Phospholipase Production Research Articles

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

Comparative analysis of extracellular enzymes and virulence exhibited by Burkholderia pseudomallei from different sources

To evaluate the potential role of extracellular proteins in the pathogenicity and virulence of Burkholderia pseudomallei, the activities of several enzymes in the culture filtrates of nine clinical and six environmental isolates were investigated in vitro and in vivo in ICR strain of mice. The production of protease, phosphatase, phospholipase C, superoxide dismutase, catalase and peroxidase were detected in the culture filtrates of all the 15 isolates at different time points of growth 4–24 h. Over time, activity of each enzyme at each time point varied. Profile of secretion was similar among the 15 isolates irrespective of source, that is clinical or environmental. Catalase, phosphatase and phospholipase C were found to be increased in 60–100% of the isolates post-passage in mice. In vivo inoculation studies in ICR mice demonstrated a wide difference in their ability to cause bacteraemia, splenic or external abscesses and mortality rate ranged from few days to several weeks.

Phenotypical characterization of Candida spp. isolated from crop of parrots (Amazona spp.)

The purpose of this study was to characterize Candida isolates from crop of parrots. Forty baby parrots of genus Amazona, species aestiva and amazonica that were apprehended from wild animal traffic were used: 18 presented ingluvitis and 22 other alterations, but showing general debilitation. Samples were seeded on Sabouraud dextrose agar with chloramphenicol after be obtained by the introduction of urethral probe through the esophagus. Based on morphology and biochemical reactions (API 20C) Candida was confirmed; it was still searched the production of proteinase and phospholipase, virulence factors for Candida species. Candida spp. were isolated from 57.5% parrots, being 72.2% from birds with ingluvitis and 45.5% from without ones. Twenty-five strains of Candida were isolated, 60% and 40%, respectively from parrots with and without ingluvitis, and were speciated: 28% C. humicola, 24% C. parapsilosis, 20% C. guilliermondii, 20% C. famata, and 8% C. albicans. These results demonstrate that C. albicans is not the most frequent species isolated, and it is the first report that shows C. guilliermondii, C. famata, and C. humicola causing infection in parrots. Many isolates presented filamentation (76%), 100% produced proteinase and 68% phospholipase. The observation of Candida spp. producing virulence factors reinforce the pathogenic role of these yeasts in the cases studied.

Mutation ofrpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence ofVibrio anguillarum

Vibrio anguillarum is a gram-negative halophilic bacterium that causes vibriosis in marine fish, freshwater fish and other aquatic animals. Bacteria have developed strategies to survive in harsh environments. The alternative sigma factor, RpoS (sigma(S)), plays a key role in surviving under stress conditions in some gram-negative bacteria. An rpoS mutant of pathogenic V. anguillarum W-1 was constructed by homologous recombination. The sensitivity of the rpoS mutant to osmotic stress [2.4 M NaCl in artificial seawater (ASW)] did not change obviously, but the sensitivity of the rpoS mutant to high temperature (45 degrees C in ASW), UV-irradiation and oxidative stress (5 mM H(2)O(2) in ASW) increased 33-fold, sixfold and 10-fold, respectively. The production of extracellular phospholipase, diastase, lipase, caseinase, hemolysin, catalase and protease of the rpoS mutant decreased markedly compared with those of the wild-type strain. Virulence of the rpoS mutant strain was also decreased when it was inoculated intraperitoneally into zebra fish; the lethal dose 50% of the wild type and the mutant was 8.66 x 10(4) and 2.55 x 10(6) CFU per fish, respectively. These results indicated that the RpoS of V. anguillarum plays important roles in bacterial adaptation to environmental stresses and its pathogenicity.

The effect of Dodonaea viscosa var. angustifolia on Candida albicans proteinase and phospholipase production and adherence to oral epithelial cells

Aim of the study This study investigated effect of a crude extract of Dodonaea viscosa on the proteinase and phospholipase production and adherence to epithelial cells by Candida albicans isolated from HIV positive and HIV negative patients. Materials and methods Twenty Candida albicans strains isolated from HIV positive and 20 from HIV negative patients were investigated. The isolates were exposed to subinhibitory concentration of crude plant extract and adherence, proteinase and phospholipase production were assessed. The results were analysed using Student's t-test and a two-way ANOVA. Results Dodonaea viscosa var. angustifolia inhibited the adherence of Candida albicans to oral epithelial cells ( p = <0.01) but no significant effect of the plant extract on proteinase and phospholipase production was observed. Results from Candida albicans strains isolated from HIV positive and HIV negative patients were similar. Conclusions Dodonaea viscosa var. angustifolia inhibited the adherence of Candida albicans to oral epithelial cells, which is the initial step of colonization in the infection process. This plant has a therapeutic potential at subinhibitory concentration.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37 degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Expression of the μ-opioid receptor onMalassezia pachydermatisand its effect in modulating phospholipase production

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

Expression of the µ-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production

Expression of the µ-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production

Environmental assessment of yield improvements obtained by the use of the enzyme phospholipase in mozzarella cheese production

Background, aim and scope Phospholipase is an enzyme which is able to increase the yield of cheese in, for instance, mozzarella production. Milk production is the most important source of environmental impacts in cheese production and it is obvious to assume that the milk saving that comes with the use of phospholipase reduces the overall environmental impacts of the final product. Production of industrial phospholipase is, however, also associated with environmental burdens and it is not known whether and to what extent the use of phospholipase is justified by overall environmental improvements. The aim of the present study is therefore to assess the environmental impacts that come with the use of industrial phospholipase in mozzarella production and compare with the savings that come with the avoided milk production. The study addresses mozzarella production in Denmark.

Actividad proteinasa y fosfolipasa como factores de virulencia en especies de Candida aisladas de sangre

The number of nosocomial blood stream infections due to Candida species has increased over the past few decades. In order to establish an infection, opportunistic pathogens have to evade the immune system, survive, divide in the host environment, and spread to other tissues. Proteinase and phospholipase secretion has been implicated as potential virulence factors for some Candida species responsible for catheter related candidemia in intensive care unit (ICU) patients with indwelling devices. We therefore have aimed at demonstrating the secretion of proteinase and phospholipase enzymes as virulent factors by Candida species isolated from blood samples collected from ICUs, dialysis units and oncology units. One hundred and fourteen isolates of Candida species were obtained from the blood samples and the isolates include 37 Candida albicans, 7 Candida glabrata, 5 Candida guilliermondii, 3 Candida kefyr, 45 Candida krusei, 5 Candida parapsilosis, and 12 Candida tropicalis. Proteinase assay was performed by using the Staib et al method. Phospholipase assay was performed by using the method of Samaranayake et al. Precipitation zone (Pz value) was determined. The percentage of isolates which produced detectable amounts of proteinase is 74.56% and 44.73% of isolates produced detectable amounts of phospholipase. We believe that production of both phospholipase and proteinase enzimes could be an important virulence factor for several Candida species.

Correlation between enzyme production, germ tube formation and susceptibility to fluconazole in Candida species isolated from patients with denture‐related stomatitis and control individuals

Phospholipase and proteinase secretion in yeasts of the genus Candida has been described as a relevant virulence factor. Also, germ tube formation by Candida albicans is associated with its invasive capacity and is considered an important pathogenic mechanism. To link the production of hydrolytic enzymes with the capacity to produce infection, 232 clinical isolates of yeasts from the oral cavity of 140 individuals wearing removable maxillary protheses were studied. The sample was composed of 70 patients with denture-related stomatitis (DRS) and 70 individuals with normal palatal mucosa. For strains identified as C. albicans, the correlation between germ tube formation and their capacity to cause infection was studied and the presence of Candida dubliniensis was investigated. Susceptibility to fluconazole was evaluated. Candida albicans was the only species producing phospholipase and germ tube. We observed a higher level of production of phospholipase in cases of infection compared with commensals. Significant differences between the two groups of C. albicans isolates were observed as to germ tube production. Only, Candida glabrata showed lower susceptibility to fluconazole. The results reinforced the idea that C. albicans is the most frequent and can be the most pathogenic yeast in oral candidosis. However, the strains isolated from DRS patients and healthy individuals showed the same virulence factors. It seems that several virulence attributes are involved in the infective process but no single factor contributes to Candida virulence. Candida dubliniensis was absent in the oral cavity of individuals with and without DRS.

Secretion of five extracellular enzymes by strains of chromoblastomycosis agents

The gelatinase, urease, lipase, phospholipase and DNase activities of 11 chromoblastomycosis agents constituted by strains of Fonsecaea pedrosoi, F. compacta, Phialophora verrucosa, Cladosporium carrionii, Cladophialophora bantiana and Exophiala jeanselmei were analyzed and compared. All strains presented urease, gelatinase and lipase activity. Phospholipase activity was detected only on five of six strains of F. pedrosoi. DNase activity was not detected on the strains studied. Our results indicate that only phospholipase production, induced by egg yolk substrate, was useful for the differentiation of the taxonomically related species studied, based on their enzymatic profile.

Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species

We investigated the production of extracellular elastase, acid proteinase and phospholipase enzyme activities in clinical isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger and any possible correlation between the existence of these enzymes and development of invasive aspergillosis. A total of 73 strains (45 A. fumigatus, 23 A. flavus, 5 A. niger) isolated from patients with invasive aspergillosis (n = 55), superficial aspergillosis (n = 5), allergic bronchopulmonary aspergillosis (n = 1), and from those colonized with Aspergillus (n = 12) were included in the study. The enzymatic activities were tested on solid media supplemented with the corresponding substrates. Elastase activity was detected in 95.6, 82.6, and 0% of A. fumigatus, A. flavus, and A. niger isolates, respectively. Acid proteinase activity was detected only for A. fumigatus and in 20 of 45 isolates belonging to this species. Phospholipase activity was present in all strains of A. fumigatus and A. niger but in none of the isolates of A. flavus. No statistical correlation could be established between the existence of elastase or acid proteinase activity and development of invasive disease (p > 0.05). While high phospholipase production was found to be associated with development of invasive aspergillosis (p < 0.01), not all isolates that caused invasive diseases showed high phospholipase activities.

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of Candida spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Cytosolic phospholipase A2 regulates viability of irradiated vascular endothelium

Radiosensitivity of various normal tissues is largely dependent on radiation-triggered signal transduction pathways. Radiation simultaneously initiates distinct signaling from both DNA damage and cell membrane. Specifically, DNA strand breaks initiate cell-cycle delay, strand-break repair or programmed cell death, whereas membrane-derived signaling through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) enhances cell viability. Here, activation of cytosolic phospholipase A(2) (cPLA(2)) and production of the lipid second-messenger lysophosphatidylcholine were identified as initial events (within 2 min) required for radiation-induced activation of Akt and ERK1/2 in vascular endothelial cells. Inhibition of cPLA(2) significantly enhanced radiation-induced cytotoxicity due to an increased number of multinucleated giant cells and cell cycle-independent accumulation of cyclin B1 within 24-48 h of irradiation. Delayed programmed cell death was detected at 72-96 h after treatment. Endothelial functions were also affected by inhibition of cPLA(2) during irradiation resulting in attenuated cell migration and tubule formation. The role of cPLA(2) in the regulation of radiation-induced activation of Akt and ERK1/2 and cell viability was confirmed using human umbilical vein endothelial cells transfected with shRNA for cPLA(2)alpha and cultured embryonic fibroblasts from cPLA(2)alpha(-/-) mice. In summary, an immediate radiation-induced cPLA(2)-dependent signaling was identified that regulates cell viability and, therefore, represents one of the key regulators of radioresistance of vascular endothelial cells.

Assessing the relationship between Malassezia and leishmaniasis in dogs with or without skin lesions

The relationship among the frequency, population size and phospholipase activity of Malassezia pachydermatis was investigated for dogs with Leishmania infantum infection (Li +) and those without evidence of this infection (Li −). A group of 188 dogs (141 without and 47 with skin lesions) was examined clinically, and samples were taken for the detection of Malassezia and L. infantum using various diagnostic methods. Malassezia was cultured from skin samples from 101 (53.7%) dogs and classified biochemically and molecularly as M. pachydermatis. A significantly higher mean population size of M. pachydermatis was cultured from the skin of L + dogs compared with L − dogs. For M. pachydermatis, most phospolipase-producing cultures and the highest phospholipase activity were recorded for L − dogs with lesions and L + dogs without lesions. The results showed that M. pachydermatis was a common commensal on dogs with or without L. infantum infection and established that L. infantum infection in dogs without skin lesions was associated with increased growth of M. pachydermatis and production of phospholipase in vitro.

Phospholipase activity of yeasts from wild birds and possible implications for human disease

Over the last decades, reports on yeast infections in humans have increased especially with respect to immunocompromised individuals. Phospholipases are enzymes which may be associated with pathogenic processes caused by opportunistic yeasts. Phospholipase activity (ph.a.) was investigated in 163 isolates of 13 species of yeasts. A total of 133 isolates were obtained through the screening of a total of 768 cloacae of wild birds (Group I: 182 birds of prey; Group II: 165 passeriformes and Group III: 421 other wild migratory birds), while 30 isolates were recovered from the droppings of birds housed in 32 distinct aviaries (Group IV). Phospholipase production was evaluated and quantified at 2 and 5 day pre-incubation (Pr.t) and incubation times (I.t). Isolates from cloacae (48.1%) and excreta (73.3%) produced ph.a. with the highest values registered after 5 days of I.t. Candida albicans, C. tropicalis, C. glabrata, C. lusitaniae, C. pelliculosa, Cryptococcus albidus, C. laurentii, Trichosporon beigelii, and Saccharomyces cerevisiae displayed the highest ph.a. after 2 days of Pr.t while Candida famata, C. guilliermondii and Cryptococcus neoformans after 5 days of Pr.t. Ph.a. was never found in Rhodotorula rubra isolates recovered from the cloacae of wild birds. Isolates (73.3%) from bird droppings showed a higher ph.a. than those from cloacae thus indicating that wild birds not only act as carriers but may also spread phospholipase-producing yeasts in the environment.

Enhanced Production of Phospholipase C and Perfringolysin O (Alpha and Theta Toxins) in a Gatifloxacin-Resistant Strain of Clostridium perfringens

Clostridium perfringens-induced gas gangrene is mediated by potent extracellular toxins, especially alpha toxin (a phospholipase C [PLC]) and theta toxin (perfringolysin O [PFO], a thiol-activated cytolysin); and antibiotic-induced suppression of toxin synthesis is an important clinical goal. The production of PLC and PFO by a gatifloxacin-induced, fluoroquinolone-resistant mutant strain of C. perfringens, strain 10G, carrying a stable mutation in DNA gyrase was compared with that of the wild-type (WT) parent strain. Zymography (with sheep red blood cell and egg yolk overlays) and time course analysis [with hydrolysis of egg yolk lecithin and O-(4 nitrophenyl-phosphoryl)choline] demonstrated that strain 10G produced more PLC and PFO than the WT strain. Increased toxin production in strain 10G was not related either to differences in growth characteristics between the wild-type and the mutant strain or to nonsynonymous polymorphisms in PLC, PFO, or their known regulatory proteins. Increased PLC and PFO production by strain 10G was associated with increased cytotoxic activity for HT-29 human adenocarcinoma cells and with increased platelet-neutrophil aggregate formation. Four other gatifloxacin-induced gyrase mutants did not show increased toxin production, suggesting that gatifloxacin resistance was not always associated with increased toxin production in all strains of C. perfringens. This is the first report of increased toxin production in a fluoroquinolone-resistant strain of C. perfringens.

Cytosolic Phospholipase A2α Regulates Cell Growth in RET/PTC-Transformed Thyroid Cells

Modulation of cytosolic phospholipase A(2) (PLA(2)) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA(2) group IV isoform alpha (PLA(2)IV alpha) controls TSH-independent growth of normal thyroid (PCCl(3)) cells, we have investigated the mitogenic role of PLA(2)IV alpha in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA(2)IV alpha acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA(2)IV alpha is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA(2)IV alpha to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA(2)IV alpha phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA(2)IV alpha and should be amenable to targeted pharmacologic intervention.

Tipranavir exhibits different effects on opportunistic pathogenic fungi

Tipranavir (TPV) is a non-peptidic protease inhibitor (PI) that represents one of the latest options approved in the salvage setting for HIV-infected multi-drug resistant patients. In this study, we explored whether TPV affects virulence of opportunistic fungi such as Cryptococcus neoformans and Candida albicans. C. neoformans and C. albicans were cocultured in the presence or absence of TPV for various time periods. Subsequently, growth inhibition, phospholipases, proteases and capsule size were examined. In selected in vivo experiments, TPV was administered in immunocompetent and immunosuppressed mice. Survival rate and colony forming units from organs were evaluated in mice systemically challenged with C. neoformans or C. albicans. Indeed, when cultured in the presence of TPV, both fungi showed significant reduction in protease and phospholipase production, but TPV showed an opposite effect on the major virulence factors of C. neoformans and C. albicans by inhibiting capsule while promoting mycelial transition, respectively. TPV impaired in vitro growth of C. neoformans, but not of C. albicans. Moreover, TPV-treated C. neoformans, but not C. albicans, resulted more susceptible to killing by human neutrophils. Finally, TPV showed a therapeutic effect in experimental systemic cryptococcosis, as evaluated by reduced fungal burden in brain and liver of immunocompetent and immunodepressed mice. These new data indicate that TPV could act in multiple ways by diversifying its effects on various opportunistic pathogenic fungi.