A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens.

INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

The effect of released new synthetic peptide from nanofibrous scaffold of peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of Candida albicans.

Secreted aspartyl proteinases (Saps), are gene products that have been shown to directly contribute to Candida albicans pathogenicity. Despite the clear difficulties of systemic C. albicans infections control, Antimicrobial peptides (AMPs) are regarded as one of the most promising alternatives in this regard. Recently, drug-loaded electrospun nanofibers have attracted a great deal of attention. In this study we have established the nanofibrous scaffold of new synthetic peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of C. albicans in comparison with free peptide. We designed new synthetic Antimicrobial peptide (AMPs) used bioinformatics tools to predict structure and stabilities. The electrospinning method was used to produce the polymeric nanofibers. Scanning Electron Microscopy (SEM) was used to measure the nanofibers diameters and morphology. To analyze the expression of SAP4, SAP5 and SAP6 genes, RNA was extracted from clinical isolates of C. albicans before and after treatment with subinhibitory concentrations of new synthetic peptide on nanofibrous scaffold of new synthetic peptide/PVA/PLLA and then the cDNA was synthesized and used for Real-time PCR assay. Scanning electron microscopy (SEM) images showed that the morphology, the diameter, are affected from peptide. Reletive quantitative Real-time PCR results revealed that the mRNA levels of SAP4, SAP5 and SAP6 genes significantly decreased after treatment with nanofibrous scaffold of new synthetic peptide/PVA/PLLA (P < .05). Electrospun nanofibrous of new Synthetic Peptide/PVA/PLLA is effective in downregulating of expression SAP4, SAP5 and SAP6 genes of C. albicans strains.

Extracellular Enzyme Composition and Functional Characteristics of Aspergillus niger An-76 Induced by Food Processing Byproducts and Based on Integrated Functional Omics.

Byproducts of food processing can be utilized for the production of high-value-added enzyme cocktails. In this study, we utilized integrated functional omics technology to analyze composition and functional characteristics of extracellular enzymes produced by Aspergillus niger grown on food processing byproducts. The results showed that oligosaccharides constituted by arabinose, xylose, and glucose in wheat bran were able to efficiently induce the production of extracellular enzymes of A. niger. Compared with other substrates, wheat bran was more effective at inducing the secretion of ß-glucosidases from GH1 and GH3 families, as well as >50% of proteases from A1-family aspartic proteases. Compared with proteins induced by single wheat bran or soybean dregs, the protein yield induced by their mixture was doubled, and the time required to reach peak enzyme activity was shortened by 25%. This study provided a technical platform for the complex formulation of various substrates and functional analysis of extracellular enzymes.

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

BACKGROUND: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AIMS: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. METHODS: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. RESULTS: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. CONCLUSIONS: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.

Candida tropicalis Biofilms: Biomass, Metabolic Activity and Secreted Aspartyl Proteinase Production.

According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24-120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation.

Discovery of a "white-gray-opaque" tristable phenotypic switching system in candida albicans: roles of non-genetic diversity in host adaptation.

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the "gray" phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus.

Impact of glucose levels on expression of hypha-associated secreted aspartyl proteinases in Candida albicans.

BACKGROUND: Ten secreted aspartyl proteinase (Sap) genes were identified in Candida albicans. The products of SAP genes are considered to be virulent factors of C. albicans that participated in causing mucocutaneous and systemic candidiasis in humans. Depending on environmental conditions, C. albicans may stay in yeast-form or convert into invasive hypha-form, and these issues may affect the expression of SAP genes. In this study we explored the component(s) of culture media that may affect the expression of hypha-associated SAP genes. RESULTS: We demonstrate that glucose levels modulate both the hyphae development and the expression strength of hypha-associated SAP genes (SAP4-6). In contrast to high glucose concentration (2%), lower glucose level (0.1%) is more potent to promote hyphae development and to promptly elicit the expression of hypha-associated Sap proteins during yeast-to-hypha transition of C. albicans. Both Cph1-mediated MAP kinase cascade and Efg1-mediated cAMP/PKA pathway, although the latter seemed dominant, participate in convey the glucose signaling to regulate the expression of hypha-associated SAP genes and this glucose level effect may perform at very early stage of yeast-to-hypha transition. In addition, when C. albicans was co-cultured with THP-1 human monocytes, the engulfed C. albicans was developing hypha efficiently within 1 hr and the expression of hypha-associated Sap proteins could be detected on the distal surface of hyphae. CONCLUSION: We propose that the glucose level of bloodstream (approximately 0.1%) may be facilitated for stimulation of C. albicans to develop invasive hypha-form and to elicit promptly production of high-level hypha-associated Sap proteins.

Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.

Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-ß-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica.

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.

Spectrophotometric analysis of the expression of secreted aspartyl proteinases from Candida in leukoplakia and oral squamous cell carcinoma.

Candida species are a normal commensal of the oral cavity in healthy individuals, but can become an opportunistic pathogen when the oral ecosystem is unbalanced. Several virulence attributes have been identified in candidal infection, among which are the hydrolases, including the secreted aspartyl proteinases (Saps). This study evaluated and compared the in vitro level of Saps from Candida albicans in nonsmokers, smokers, and patients with leukoplakia and oral squamous cell carcinoma (OSCC). Candida cell count (CCC) at 48 h was also assessed. The Sap level was measured by spectrophotometry in 38 clinical isolates of C. albicans obtained from the oral cavity of the four different groups. Culturing was done in yeast carbon base-bovine serum albumin. Speciation of Candida was performed by using a Candida identification kit, and CCC was measured by hemocytometer. Sap levels and CCC were higher in individuals with leukoplakia and OSCC than in nonsmokers or smokers (P = 0.001); however, there was no significant difference in Sap levels or CCC between smokers and nonsmokers (P = 0.529). Further, an intragroup correlation between CCC and Sap level was also observed. The higher level of Saps from C. albicans in individuals with leukoplakia and OSCC suggests that this pathogen plays a role in disease development and could aid in identifying the pathogenic commensal.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity.

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1% and 55.9% of Candida albicans and 69.8% and 37.7% of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity / Diferenças na produção de exoenzimas e habilidade de aderência entre isolados de espécies de Candida provenientes do cateter, sangue e cavidade bucal

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1 percent and 55.9 percent of Candida albicans and 69.8 percent and 37.7 percent of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

A produção de proteinase e fosfolipase e habilidade de adesão à célula epitelial bucal de 112 isolados de Candida originadas da cavidade bucal de pacientes infectados pelo HIV e de sangue e cateter de pacientes hospitalizados foram investigados. A produção de proteinase foi detectada por inoculação em ágar soro albumina bovina e a atividade de fosfolipase foi realizada usando emulsão de gema de ovo. A suspensão de levedura de cada isolado foi incubada com célula epitelial e o número de leveduras aderidas a célula epitelial foi contada. Uma porcentagem de 88,1 e 55,9 por cento de C. albicans e 69,8 e 37,7 por cento de isolados de Candida não albicans produziram proteinase e fosfolipase, respectivamente. Candida não albicans obtidas do cateter foram mais proteolíticos que isolados de Candida albicans (p < 0,001). Isolados do sangue foram mais proteolíticos do que isolados do cateter e cavidade bucal, enquanto isolados da cavidade bucal produziram mais fosfolipase do que aqueles isolados do sangue e cateter. C. albicans isoladas da cavidade bucal e do cateter foram mais aderentes à célula epitelial bucal do que isolados de Candida não albicans, mas não houve diferença na adesão entre os três locais analisados. Os resultados indicaram diferenças na produção de fosfolipase e proteinase e na habilidade de adesão à célula epitelial bucal entre os isolados de Candida das diferentes fontes. Este estudo sugere que a patogenicidade de Candida spp pode estar correlacionada ao local infectado.

Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance.

The Oryza sativa constitutive disease resistance 1 (OsCDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling. This apoplastic enzyme is a member of the group of 'atypical' plant aspartic proteinases. Recombinant OsCDR1 expressed in Escherichia coli exhibited protease activity against succinylated-casein substrate. Inactivating the enzyme through modification of an aspartate residue present in the deduced active site completely abolished its proteinase activity. Infiltration of the OsCDR1 fusion protein into leaves of Arabidopsis plants induced PR2 transcripts in both the infiltrated leaf (primary) and in non-treated secondary leaves while the inactive recombinant protein failed to induce either local or systemic PR2. These findings demonstrate that OsCDR1 is capable of inducing systemic defense responses in plants.

Alterations in the expression level of a putative aspartic protease in the development of Angiostrongylus cantonensis.

Aspartic proteases are a family of proteinases with catalytic aspartate residues in the active site. These enzymes have been reported to initialize the degradation of host hemoglobin in blood-feeding helminths. After identifying an expressed sequence tag representing an aspartic protease from an Angiostrongylus cantonensis young adult dataset, this sequence was found to encode a protein with a predicted molecular mass of 46 kDa. It also showed good homologies to aspartic proteases from Caenorhabditis elegans (50.7% identity), Haemonchus contortus (43.0% identity), Necator americanus (41.5% identity), Strongyloides stercoralis (35.9% identity), and Burgia malayi (29.6% identity). This putative aspartic protease was determined to be expressed in the infective larvae, young adults, and adult worms of A. cantonensis by quantitative real-time PCR. Among male worms, the expression level was determined to increase by 223.0 + or - 24.2 fold in young adults relative to the infective larvae and then decreased to 7.1 + or - 0.2 fold in adult worms. In female worms, the expression level was observed to increase by 118.5 + or - 10.1 fold in young adults and by 277.5 + or - 29.2 fold in the adults, when compared with infective larvae. These findings not only indicate that the expression level of aspartic protease gene in A. cantonensis changes with development but also has a sexual difference in individual developmental stages in the final host.