Extracellular enzyme production in <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> isolates

Extracellular enzymes produced by Metarhizium anisopliae are believed to play a key role in cuticle hydrolysis. The in-vitro production of cuticle-degrading enzymes, such as chitinase, proteinase, caseinase, lipase and amylase in fourteen isolates of M. anisopliae exhibited significant natural isolate variability. The isolates were also evaluated for chitinase and proteinase enzyme assays in order to quantify the enzyme production. The growth characteristics and colony morphology of the isolates showed variation and few isolates formed sectors and the colonies were either fluffy or powdery. Among the isolates studied, isolate UM2 was found to show good consistence with the results on enzyme measurements as well as the growth characteristics and colony morphology. Such characterization of isolate variability could rationally be used in the selection of isolates for the production of improved myco-pesticides in the integrated pest management programs.     

Proteolytic activity of clinical Candida albicans isolates in relation to genotype and strain source

Proteolytic activity is regarded as one of the most important virulence factors of Candida albicans. Several authors recently demonstrated that some karyotypes and genotypes harbouring a group I self-splicing intron (CaLSU) located in the gene encoding the large rRNA subunit showed a high level of proteinase production. The aim of this study was to investigate the correlation between the level of proteinase production and the presence of the CaLSU intron in C. albicans isolates originating from the blood and respiratory tracts (sputum/pharyngeal swabs) of patients with and without oropharyngeal candidosis. The results revealed statistically significant differences in genotype distribution and the level of proteinase production between the C. albicans isolates obtained from blood and from the respiratory tract. Genotype A, without the intron, was prevalent in all groups of strains and its prevalence was higher among isolates from blood (75%) and from patients with candidosis (80%) compared with strains from colonisation (as opposed to infection) (57.8%). Isolates from blood produced significantly less proteinase than isolates from the respiratory tract (p<0.02), and this difference should be attributed to lower proteinase production of genotypes B and C from blood compared with genotypes B and C from the respiratory tract (p<0.01). The higher proteinase production of genotype B than of genotype A was found among respiratory tract isolates only. The presented data indicate that the association between proteinase production and the CaLSU intron depends on the strains' population. Further study is needed on well-defined groups of clinical isolates to elucidate whether the observed diversity in proteinase production plays a role in the selection of strains inducing bloodstream infections.     

Effect of the incubation conditions on the production of patulin by Penicillium griseofulvum isolated from wheat

A total of 290 Candida isolates from patients were investigated for in vitro proteinase production. Overall, sixty percent of these strains were found to be proteinase producers. Of the C. albicans strains, 81.4% of the significant isolates in contrast to 19.7% of nonsignificant isolates were proteinase producers, the difference being statistically significant (P<0.001). Amongst the different Candida species, the proteinase production was found not only in Candida albicans, but also in C. tropicalis, C. parapsilosis and C. glabrata. Thus this in vitro method of demonstration of proteinase may be a good adjunct to smear and culture examination in identifying pathogenic Candida species from anatomical sites where they can also be present as commensals.     

Influence of various nitrogen and carbon sources on the production of pectolytic,cellulolytic and proteolytic enzymes byAspergillus niger

Three isolates ofAspergillus niger produced polygalacturonase (PG) and pectin methyl galacturonase (PMG) in the presence of organic and inorganic nitrogen sources. Complete inhibition of PG PMG cellulase (C_x) and proteinase synthesis was found in the presence of cystine in all isolates. Maximum biomass was found in sodium nitrate whereas no isolate could grow in the presence of cystine. A correlation between biomass and enzyme production could be obtained when sodium nitrate and cystine were added to the medium separately. All isolates produced pectic cellulolytic and proteolytic enzymes in the presence of various native carbon sources. Sodium polypectate was found to be the best carbon source for the production of PG and PMG; pectin inhibited completely the production of PG and PMG. Maximum cellulase production was brought about by cotton in all three isolates. Maximum proteinase production was observed with gelatin which served as poor substrate for fungal growth. Sucrose supported maximum fungal growth in comparison with all other native carbon sources. The increased production of pectolytic cellulolytic and proteolytic enzymes in the presence of sodium polypectate reflected a stimulation rather than an induction of synthesis of these enzymes.     

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) rhizosphere

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099^T), Pseudomonas aureofaciens (NCIM2026^T), and Pseudomonas aeruginosa (MTCC2582^T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH₄)₂SO₄ precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn²⁺, Cu²⁺, and Ni²⁺ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.     

Molecular Characterization of a Novel Bacteriocin and an Unusually Large Aggregation Factor of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BGSJ2-8, a Natural Isolate from Homemade Cheese

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.     

In Vitro Evaluation of Phospholipase,Proteinase, and Esterase Activities of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida metapsilosis</Emphasis>

The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR–RFLP) of the secondary alcohol dehydrogenase (SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue.     

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.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Quantitative determination of gelatinase activity among enterococci

Gelatinase, hemolysin and aggregation substance have all been reported to be virulence factors of enterococci. In this study, gelatinase production was investigated in isolates of Enterococcus faecalis (n=93), E. faecium (n=49) and E. avium (n=36) recovered from hospitalized patients. Gelatinase was detected in 45% of E. faecalis isolates, but could not be detected in E. faecium and E. avium. Gelatinase activity was then measured by radial diffusion for the 42 gelatinase-positive E. faecalis isolates. To convert gelatinase activity into proteinase K activity, a standard curve was produced by placing different concentrations of proteinase K into wells in the gelatine plate. Gelatinase activity per E. faecalis colony ranged from 2.6 x 10(-7) to 2.2 x 10(-5) microg/ml, proportionate to the activity of proteinase K. An approximately 84-fold difference in gelatinase concentration was observed between the colony producing the highest amount and that producing the lowest amount. This method may be useful for determining the virulence of given isolates in relation to gelatinase production as it is quick, easy and inexpensive to perform.     

Effects of Temperature and Nutrients on Proteinase Production by Pseudomonas fluorescens and Ps. aeruginosa in Broth and Milk

Temperature and the composition of the medium influenced the production of proteinase by Pseudomonas fluorescens and Pseudomonas aeruginosa isolated from raw milk. Many isolates of Ps. fluorescens digested litmus milk at 10° but not at 5° or 2°. With Ps. fluorescens proteinase production per unit of growth in a Peptone–Yeast Extract broth declined progressively as the incubation temperature was reduced from 20° to 5°. At 30° there was heavy growth in the same medium but only slight proteinase production whereas enzyme production by Ps. aeruginosa was maximal at this temperature. Proteinase production by both species in semi-defined media was essentially a function of the organic nitrogen content of the medium; there was evidence of catabolite repression by glucose and, to a lesser extent, lactate. In milks seeded with these pseudomonads, the extent of proteolysis was either increased markedly or slightly decreased when glucose was included.     

Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral <Emphasis Type="Italic">Candida albicans</Emphasis> and Proteinases

Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.     

Lantibiotics biosynthesis genes and bacteriocinogenic activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. isolated from raw milk and cheese

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.     

Physiological dendrogram of<Emphasis Type="Italic">Claviceps</Emphasis> spp. based on sucrose metabolism in submerged cultures and its comparison with phylogenetic tree

Sixteen isolates of Claviceps spp. were analyzed for the production of polysaccharides, oligosaccharides, and sucrose metabolism under conditions of submerged fermentation. Physiological markers calculated by the Verhulst-Pearl law were used for hierarchical cluster analysis. Low correlation was found between physiologically based dendrogram and phylogenetic analysis constructed from an alignment of rDNA sequences. To confirm the intraspecific uniformity of physiological markers three isolates of C. africana from different hosts and locations were included. The influence of genotype, physiological variability, environmental location and habitat on metabolite production is discussed.     

Proteinase zymograms ofSerratia marcescens as an epidemiological tool

Six hundred and fifty-one clinicalSerratia marcescens isolates from Pellegrin Hospital (Bordeaux, France) were submitted to proteinase agar gel electrophoresis. Thirty-three different proteinase patterns (zymotypes) were found, and the distribution of zymotypes among ten clinical departments of the hospital from July, 1968, to June, 1974, was studied. Six major zymotypes, accounting for 76% of theS. marcescens isolates, were shown to have their own distribution patterns in time and space. This correlation suggests that determination of proteinase patterns might be a useful epidemiological tool.     

Oxidative stress response and virulence factors in <Emphasis Type="Italic">Candida glabrata</Emphasis> clinical isolates

We determined the susceptibility to oxidative stress and assessed the four virulence factors of the 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. All the isolates were susceptible to hydrogen peroxide, diamide, and 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) inducing an increased formation of reactive oxygen species in fungal cells. The mean relative cell surface hydrophobicity (CSH) of isolates was 21.9, ranging from 1.92 to 56.96. All isolates showed biofilm formation. A high biofilm formation was observed among 60.5% of isolates. Positive correlations were observed between biofilm formation and moderate values of CSHs. The 76.3% and 84.2% of isolates displayed varying degrees of proteinase and phospholipase activity, respectively. These results demonstrate a differential distribution of factors contributing to virulence of C. glabrata clinical isolates and point to their significance in pathogenesis that would be targeted by novel antifungals.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.     

Cysteine proteinase 30 in clinical isolates of T. vaginalis from symptomatic and asymptomatic infected women

A cysteine proteinase of 30 kDa (CP30) of Trichomonas vaginalis, is known to play a role in cytoadherence of the parasite to host cells. However, the CP30 activity in clinical isolates from symptomatic and asymptomatic patients has not been analyzed. In the present study, CP30 was detected in 20 fresh and long-term culture maintained T. vaginalis isolates each from symptomatic and asymptomatic women by substrate gel electrophoresis and immunoblotting. Though CP30 was detected in all the fresh isolates from 20 symptomatic and 20 asymptomatic women, the intensity of CP30 band was significantly higher in isolates from symptomatic as compared to asymptomatic women indicating higher expression in former. CP30 was found in all the 20 long-term cultured isolates from symptomatic whereas only in 70% of asymptomatic women indicating that CP30 expression is a more stable characteristic of symptomatic isolates. The isolates from symptomatic women, demonstrated significantly higher cytoadherence to VECs as compared to asymptomatic women. In both the types of isolates, this cytoadherence was inhibited significantly by CP30 specific hyperimmune serum. These results confirm that CP30 is an important virulence factor of T. vaginalis and has an important role in cytoadherence to VECs and thus has a role in pathogenesis of trichomoniasis.     

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.

Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a Prt- derivative of L. lactis subsp. cremoris SK112. In contrast, an approximately threefold overproduction of the cell envelope-located or fully secreted proteinase was found in strain MG1820 compared with that of its parental strain L. lactis subsp. lactis SH4109. In all strains proteinase production appeared to be regulated by the medium composition. Highest proteinase production of the SK11 derivatives was found in milk, in contrast to derivatives of SH4109 that produced most proteinase in whey permeate medium. Analysis of single strains with different levels of proteinase production or mixed cultures containing various ratios of Prt+ and Prt- cells indicated that the amount of proteinase produced per cell or culture determines the specific growth rate in milk. Overproduction of cell envelope-located or secreted proteinase in strain MG1820 resulted in a 20%-higher specific growth and acidification rate in milk compared with that in the wild-type strain SH4109. These results indicate that the growth of lactococci in milk is limited by the caseinolytic activity of the proteinase.