Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 μM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S. cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.     

Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a

SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements. To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation. Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R₁ progeny. SaPIN2a mRNA was detected in both the R₀ and R₁ transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a. Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited. Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.Abbreviations CaMV cauliflower mosaic virus - cDNA complementary DNA - NOS nopaline synthase - PAGE polyacrylamide gel electrophoresis - PI proteinase inhibitor - SaPIN2a Solanum americanum proteinase inhibitor IIa - SDS sodium dodecyl sulphate - T-DNA transferred DNA     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.     

Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus

Three variants of a chymotrypsin-like protease were purified from scallop digestive glands successively by ion-exchange, gel filtration and high-performance liquid chromatographies. Enzyme activity was detected using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a specific synthetic substrate for chymotrypsin. This proteinase was inhibited by chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. Estimated molecular mass of the purified enzyme is around 32 kDa. These isoenzymes exhibit very low activities in hydrolyzing small synthetic specific substrates used for trypsic, elastolytic and collagenolytic measurements and referred mainly to a chymotrypsin-like proteinase. Very few differences were measured concerning pH profiles among the three isoenzymes. Stability is higher at low temperature for two variants. An N-terminal analysis was performed on one variant (B) among the three isoenzymes. The alignment of the N-terminal amino acid sequence indicates some homologies with abalone chymotrypsin-like protein and arthropod chymotrypsin proteases as well as with vertebrate serine protease counterparts (trypsin, chymotrypsin and elastase).     

Phospholipase and Proteinase Activities of Clinical Isolates of Candida from Immunocompromised Patients

Sixty-one isolates of Candida recovered from HIV seropositive and cancer patients were studied for elaboration of putative virulence determinants – phospholipase (PL) and secreted aspartyl proteinase (Sap). Forty two (68.85%) isolates examined were PL producers and 51 (83.6%) were positive for Sap. 57.37% (35/61) isolates produced both enzymes. Enzymatic activity was more pronounced in Candida albicans with 100% PL and 94.1% Sap activity. In contrast, non-C. albicans species demonstrated only 29.6% PL and 70.3% Sap activity, indicating interplay of other virulence determinants in these yeasts in colonization and disease.     

Identification of LY83583 as a specific inhibitor of Candida albicans MPS1 protein kinase

Candida albicans is the most common and virulent fungus causing candidiasis in various parts of the body and can be lethal to immunocompromised patients. All currently known antifungal therapies are drugs which cause serious side effects in the host. An inhibitor specific for fungus survival is an ideal therapeutic. C. albicans MPS1 (monopolar spindle 1) has been reported as a kinase essential to its survival. Because CaMps1p shares limited sequence homology with the human ortholog (hMps1p), we screened for a chemical inhibitor in anticipation of finding one with Candida specific cytotoxicity. In vitro screening using a recombinant catalytic domain of CaMps1p identified LY83583 (6-anilino-5,8-quinolinedione), known as a guanylate cyclase inhibitor, to be blocking CaMps1p kinase activity. In addition to its in vitro kinase inhibition, LY83583 reduced the growth rate of C. albicans. Finally, we compared the inhibitory activity on CaMps1p and hMps1p among inhibitors against those kinases. LY83583 showed specific inhibition for CaMps1p with no effect on hMps1p activity. Conversely, the CaMps1p activity was not affected by known hMps1p inhibitors. These findings suggest that CaMps1p may well be an ideal target molecule for antifungal therapy.     

The influence of Zn(II) and Mn(II) on catalytic activity of C. albicans aspartic proteinases

The interaction of secreted aspartic proteinases from C. albicans (C. albicans SAP) with ZnCl2 and MnCl2 has been studied. Logarithms of stability constant from the data of electronic spectroscopy were calculated for the complexes of SAP with Zn (II) (SAP-ZnII, logβ = 4.73 ± 0.20) and with Mn(II) (SAP-MnII, logβ = 7.02 ± 0.20). The composition and maximum accumulation of complexes in solution were calculated. The optimal conditions of hydrolysis of the substrate, HAS (human serum albumin) in the presence of C. albicans SAP-MnII and SAP-ZnII proteinases were determined. These were: [HSA] = 1 mg/ml, [SAP] = 2.33 μM, pH = 4.5, incubation time of 25 min. The activity of C. albicans SAP in the presence of different concentrations of ZnCl₂ and MnCl₂ was evaluated under optimal conditions of enzymatic hydrolysis. For the first time the activating action of 0.5 μM ZnCl₂ on catalytic activity of C. albicans SAP proteinase has been demonstrated. The maximal rate of enzymatic reaction (V _max), the Michaelis constant (K _m ) and constants of effects in the presence and in the absence of the effector, ZnCl₂, were calculated. The albuminase activity of C. albicans pathogenic strains of different localization was evaluated in the presence and the absence of the effector of ZnCl₂.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Induction of secretory aspartyl proteinase of <Emphasis Type="Italic">Candida albicans </Emphasis>by HIV-1 but not HSV-2 or some other microorganisms associated with vaginal environment

The most common type of candidiasis involves mucosal sites such as the oral cavity, the gastrointestinal tract and the vagina. Among many of virulence factors, the production of secretory aspartyl proteinase (Sap) by Candida albicans (C. albicans) has gained much attention, and factors leading to Sap induction are thus under intense study. The aim of this study was to examine whether some microorganisms such as Lactobacillus, Gardnerella vaginalis (G. vaginalis), human immunodeficiency virus type-1 (HIV-1) and human herpes simplex virus type-2 (HSV-2) had any Sap inducing effect on C. albicans. Here we showed that among the microorganisms tested in vitro only HIV-1 induced Sap production from C. albicans.     

Characterization of a collagenolytic serine proteinase from the Atlantic cod (gadus morhua)

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH₄)₂SO₄ fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (±0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-l-Ala-l-Ala-l-Pro-l-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50°C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30°C.     

A double WAP domain (DWD)-containing protein with proteinase inhibitory activity in Chinese white shrimp, Fenneropenaeus chinensis

The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78 kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillus subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens.     

Does host range influence susceptibility of herbivorous insects to non-host plant proteinase inhibitors?

We examined the influence of proteinase inhibitors on digestive enzymes and development of oriental beetle,Exomala orientalis Waterhouse, European chafer,Rhizotrogus majalis (Razoumowsky),Phyllophaga white grub,Phyllophaga anxia (LeConte), cranberry root grub,Lichnanthe vulpina (Hentz), Japanese beetle,Popillia japonica Newman, Asiatic garden beetle, Maladera castanea (Arrow) (Coleoptera: Scarabaeidae), and the black cutworm,Agrotis ipsilon (Rottemburg) (Lepidoptera: Noctuidae). We demonstrated that all species within our test group had alkaline midguts that contained proteinase activity that could be inhibited,in vitro with serine proteinase inhibitors. Our data suggests that host range may influence the susceptibility to non-host inhibitors. Chronic ingestion of the serine proteinase inhibitor, Kunitz-soybean trypsin inhibitor (STI), significantly reduced proteolytic activityin vivo in those species with relatively specialized feeding habits (i.e., cranberry root grub, Japanese beetle, Asiatic garden beetle, and black cutworm). Chronic ingestion of STI also resulted in reduced larval growth and delayed pupation for black cutworm, and elevated larval mortality for Japanese beetle. However, chronic ingestion of STI did not influence larval survival for those species with relatively generalized feeding habits (i.e., oriental beetle, European chafer). Based on these results, we propose mechanistically-based criteria for selecting proteinase inhibitors for phytochemical defense against herbivorous insects.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.     

cAMP regulates vegetative growth and cell cycle in <Emphasis Type="Italic">Candida albicans</Emphasis>

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.