIPK3/MLKL signaling pathway, exacerbating the inflammatory response and accelerating alveolar bone destruction. Stimuli such as pathogenic microorganisms and cellular injury may also activate caspase-1-dependent or independent signaling pathways and gasdermin D family proteins, promoting osteoblast pyroptosis and releasing pro-inflammatory cytokines to mediate alveolar bone damage. Iron overload and lipid peroxidation in periodontitis can trigger ferroptosis in osteoblasts, impacting their survival and function, ultimately leading to bone homeostasis imbalance. This article focuses on the mechanism of periodontal disease affecting bone homeostasis through regulatory cell death, aiming to provide research evidence for the treatment of periodontitis and alveolar bone homeostasis imbalance.

Ume6-dependent pathways of morphogenesis and biofilm formation in Candida auris.

Candida auris is a yeast pathogen causing nosocomial outbreaks of candidemia. Its ability to adhere to inert surfaces and to be transmitted from one patient to another via medical devices is of particular concern. Like other Candida spp., C. auris has the ability to transition from the yeast form to pseudohyphae and to build biofilms. Moreover, some isolates have a unique capacity to form aggregates. These morphogenetic changes may impact virulence. In this study, we demonstrated the role of the transcription factor Ume6 in C. auris morphogenesis. Genetic hyperactivation of Ume6 induced filamentation and aggregation. The Ume6-hyperactivated strain (UME6HA) also exhibited increased adhesion to inert surface and formed biofilms of higher biomass compared to the parental strain. Transcriptomic analyses of UME6HA revealed enrichment of genes encoding for adhesins, proteins involved in cell wall organization, sterol biosynthesis, and aspartic protease activities. The three most upregulated genes compared to wild-type were those encoding for the agglutin-like sequence adhesin Als4498, the C. auris-specific adhesin Scf1, and the hypha-specific G1 cyclin-related protein Hgc1. The deletion of these genes in the UME6HA background showed that Ume6 controls filamentation via Hgc1 and aggregation via Als4498 and Scf1. Adhesion to inert surface was essentially triggered by Scf1. However, Als4498 and Hgc1 were also crucial for biofilm formation. Our data show that Ume6 is a universal regulator of C. auris morphogenesis via distinct modulators.IMPORTANCEC. auris represents a public health threat because of its ability to cause difficult-to-treat infections and hospital outbreaks. The morphogenetic plasticity of C. auris, including its ability to filament, to form aggregates or biofilms on inert surfaces, is important to the fungus for interhuman transmission, skin or catheter colonization, tissue invasion, antifungal resistance, and escape of the host immune system. This work deciphered the importance of Ume6 in the control of distinct pathways involved in filamentation, aggregation, adhesion, and biofilm formation of C. auris. A better understanding of the mechanisms of C. auris morphogenesis may help identify novel antifungal targets.

Evidence of γ-secretase complex involved in the regulation of intramembrane proteolysis in Entamoeba histolytica

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites.

Candida spp. isolated from recreational coastal waters of Rio de Janeiro – Brazil: Focus on antifungal resistance and virulence attributes

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1–5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87–3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

Estimating the expression levels of genes controlling biofilm formation and evaluating the effects of different conditions on biofilm formation and secreted aspartic proteinase activity in Candida albicans and Saccharomyces cerevisiae: a comparative study

BackgroundCharacterization of yeast virulence genes is an important tool for identifying the molecular pathways involved in switching yeast virulence. Biofilm formation (BF) and secreted aspartic proteinase (SAP) activity are essential virulence factors that contribute to yeast pathogenicity.ResultsFour Candida albicans and two Saccharomyces cerevisiae strains were tested for BF and SAP activity under optimum conditions, and the expression levels of several genes controlling BF were quantified under the optimal conditions. Biofilm formation was assessed by the microplate method at different pH values, incubation times and culture media. Similarly, SAP activity was assessed at different pH values and incubation periods. The expression levels of nine genes were determined via qRT-PCR technique. All tests were carried out in triplicate, and the values presented as the means ± standard deviations and were analysed with the SPSS programme. Only C. albicans (1), C. albicans (2) and S. cerevisiae 43 formed biofilms. The optimal BF was obtained after culture in sabouraud dextrose broth with 8% glucose at pH 7.5, 4 and 6, respectively, for 48h. Candida albicans biofilm production was more significant than that of S. cerevisiae 43. Moreover, the SAP activity was estimated under the optimum conditions. All yeasts showed optimal SAP activity at pH 4, but astonishingly the SAP activity of S. cerevisiae 44 was higher than that of C. albicans. The expression levels of EFG1 and ZAP1 (transcription factors); ALS3, HWP1and YWP1 (adhesion genes); SAP1 and SAP4 (aspartic proteinase) in C. albicans (1); and FLO11 (adhesion gene) and YPS3 (aspartic proteinase) in S. cerevisiae 43 were quantified during biofilm development at different time intervals. The expression levels of EFG1, ALS3, YWP1, SAP1, SAP4, FLO11 and YPS3 were upregulated at 8 h, while that of ZAP1 was upregulated at 48 h. Only HWP1 was downregulated.ConclusionsThe findings of the present study may provide information for overcoming yeast BF and pathogenicity by regulating specific genes at specific times. Additionally, this study revealed the virulence of the commensal S. cerevisiae, which may take the pathogenicity direction as C. albicans.

Pathogen-induced methylglyoxal negatively regulates rice bacterial blight resistance by inhibiting OsCDR1 protease activity

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), a globally devastating disease of rice (Oryza sativa) that is responsible for significant crop loss. Sugars and sugar metabolites are important for pathogen infection, providing energy and regulating events associated with defense responses; however, the mechanisms by which they regulate such events in BB are unclear. As an inevitable sugar metabolite, methylglyoxal (MG) is involved in plant growth and responses to various abiotic stresses, but the underlying mechanisms remain enigmatic. Whether and how MG functions in plant biotic stress responses is almost completely unknown. Here, we report that the Xoo strain PXO99 induces OsWRKY62.1 to repress transcription of OsGLY II genes by directly binding to their promoters, resulting in overaccumulation of MG. MG negatively regulates rice resistance against PXO99: osglyII2 mutants with higher MG levels are more susceptible to the pathogen, whereas OsGLYII2-overexpressing plants with lower MG content show greater resistance than the wild type. Overexpression of OsGLYII2 to prevent excessive MG accumulation confers broad-spectrum resistance against the biotrophic bacterial pathogens Xoo and Xanthomonas oryzae pv. oryzicola and the necrotrophic fungal pathogen Rhizoctonia solani, which causes rice sheath blight. Further evidence shows that MG reduces rice resistance against PXO99 through CONSTITUTIVE DISEASE RESISTANCE 1 (OsCDR1). MG modifies the Arg97 residue of OsCDR1 to inhibit its aspartic protease activity, which is essential for OsCDR1-enhanced immunity. Taken together, these findings illustrate how Xoo promotes infection by hijacking a sugar metabolite in the host plant.

Investigation of optimal parameters for aspartic protease production from Aspergillus terreus using solid-state fermentation

The current study presents extracellular Aspartic protease production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus terreus, and parameters that largely affect enzyme production, namely fermentation time, pH, moistening solution, temperature, inoculum volume, and different solutions, were utilized for enzyme extraction. The isolated fungus was identified as A. terreus (registered as A. terreus SFH5 in the gene bank) by phenotypic and molecular characterization, showed the highest aspartic protease activity of 258.6 units/mL and 272.2 units/mg using a solid-state fermentation technique during screening, and was further used for media component screening and optimization studies. The study employed wheat bran as the preferred production medium and casein as the nitrogen source. The fermentation process was conducted at a temperature of 30°C and a pH of 5. The moistening solution to fermentation medium ratio was set at 3:1, while an inoculum volume of 2 mL (containing 1 × 106 spores/10 g solid) was used. Distilled water was selected as the most effective solution for extracting the enzyme. It has been discovered that the locally isolated novel strain of A. terreus, SFH5, can efficiently produce a substantial amount of aspartic protease by utilizing agro-industrial waste. This makes it a viable candidate for large-scale optimized production of the enzyme to satisfy industrial requirements.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

BackgroundAdding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied.ResultsIn our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0–5.0 and 30–60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below.ConclusionsIn this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening.

Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms. In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors. Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays. Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 μM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 μM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h). Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Cell progression of biofilm formation in Candida albicans and estimation of aspartic proteinase activity

Cell progression of biofilm formation in Candida albicans and estimation of aspartic proteinase activity

Distinct Phenotypic Variation of Blastocystis sp. ST3 from Urban and Orang Asli Population-An Influential Consideration during Sample Collection in Surveys.

Simple SummaryBlastocystis sp. is a common intestinal protozoan of humans with the phenotypic characteristics strongly associated with its activity, including pathogenicity. This characteristic varies, but the variation has not been clearly understood. The present study evaluates the variation when a single subtype of Blastocystis sp. was isolated from a population with distinct gut microbial composition, namely, the urban and orang asli(indigenous) population. Blastocystis sp. cells isolated from orang asli individuals had a higher growth rate with elevated resistance to harsh conditions. Distinct surface coats with amoebic forms were noticed in parasite cells from urban individuals. Proteases, commonly a virulent factor in other parasites, showed variation depending on the isolation source. Stimulation of cancer cell proliferation by only Blastocystis sp. isolated from urban individuals is suggestive of the variation at the antigenic level. This phenotypic variation suggests that implicating subtype to pathogenicity may be too early, and a deeper understanding of Blastocystis sp. and microenvironment interaction is essential.Blastocystis sp. is a globally distributed protozoan parasite with uncertain pathogenicity. Phenotypic variation in Blastocystis sp. suggests its adaptation; however, the phenotypic features of Blastocystis sp. ST3 from a distinct source of isolation is unknown. Blastocystis sp. isolated from individuals in urban and orang asli (indigenous population in Selangor, Malaysia) settlements were studied for phenotypic characteristics such as growth profile, morphology, ultrastructure, and resistance to harsh conditions. Subsequently, pathogenic potentials, such as in protease activity and the ability to stimulate the proliferation of cancer cells, were assessed. Higher parasite counts with granular and apoptotic forms were found in Blastocystis sp. from orang asli individuals. Cells with fuzzy coats and amoebic structures which seemingly implicate increased interaction with bacteria were seen predominantly in urban symptomatic persons. Also, Blastocystis sp. from orang asli isolates resisted harsh environments, suggesting longer co-adaptation to the hosts. Urban and orang asli symptomatic isolates possessed a predominance of only cysteine protease, whereas all the asymptomatic isolates showed significantly higher cysteine, serine, or aspartic protease activity. However, only solubilized antigen from urban symptomatic isolates showed significant stimulation of cancer cell proliferation. For the first time, our findings demonstrate significant phenotypic variation in a single subtype, ST3 of Blastocystis sp., isolated from urban and orang asli populations that are known to have distinct gut microbial compositions. The outcome emphasizes the importance of identifying people’s locations and lifestyles during sample collection before forming conclusions on the prevailing data and implicating subtypes to pathogenicity. The environment plays a significant role in Blastocystis sp. infection.

Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15µg of protein/mg and specific protease activity of 3.87 ± 0.52mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50°C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072μM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589mM and Vmax 1.909mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

Improving the alkaline stability of pepsin through rational protein design using renin, an alkaline-stable aspartic protease, as a structural and functional reference

The present study sought to identify the structural determinants of aspartic protease structural stability and activity at elevated pH. Various hypotheses have been published regarding the features responsible for the unusual alkaline structural stability of renin, however, few structure-function studies have verified these claims. Using pepsin as a model system, and renin as a template for functional and structural alkaline stability, a rational re-design of pepsin was undertaken to identify residues contributing to the alkaline instability of pepsin-like aspartic proteases in regards to both structure and function. We constructed 13 mutants based on this strategy. Among them, mutants D159 L and D60A led to an increase in activity at elevated pH levels (p ≤ 0.05) and E4V and H53F were shown to retain native-like structure at elevated pH (p ≤ 0.05). Previously suggested carboxyl groups Asp11, Asp118, and Glu13 were individually shown not to be responsible for the structural instability or lack of activity at neutral pH in pepsin. The importance of the β-barrel to structural stability was highlighted as the majority of the stabilizing residues identified, and 39% of the weakly conserved residues in the N-terminal lobe, were located in β-sheet strands of the barrel. The results of the present study indicate that alkaline stabilization of pepsin will require reduction of electrostatic repulsions and an improved understanding of the role of the hydrogen bonding network of the characteristic β-barrel.

Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration.

Dysfunction in the neurovascular unit (NVU) is a key component in the progressive deterioration of Alzheimer’s disease (AD) and is critical in vascular dementia. Recent studies have shown that inflammation plays early and perhaps causal roles in the pathogenesis of AD related to NVU damage, possibly in part by overactivating the aspartic acid protease activity of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which until now has almost solely been studied in the context of the β-amyloid cascade. In this study, we analyzed the relationship of BACE1 with astrocytes and blood vessels in human brains with sporadic and familial dementia [Autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), sporadic Alzheimer’s disease (SAD), and familial Alzheimer’s disease (FAD)] and how BACE1 inhibition affects astrocytes and endothelial cells under conditions of glutamate toxicity. Our results show increased BACE1, PHF (Paired helical filaments)-tau and GFAP (Glial Fibrillary Acid Protein) immunoreactivity (IR) in the CA1 hippocampal regions of FAD and SAD brains. Furthermore, BACE1 immunoprecipitated with GFAP in tissue samples from all study cases, but their immunofluorescence close to (10 μm3) or overlapping blood vessels was only increased in FAD and SAD brains, and PHF-tau was present around the vessels mainly in FAD brains. Interestingly, the increased BACE1 levels were associated with reactive astrocytes, characterized by morphological changes and upregulation of GFAP under pathological and stressful conditions, and endothelial disruption by glutamate excitotoxicity, and these effects were reversed by BACE1 inhibition; further, BACE1-inhibited astrocytes protected endothelial cell integrity by preserving zonula occludens-1 (ZO-1) distribution and decreasing the expression of inflammatory markers. Taken together, these findings suggest that BACE1 dysregulation in astrocytes may have a role in the alterations in NVU integrity implicated in neurodegeneration.

Toxoplasma gondii UBL-UBA shuttle proteins contribute to the degradation of ubiquitinylated proteins and are important for synchronous cell division and virulence.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes lethal diseases in immunocompromised patients. Ubiquitin-proteasome system (UPS) regulates many cellular processes by degrading ubiquitinylated proteins. The UBL-UBA shuttle protein family, which escorts the ubiquitinylated proteins to the proteasome for degradation, are crucial components of UPS. Here, we identified three UBL-UBA shuttle proteins (TGGT1_304680, DNA damage inducible protein 1, DDI1; TGGT1_295340, UV excision repair protein rad23 protein, RAD23; and TGGT1_223680, ubiquitin family protein, DSK2) localized in the cytoplasm and nucleus of T gondii. Deletion of shuttle proteins inhibited parasites growth and resulted in accumulation of ubiquitinylated proteins. Cell division of triple-gene knockout strain was asynchronous. In addition, we found that the retroviral aspartic protease activity of the nonclassical shuttle protein DDI1 was important for the virulence of Toxoplasma in mice. These results showed the critical roles of UBL-UBA shuttle proteins in regulating the degradation of ubiquitinylated proteins and cell division of T gondii.

Comparative Analysis of Putative Virulence-Associated Factors of Microsporum canis Isolates from Human and Animal Patients.

Microsporum canis is a zoophilic dermatophyte and the most common fungus isolated from dogs and cats worldwide. To invade skin, this pathogen uses different enzymes, which may be associated with virulence, that contribute to the fungal pathogenicity. The aim of this study is to compare the expression of enzymes that may be associated with virulence, and thermotolerance of M. canis strains isolated from dogs, cats, and humans. The in vitro expression of the enzymes keratinase, catalase, urease, hemolysin, and aspartic protease was evaluated in 52M. canis strains recently isolated from 14 human patients, 12 dogs, 15 symptomatic, and 11 asymptomatic cats. In addition, thermotolerance was assessed by comparative analysis of fungal growth at 25°C and 35°C. Keratinase activity was low in 34 and moderate in 18 strains. Aspartic-protease activity was low in 7, moderate in 33, and high in 12 strains. Hemolysin activity was low in 44 and moderate in 8 strains. All strains were classified as low producers of catalase. All but three strains produced urease in vitro, with a broad range of activity. The strains presented in vitro growth at the two studied temperatures were classified as presenting low (36.5%), medium (44.3%), or high (19.2%) thermotolerance. There was no statistically significant difference in the new putative virulence-associated factors studied among the different hosts, which suggests that they may have a similar role on human, cat, and dog infection. Also, no difference was observed between strains isolated from symptomatic and asymptomatic cats. This suggests that these factors have a limited impact on the fate of feline dermatophytosis caused by M. canis.

In vitro and in vivo Effect of Exogenous Farnesol Exposure Against Candida auris.

The spreading of multidrug-resistant Candida auris is considered as an emerging global health threat. The number of effective therapeutic regimens is strongly limited; therefore, development of novel strategies is needed. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment against Candida species including C. auris. To examine the effect of farnesol on C. auris, we performed experiments focusing on growth, biofilm production ability, production of enzymes related to oxidative stress, triazole susceptibility and virulence. Concentrations ranging from 100 to 300 μM farnesol caused a significant growth inhibition against C. auris planktonic cells for 24 h (p < 0.01–0.05). Farnesol treatment showed a concentration dependent inhibition in terms of biofilm forming ability of C. auris; however, it did not inhibit significantly the biofilm development at 24 h. Nevertheless, the metabolic activity of adhered farnesol pre-exposed cells (75 μM) was significantly diminished at 24 h depending on farnesol treatment during biofilm formation (p < 0.001–0.05). Moreover, 300 μM farnesol exerted a marked decrease in metabolic activity against one-day-old biofilms between 2 and 24 h (p < 0.001). Farnesol increased the production of reactive species remarkably, as revealed by 2′,7′-dichlorofluorescein (DCF) assay {3.96 ± 0.89 [nmol DCF (OD640)–1] and 23.54 ± 4.51 [nmol DCF (OD640)–1] for untreated cells and farnesol exposed cells, respectively; p < 0.001}. This was in line with increased superoxide dismutase level {85.69 ± 5.42 [munit (mg protein)–1] and 170.11 ± 17.37 [munit (mg protein)–1] for untreated cells and farnesol exposed cells, respectively; p < 0.001}, but the catalase level remained statistically comparable between treated and untreated cells (p > 0.05). Concerning virulence-related enzymes, exposure to 75 μM farnesol did not influence phospholipase or aspartic proteinase activity (p > 0.05). The interaction between fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole and farnesol showed clear synergism (FICI ranges from 0.038 to 0.375) against one-day-old biofilms. Regarding in vivo experiments, daily 75 μM farnesol treatment decreased the fungal burden in an immunocompromised murine model of disseminated candidiasis, especially in case of inocula pre-exposed to farnesol (p < 0.01). In summary, farnesol shows a promising therapeutic or adjuvant potential in traditional or alternative therapies such as catheter lock therapy.

Molecular characterization of a Trichinella spiralisaspartic protease and its facilitation role in larval invasion of host intestinal epithelial cells.

BackgroundT. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi.Methodology/Principal findingsRecombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 μM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group,ConclusionsrTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs.

Intramolecular chaperone-mediated secretion of an Rhs effector toxin by a type VI secretion system

Bacterial Rhs proteins containing toxic domains are often secreted by type VI secretion systems (T6SSs) through unclear mechanisms. Here, we show that the T6SS Rhs-family effector TseI of Aeromonas dhakensis is subject to self-cleavage at both the N- and the C-terminus, releasing the middle Rhs core and two VgrG-interacting domains (which we name VIRN and VIRC). VIRC is an endonuclease, and the immunity protein TsiI protects against VIRC toxicity through direct interaction. Proteolytic release of VIRC and VIRN is mediated, respectively, by an internal aspartic protease activity and by two conserved glutamic residues in the Rhs core. Mutations abolishing self-cleavage do not block secretion, but reduce TseI toxicity. Deletion of VIRN or the Rhs core abolishes secretion. TseI homologs from Pseudomonas syringae, P. aeruginosa, and Vibrio parahaemolyticus are also self-cleaved. VIRN and VIRC interact with protein VgrG1, while the Rhs core interacts with protein TecI. We propose that VIRN and the Rhs core act as T6SS intramolecular chaperones to facilitate toxin secretion and function.