Regulation of ERG3, ERG6, and ERG11 genes in antifungal-resistant isolates of candida parapsilosis

Background: Candida parapsilosis is one of the five common strains of yeasts involved in invasive candidiasis. The expression analysis of sterol biosynthesis pathway genes, which are associated with resistance, can assist the better understanding of antifungal resistance mechanisms

Methods: The antifungal susceptibility of 120 clinical C. parapsilosis isolates was examined. The changes in the gene expression related to resistance were analyzed

Results: Eight strains were resistant to fluconazole [FLC], itraconazole [ITC], and amphotericin B [AMB]. The regulation variations included increased mRNA levels of ERG3, ERG6, and ERG11 and decreased mRNA levels of ERG3 and ERG6 in response to FLC. ERG11 mRNA level increases in response to ITC and AMB

Conclusion: The mechanism of resistance to azoles in C. parapsilosis is very similar to C. Albicans. This feature may help to design new treatment strategy for candidiasis

Genetic identification of trichomonas vaginalis by using the actin gene and molecular based methods

Trichomonas vaginalis is the agent of urogenital tract infection that causes human trichomoniasis with some serious health complications. More under-standing about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection. For this end, we conducted analysis of the actin gene of T. vaginalis by applying the PCR-SSCP [PCR-Single Stranded Conformational Polymorphism] and nucleotide sequencing method. Fifty T. vaginalis samples were collected from 950 women attending gynecology clinics in two cities of Iran, Hamadan and Tehran, from November 2010 to July 2011. After axenisation of isolates, all samples subjected to PCR-SSCP and nucleotide sequencing. According to the SSCP banding patterns and nucleotide sequencing, seven sequence types were detected among the isolates. Alignment of the nucleotide sequences showed five polymorphic sites in the different strain types. Amino acid substitution was not observed in the nucleotide sequence translation of the all sequences. The actin gene analysis represents genetic diversity of T. vaginalis and it suggests that various strains can be responsible for clinically different trichomoniasis in infected individuals. It is expected that further studies will be conducted to increase our knowledge about relationship between the actin gene polymorphism and different biological behavior of the parasite

Down regulation of sidB gene by use of RNA interference in aspergillus nidulans

Introduction of the RNA interference [RNAi] machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans. The 21-nucleotide small interfering RNA [siRNA] was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay. Compared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA [42 VS 21 microM]. In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed. Usage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems

Green synthesis of silver nanoparticles: the reasons for and against Aspergillus parasiticus

The enzymatic activity of fungi has recently inspired the scientists with re-explore the fungi as potential biofactories rather than the causing agents of humans and plants infections. In very recent years, fungi are considered as worthy, applicable and available candidates for synthesis of smaller gold, silver and other nano-sized particles. A standard strain of Aspergillus parasiticus was grown on a liquid medium containing mineral salt. The cell-free filtrate of the culture was then obtained and subjected to synthesize SNPs while expose with 1mM of AgNO[3]. Further characterization of synthesized SNPs was performed afterward. In addition, antifungal activity of synthesized SNPs was evaluated against a standard strain of Candida albicans. The reduction of Ag+ ions to metal nanoparticles was investigated virtually by tracing the color of the solution which turned into reddish-brown after 72 h. The UV-vis spectra demonstrated a broad peak centering at 400 nm which corresponds to the particle size much less than 70 nm. The results of TEM demonstrated that the particles were formed fairly uniform, spherical, and small in size with almost 90% in 5-30 nm range. The zeta potential of silver nanoparticles was negative and equal to 15.0 which meets the quality and suggested that there was not much aggression. Silver nanoparticles synthesized by A. parasiticus showed antifungal activity against yeast strain tested and exhibited MIC value of 4 microg/mL. The filamentous fungus, A. parasiticus has successfully demonstrated potential for extra cellular synthesis of fairly monodispersed, tiny silver nanoparticles

effect of EFG1 gene silencing on down-regulation of SAP5 gene, by use of RNAi technology

Efg1 transcription factor is believed to be the main regulator of hyphal formation under many different conditions. In addition, it is responsible for positive regulation of the expression of several hyphalspecific genes. SAP5, which encodes secreted aspartic proteinase, is one of the mentioned genes and is crucial for pathogenicity properties. In the present work we have established the experimental conditions for the use of siRNA in the diploid yeast Candida albicans in order to knock-down the EFG1 gene expression as well as the Efg1-dependent gene, SAP5. The 19-nucleotide siRNA was designed according to cDNA sequence of EFG1 gene in C. albicans and modified-PEG/LiAc method was applied for yeast transfection. To quantify the level of both EFG1 and SAP5 gene expression, the cognate mRNAs were measured in C. albicans by quantitative real-time RT-PCR and data was consequently analyzed by use of REST[registered mark] software. Images taken by fluorescent microscopy method indicated the effectiveness of transfection. According to REST[registered mark] software data analysis, expression of EFG1 gene decreased about 2.5-fold using 500 nM of siRNA. A 7-fold decrease in EFG1 gene expression was observed when applying 1 micro M of siRNA [P<0.05]. Consequently, the expression of SAP5 was significantly down-regulated both in yeast treated with 500 and 1000 nM of siRNA [P<0.05]. In conclusion, post-transcriptional gene silencing [PTGS] is likely to be considered as a promising approach to discover new gene targets so as to design fungal-specific antifungal agents, and it is strongly possible that we are taking the right way to battle with C. albicans-associated infections

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Down-regulation of the ALS3 gene as a consequent effect of RNA-mediated silencing of the EFG1gene in candida albicans

The most important virulence factor which plays a central role in Candida albicans pathogenesis is the ability of this yeast to alternate between unicellular yeast and filamentous hyphal forms. Efg1 protein is thought to be the main positive regulating transcription factor, which is responsible for regulating hyphal-specific gene expression under most conditions. ALS3 is one of the Efg1-associated genes encoding a multi-functional adhesive polypeptide, which mediates adherence to diverse host substrates. In this study, the EFG1 gene was knocked down by using synthetic siRNA in C. albicans and the regulation in ALS3 as one of the Efg1-dependent genes was investigated. The 19-nucleotide siRNA was designed based on cDNA sequence of EFG1 gene in C. albicans. Transfection was performed using modified- plyethylen glycol/LiAc method. To quantify the level of EFG1 and the hyphal-specific ALS3 gene expression, the cognate EFG1 and ALS3 mRNA were measured in C. albicans by quantitative real-time RT-PCR. Fluorescent microscopy pictures indicated that transfection was performed successfully. Also, according to relative expression software tool, expression of EFG1 gene was decreased significantly with 500 nM siRNA as well as 1 micro M siRNA [P<0.05]. However, more significant downregulations were observed in the expression of ALS3 in both concentrations of 500 nM and 1 micro M siRNA [P<0.05]. In conclusion, we demonstrated the down-regulation of ALS3 gene as a consequent of applying EFG1-specific siRNA in C. albicans. This may lead us to design anti-fungal-specific agents in order to face with C. albicans-associated infections

Extracellular production of silver nanoparticles by using three common species of dermatophytes: trichophyton rubrum, trichophyton mentagrophytes and microsporum canis

To develop a new green approach for biosynthesis of silver nanoparticles, myconanotechnology has been represented as a novel field of study in nanotechnology. In this study, we have reported the extracellular synthesis of highly stable silver nanoparticles using three species of dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. Clinical strains of these species were grown in a liquid medium containing mineral salt and incubated at 25[degree sign]C for 5-7 days. The cell-free filtrate of each culture was obtained and subjected to synthesize silver nanoparticles in the presence of 1 mM AgNO[3]. The reduction of Ag+ ions in metal nanoparticles was investigated virtually by tracing the solution color which was switched into reddish-light brown after 72 h. For T. mentagrophytes, a UV-visible spectra demonstrating a strong, quite narrow peak located between 422 and 425 nm was obtained. For M. canis, a fairly wide peak centering at 441 nm and for T. rubrum, a weak spectrum to decipher were observed. According to transmission electron microscopy [TEM] results, fairly uniform, spherical, and small in size with almost less than 50 nm particles were forms in case of T. mentagrophytes. For the other two species, TEM images showed existence of small spherical nanosilvers but not as small as nanoparticles synthesized by T. mentagrophytes. We observed that species belong to a single genus of the fungi have variable ability to synthesize silver nanoparticles extracellulary with different efficiency. Furthermore, the extracellular synthesis may make the process simpler and easier for following processes