PLB1 Gene Research Articles

Diagnosis of Oral candidiasis in Patients under 12 Years: 18S rRNA as a Marker of Molecular Characterization of Candida tropicalis.

Candida tropical has been found as the most abundant pathogenic yeast species under the group Candida-non-albicans. Despite this, it is taxonomically related to C. albicans and has many of its pathogenic characteristics. Infection with Candida tropicalis is closely associated with many virulence factors encoded by multiple virulence genes. This study aims to diagnose C. tropicalis based on the presence of 18SrRNA and to detect many virulence genes. C. tropicalis isolates were collected from oral candidiasis patients. Children infected with oral thrush ranging in age from infants to 12 years old provided 150 samples. C. albicans (66.68 %), C. tropicalis (13.21 %), C. krusie (9.43 %), C. parapsilosis (7.55 %), and C. glarata were isolated as C. tropicalis types, according to the findings of the present study (2.83%). The presence of the 18SrRNA gene was confirmed in the isolates. All isolates were positive for cph1 and hwp1, while some were positive for sap1 (78.5%) and plb1 genes (71.4%). Using sequences and phylogenetic trees, it was determined that there was negligible genetic variation between local isolates and global strains. These virulence factor genes play a crucial role in developing infections.

Determination of Virulence Factors in Candida albicans isolated from Cattle with Mastitis

In recent years, the importance of yeast in the etiology of bovine mastitis is increasing. The aim of this study was to determine the prevalence of Candida albicans in milk samples collected from cows with mastitis, to identify the phenotypic and genotypic methods and to determine the virulence factors. According to the results of the surveys, 686 milk samples were collected from 178 animals with mastitis problems from 20 dairy cattle farms. Forty nine yeast isolates were obtained from these samples. Five isolates were identified as C. albicans with phenotypic tests (germ tube test, chlamydospor formation, chromogenic medium and reproduction at 45°C). Five isolates that were found to be phenotypically C. albicans were confirmed by PCR using specific primers for the C. albicans CALB1 gene. It was determined that 2 (%40) of the C. albicans isolates had ALS1 and PLB1, 1 (%20) had ALS1 and 1 (%20) had PLB1 genes and no gene were not found in 1 (%20) isolate. When the biofilm formation properties of C. albicans isolates were examined by tube adherence method, it was determined that 3 (%60) isolates were strong, 1 (%20) isolate was weak positive and 1 (%20) isolate was negative. In conclusion, in the C. albicans isolates which were isolated from cows with mastitis had ALS1 and PLB1 genes and biofilm formation.

In situ analysis of variations of arsenicals, microbiome and transcriptome profiles along murine intestinal tract

In situ-based studies on microbiome-host interactions after arsenic exposure are few. In this study, the variations in arsenics, microbiota, and host genes along murine intestinal tracts were determined after arsenic exposure for two months. There was a gradual increase in the concentration of total As (CtAs) in feces from ileum to colon, whereas CtAs in the corresponding tissues were relatively stable. Differences in arsenic levels between feces and tissues were significantly different. The proportion of arsenite (iAsⅢ) in feces gradually decreased, however, it gradually increased in tissues. After arsenic exposure, the diversity and abundance of microbial community and networks in each segment were significantly dysregulated. Notably, 328, 579 and 90 differently expressed genes were detected in ileum, cecum, and colon, respectively. In addition, microbiome and transcriptome analyses showed a significant correlation between the abundance of Faecalibaculum and expressions of Plb1, Hspa1b, Areg and Duoxa2 genes. This implies that they may be involved in arsenic biotransformation. In vitro experiments using Biofidobactrium and Lactobacillus showed that probiotics have arsenic transformation abilities. Therefore, gut microbiome may modulate arsenic accumulation, excretion and detoxification along the digestive tract. Moreover, the abundance and diversity of gut microbiome may be related to the changes in host health.

Two distinct mechanisms underlie estrogen-receptor-negative breast cancer susceptibility at the 2p23.2 locus.

Genome wide-association studies (GWAS) have established over 400 breast cancer risk loci defined by common single nucleotide polymorphisms (SNPs), including several associated with estrogen-receptor (ER)-negative disease. Most of these loci have not been studied systematically and the mechanistic underpinnings of risk are largely unknown. Here we explored the landscape of genomic features at an ER-negative breast cancer susceptibility locus at chromosome 2p23.2 and assessed the functionality of 81 SNPs with strong evidence of association from previous fine mapping. Five candidateregulatory regions containing risk-associated SNPs were identified. Regulatory Region 1 in the first intron of WDR43 contains SNP rs4407214, which showed allele-specific interaction with the transcription factor USF1 in in vitro assays. CRISPR-mediated disruption of Regulatory Region 1 led to expression changes in the neighboring PLB1 gene, suggesting that the region acts as a distal enhancer. Regulatory Regions 2, 4, and 5 did not provide sufficient evidence for functionality in in silico and experimental analyses. Two SNPs (rs11680458 and rs1131880) in Regulatory Region 3, mapping to the seed region for miRNA-recognition sites in the 3' untranslated region of WDR43, showed allele-specific effects of ectopic expression of miR-376 on WDR43 expression levels. Taken together, our data suggest that risk of ER-negative breast cancer associated with the 2p23.2 locus is likely driven by a combinatorial effect on the regulation of WDR43 and PLB1.

Genotyping, drug resistance and virulence factors of Candida species isolated from patients using long-term inhaled steroids.

In this study, it was aimed to determine the isolation frequency and species distribution of Candida species isolated from asthmatic patients using long-term inhaled steroids. It was also aimed to determine the drug resistance patterns and the frequency of erg11, HWP1, ALS1, INT1, SAP1 PLB1 genes in isolates. Genotyping of Candida strains isolated from patients and healthy control group was performed by PCR-RFLP method. Drug resistance was investigated phenotypically, and the presence of erg11 resistance genes and HWP1, ALS1, INT1, SAP1 PLB1 virulence genes were investigated by PCR method. C albicans was the most isolated species in steroid-using patients and healthy control groups (patients: 44.2%; control group: 30.8%). C tropicalis and C glabrata were found to have the highest rates of non-albicans Candida in patients with 17.4% and 13.77%, respectively. Azole resistance was found to be significantly higher in isolates isolated from patients compared to the control group. Similarly, the presence of erg11 resistance gene was highest in C albicans (17.65%), C glabrata (12.5%) and C tropicalis (8.3%) strains in the control group, while C parapsilosis was highest in patients. (57.1%) and C glabrata (54.2%) strains. Compared to the control group, the virulence of Candida strains isolated from the patients was found to be higher. Presence of HWP1, ALS1, INT1, SAP1 and PLB1 genes in patients were determined as 72.1%, 63.9%, 68.9%, 57.38% and 54.5%, respectively. These rates were 29.4%, 35.3%, 25.5%, 17.7% and 23.5% in the healthy control group, respectively. In asthma patients using long-term inhaled steroids, both Isolation rates of Candida species, drug resistance rates, presence of virulence genes were found to be significantly higher in patients than in the control group. We think that this may be due to the suppression of cellular immunity by long-term steroid use.

Detection of Phospholipase and Asparty1 Protaines gens in Candida Species Isolated From Mammary Candidiasis in Lactating Women

Breast and nipple thrush (BNT) is a yeast infection of the nipple and breast caused by a fungal organism knownas Candida albicans, a common cause of all thrush infections. It occurs most commonly in breastfeedingmothers. The infection may lead to severe nipple and breast pain, especially during breastfeeding. Thecurrent study was conducted at Al-Kut hospital for Gynecology, obstetrics and pediatrics, and TaibaSpecialist Centerin Wasit province. (162) milk samples were collected from lactating wommen suspected tohave mammary candidiasis infections according to the clinical manifestations diagnosed by the examiningphysician and patient with symptom , then, the samples were examined by optical microscopy and celldetection which show evidence of the presence of pathogenic yeast. Samples were cultured in sabouraudagar and then in chrom agar to classify the organisms, and they were held for further conformation and todetect the most common virulence factor gen by polymerase chain reaction (PCR) Molecular identificationof Candida spp in this study, done by conventional PCR by partial amplification gene encoding for largesubunit of 18S rRNA gene by specific primer sequences. The yield of the detection C. albicans and C.parapsilosis, was 37/37(100%) (100%) and 15/15 (100%), respectively. The results of PCR amplificationof( SAP1-8)genes in Candida isolates (61) were SAP1 gen was present in 61/61(100%) , SAP2 gen was54/61(96%), SAP3 gen was 45/61(86%), SAP4 gen was 35/61(76%), SAP5 gen was 32/61(66%), SAP6 genwas 45/61(60%), SAP7 gen was 35/61(30%) and SAP8 gen was 21/61(34%). By molecular technique usingPCR (PLB1-2 gene) and sequencing, it was seen that Seventeen isolates belong C. albicans were positivefor PLB1 gene 17/61 (91.6%). Fourteen isolates belong C. albicans were positive for PLB1 gene 14/61.

Determination the Gene Expression Levels of adhesins and Extracellular Enzymes Genes in Candida albicans biofilm producer by Quantitative Real Time PCR Technique (qRT-PCR)

In the present study, all C. albicans were biofilm producers with variable strength. Molecular study ofvirulence genes correlated with biofilm C. albicans showed that HWP1, ALS1, ALS3, SAP5, PLB1 and LIP8genes were detected in 100% of C. albicans from vaginal and oral infections. In addition to that quantitativereal time PCR technique show that ITS1 was used as the housekeeping gene and was shown to be stablyexpressed throughout all biofilm conditions and HWP1, ALS1, ALS3, SAP5, PLB1 and LIP8 where 100%expressed in all ten tested C. albicans. It was appeared that adhesins genes such as HWP1, ALS1 and ALS3were highly expressed in HBF and less expressed in LBF, whereas in very low BF producers the fold upregulation were not particularly high, but they were over expressed in strong biofilm producers in both ofvaginal and oral isolates. The average of gene expression folding show that SAP5 and PLB1 were highlyexpressed among vaginal isolates rather than oral ones, in contrast, ALS1, HWP1 and LIP8 were predominantexpressed among oral isolates, whereas ALS3 was higher in vaginal isolates than oral with little differences.Gene expression levels by q RT-PCR show that SAP5 and PLB1 were up-regulated in both of HBF andLBF producers, and they were highly expressed among the ten tested isolates. The over expressions ofSAP5 and PLB1 genes were observed in vaginal isolates with high biofilm. The current study found anoverexpression of adhesins such as HWP1, ALS1, ALS3, are responsible in strength of biofilm formationin C. albicans isolated from oral and vaginal infection with different ages and different clinical states,in addition to upregulated of extracellular hydrolytic enzymes genes belong to SAP, PLB and LIP wasimportant in biofilm formation.

Alternation of HWP1 and PLB1 mRNA Expression Level in Progression of Candida albicans in Different Anatomical Sites

Background: Candida albicans can cause oral, vaginal, and cutaneous infections, as well as systemic candidiasis. It has been recently documented that molecular factors play significant roles in the pathogenesis of Candida albicans in various anatomical sites of the host. Objectives: The present study was designed to answer the hypothesis of whether PLB1 and HWP1 mRNA expression patterns are related to the progression of infection in different anatomical sites of the body. Methods: The experimental study was performed on 120 clinical isolates of C. albicans obtained from various sites of non-immune-compromised and immune-compromised patients. Initially, all samples were cultured on Sabouraud-dextrose agar and then CHROM agar Candida medium to isolate and obtain a pure colony of yeasts. Quantitative real-time PCR was carried out for the quantitative evaluation of HWP1 and PLB1 mRNA expression in all clinical samples. The frequency of the PLB1 and HWP1 genes among C. albicans strains isolated from four clinical sites was analyzed using Fisher’s exact test with a significance threshold of P < 0.05. Finally, data obtained from real-time PCR was interpreted using the comparative Ct method (∆∆Ct) by REST© software. Results: The HWP1 gene was detected at a higher frequency than the PLB1 gene in C. albicans strains. The HWP1 mRNA expression level of clinical samples was upregulated by 70, 83.3, 43.3, and 33.3% in four sites (oral, vaginal, BAL, and cutaneous sites), respectively. The PLB1 mRNA expression level of all samples was upregulated by 46.7, 53.3, 40, and 3.3% (P < 0.001) in four sites compared to the control group. Conclusions: The PLB1 and HWP1 genes were expressed predominantly in mucosal (oral, vaginal, and BAL) specimens. This clearly shows that the expression pattern of these candidate genes depends on the organ localization. Furthermore, the presence of samples with no expression of HWP1 and PLB1 genes mRNA confirmed the recent hypothesis that there is a meaningful relationship between the higher expression level of candidate genes mRNA and the presence of infections in a specific site of the body. However, more studies are required on larger samples to characterize the exact molecular mechanism of candidate genes involved in the severity of symptoms, as well as their contribution to the site of infection.

Study of Antifungal Susceptibility, Virulence Genes and Biofilm Formation in Candida albicans

Background: Candida albicans has emerged as an important nosocomial pathogen. The morbidity and mortality associated with this pathogen are related to the presence of virulence genes and antifungal resistance. The objective of the present study was to investigate the prevalence of antifungal resistance, biofilm formation and some virulence genes such as ALS1, PLB1, INT1, SAP1 and HWP1, among clinical isolates of Candida albicans recovered from immunocompromised patients. Methods: The study included one hundred C. albicans isolates identified phenotypically and by a molecular technique using Polymerase Chain Reaction (PCR). The identified C. albicans was further subjected to antifungal study by the microdilution method, biofilm study and molecular study for virulence genes by PCR. Results: The resistance to antifungal drugs, fluconazole, caspofungin and itraconazole was 8% for each of them and for amphotericin B, it was 9%. The prevalence of the studied virulence genes was HWP1 77%, INT1 72%, ALS1 65%, SAP1 65% and PLB1 52%. The biofilm capacity was identified by the microplate method in 58% of C. albicans. The OD was intense in 20 isolates, moderate in 21 isolates and mild in 17 isolates. There was a statistically significant increase in the prevalence of the studied virulence genes INT1, ALS1, HWP1, SAP1 and PLB1 among biofilm forming C. albicans as compared to non-biofilm forming isolates (P=0.0001). Additionally, the resistance to fluconazole, itraconazole and caspofungin was statistically, significantly higher in C. albicans with the capacity to form biofilm as compared to non-biofilm forming C. albicans. Conclusion: The present study highlights the prevalence of resistance to antifungal drugs among C. albicans which are not uncommon. Moreover, there was a high prevalence of INT1, ALS1, HWP1, SAP1 and PLB1 genes in C. albicans. The resistance to antifungal drugs was common among isolates with the capacity to form the biofilm. There was an association between the biofilm formation and virulence genes.

In vitro expression of vital virulent genes of clinical and environmental isolates of Cryptococcus neoformans/gattii in endothelial cells of human blood-brain barrier

BackgroundEvaluation of the pathogenesis of clinical and environmental cryptococcal isolates to the central nervous system is necessary for understanding the risk. This study was designed to determine the in vitro expression of six important virulent genes of Cryptococcus neoformans/gattii in Human Brain Microvascular Endothelial cells (hBMEC). MethodsThe hBMEC were infected with Cryptococcus to determine invasion and survival rate at 3, 12 and 24hours by subsequent colony count of internalized yeasts. The whole RNA of the intracellular Cryptococcus was extracted to quantify the expression of CAP10, PLB1, ENA1, URE1, LAC1, and MATα genes by real-time quantitative PCR for 3 and 12hours of infection. ResultsInvasion and survival rates were higher in clinical and standard strains of C. neoformans. A significant difference was observed among the clinical and environmental isolates for the expression of CAP10, ENA1, LAC1, MATα and URE1 at 3hours, and ENA1, LAC1, MATα, PLB1 and URE1 at 12hours. Clinical isolates showed significant upregulation of all the genes except PLB1, which was higher in environmental isolates. Relative expressions at the two time-points showed statistically significant (P=0.043) changes for the clinical isolates and no significance (P=0.063) for environmental isolates. ConclusionThe C. gattii (VGI) isolates showed significantly lower invasion and survival than C. neoformans (VNI, and VNII) irrespective of their sources. Clinical isolates exhibited higher expression for the majority of the virulent genes until 12hours of infection, probably due to their better adaptation in the host system and enhanced pathogenicity than the environmental counterparts.

Identification of the major molecular types of Cryptococcus neoformans and C. gattii by Hyperbranched rolling circle amplification.

The agents of cryptococcosis C. neoformans and C. gattii are important agents of meningoencephalitis in immunocompromised and immunocompetent hosts, respectively. They are grouped into eight major molecular types, VNI-VNIV for C. neoformans and VGI-VGIV for C. gattii. These major molecular types differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. To enable a rapid identification of the major molecular types and potential hybrids within the two species specific probes based on the PLB1 gene in combination with hyperbranched rolling circle amplification (HRCA) were developed. HRCA was applied to 76 cryptococcal strains, 10 strains each representing the 7 haploid major molecular types, 4 VNIII hybrid strains and 2 inter-species hybrid strains. All strains were correctly identified to the major molecular type and or hybrid type using HRCA alone. To increase the sensitivity a semi-nested PCR step was developed, which will enable the identification of the molecular types/hybrids directly from clinical samples, harboring a low copy number of DNA (40 copies). Thus, HRCA based on the PLB1 locus alone and in combination with a semi-nested PCR showed to be a specific and sensitive methodology, with a great potential to be used on clinical specimens for the direct diagnosis of the agents of cryptococcosis, including hybrid strains, enabling a rapid and patient tailored treatment choice of this disease.

Phospholipase gene expression during Paracoccidioides brasiliensis morphological transition and infection

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

Subgenotyping analysis of pathogenic Cryptococcus gattii isolates from China.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands. Key words: Cryptococcus neoformans; Genes, mating type, fungal; Cluster analysis10.3760/cma.j.issn.0412-4030.2009.02.026

LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae

The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Pruger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121-28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5-3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells.

The phospholipase B homolog Plb1 is a mediator of osmotic stress response and of nutrient-dependent repression of sexual differentiation in the fission yeast Schizosaccharomyces pombe.

Although phospholipase B (PLB) enzymes have been described in eukaryotes from yeasts to mammals, their biological functions are poorly understood. Here we describe the characterization of plb1, one of five genes predicted to encode PLB homologs in the fission yeast, Schizosaccharomyces pombe. The plb1 gene is dispensable under normal growth conditions but required for viability in high-osmolarity media and for normal osmotic stress-induced gene expression. Unlike mutants defective in function for the stress-activated MAP kinase Spc1, plb1Delta cells are not hypersensitive to oxidative or temperature stresses, nor do they undergo a G2-specific arrest in response to osmotic stress. In addition to defects in osmotic stress response, plb1Delta cells exhibit a cold-sensitive defect in nutrient-mediated mating repression, a phenotype reminiscent of mutants in the cyclic AMP (cAMP) pathway. We show that, like plb1Delta cells, mutants in the cAMP pathway are defective for growth in high-osmolarity media, demonstrating a previously unrecognized role for the cAMP pathway in osmotic stress response. Furthermore, we show that gain-of function in the cAMP pathway can rescue the osmosensitive growth defect of plb1Delta cells, suggesting that the cAMP pathway is a potential downstream target of the actions of Plb1 in S. pombe.

Differential expression of Candida albicans phospholipase B (PLB1) under various environmental and physiological conditions

Candida is the fourth most common organism responsible for bloodstream infections in many intensive care units, with Candida albicans being the most predominant species isolated in such cases. It has previously been shown that candidal phospholipase B, encoded by the PLB1 gene, is an important virulence factor for C. albicans pathogenesis. In this study, the effects of environmental factors (carbohydrate source and pH) and physiological conditions (serum, phospholipids and temperature) on the expression of PLB1 by C. albicans cells grown in rich [Sabouraud dextrose broth (SB) or yeast extract/peptone/dextrose] or chemically defined [Lee's, RPMI-1640 or yeast nitrogen base (YNB)] media were investigated. Northern blot analyses revealed that PLB1 mRNA was expressed in C. albicans cells grown in rich media at 30 degrees C but not at 37 degrees C. However, the protein Plb1p was detected in fungal cells growing at 37 degrees C in SB, as determined by Western blot analysis, indicating that although the mRNA for this gene was not detected, the actual gene product was present at this temperature. Expression of PLB1 was detected in cells grown in YNB/glucose at 30 degrees C but not at 37 degrees C. However, growth of C. albicans in YNB/glucose supplemented with serum and phospholipids resulted in expression of PLB1 at 37 degrees C also. Additionally, acidic pH induced higher levels of PLB1 mRNA expression compared to neutral pH, while the morphological form of C. albicans did not have any influence on the expression of this gene. The studies described here show that the expression of PLB1 is regulated by nutritional supplementation, environmental factors and the growth phase of the C. albicans cells, as well as by physiological conditions. The differential expression of PLB1 in response to environmental factors may be correlated to host-specific components available to C. albicans during infection.

Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo.

Phospholipases have been proposed to contribute to the virulence of Candida albicans. Recently, a candidal strain deleted for PLB1, the gene encoding the predominant phospholipase B (Plb1) secreted by C. albicans, was constructed and its virulence in an intravenous murine model of disseminated candidiasis was evaluated. In the present study, the PLB1 gene was reintroduced back into the plb1 null mutant to generate the revertant strain, which showed similar growth and morphology to its isogenic parent strain. Virulence of the revertant strain was found to be comparable to that of the parent strain in an intravenous murine model of disseminated candidiasis. To compare the abilities of the plb1 null mutant, the revertant and the isogenic parent strains to cross the gastrointestinal (GI) tract and cause systemic infection, an oral-intragastric infant mouse model of candidiasis was used. Histological examinations and analysis of c.f.u. of the pathogen in liver homogenates revealed that the parental and revertant strains were able to invade and traverse the GI mucosa to a significantly greater extent than the plb1 null mutant. Immunofluorescence and immunoelectron microscopic studies of infected host tissue using anti-Plb1 antibody showed that Plb1 is secreted during invasion of the gastric mucosa by the parental and revertant strains. In contrast, little or no labelling was observed in the null mutant strain. The results indicate that the Plb1 secreted by C. albicans enhances the ability of this organism to cross the GI tract and disseminate haematogenously. These studies provide unequivocal evidence supporting a role for Plb1 during the course of infection by C. albicans.

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase.

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.

Cloning and regulated expression of the Candida albicans phospholipase B ( PLB1) gene

Degenerate oligonucleotides (derived from conserved regions of PLB1 genes from S. cerevisiae and other fungi) were used to amplify PLB1 homolog fragments from C. albicans and C. tropicalis by using the polymerase chain reaction. The C. albicans PLB1 fragment was then used as a probe to clone the full-length gene and to monitor PLB1 mRNA expression. The C. albicans PLB1 gene consists of a 1815-bp open reading frame encoding a putative protein of 605 amino acids. It contains the highly conserved Gly-X-Ser-X-Gly catalytic motif, found in all lipolytic enzymes, and exhibits significant homology with other fungal PLB1 gene products (∼63% similarity, ∼45% identity). Blastospores and pseudohyphae expressed higher levels of PLB1 mRNA than germ-tube-forming cells. TUP1, a general transcriptional repressor, may regulate PLB1 expression in C. albicans, since PLB1 expression was the highest in tup1Δ mutants and did not vary in response to environmental stimuli. Together, these results suggest that expression of the C. albicans PLB1 gene is regulated as a function of morphogenic transition.