RESUMO
Protein translocating Cag type IV secretion system of Helicobacter pylori is a diverse multi-protein complex. Here, we have characterized one of its key subunit CagW to identify its interacting partners. Our results demonstrate for the first time that this VirB6 homologue interacts with the substrate of the secretion system CagA. CagW forms multimer and its absence affects cellular levels of pilus forming components, CagL, CagI and CagH. Our results support the notion that the protein is essential for the transport of CagA across the bacterial membrane barrier and would aid in improving our understanding of structural and functional aspects of the inner membrane part of Cag-T4SS channel complex for the passage of substrate CagA.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Transporte Biológico , Brucella/genética , Brucella/metabolismo , Fímbrias Bacterianas/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Mutação , Fosforilação , Transporte ProteicoRESUMO
The type IV secretion system of Helicobacter pylori (Cag-T4SS) is composed of ~ 27 components including a VirB8 homolog, CagV. We have characterized CagV and reported that it is an inner membrane protein and, like VirB8, forms a homodimer. Its stability is not dependent on the other Cag components and the absence of cagV affects the stability of only CagI, a protein involved in pilus formation. CagV is not required for the stability and localization of outer membrane subcomplex proteins, but interacts with them through CagX. It also interacts with the inner membrane-associated components, CagF and CagZ, and is required for the surface localization of CagA. The results of this study might help in deciphering the mechanistic contributions of CagV in the Cag-T4SS biogenesis and function.
RESUMO
Ncb2, the ß subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.
Assuntos
Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Regiões Promotoras Genéticas , Estresse Fisiológico , Transcrição Gênica , Azóis/farmacologia , Sequência de Bases , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , TATA Box/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Many strains of Helicobacter pylori encode a protein secreting type IV secretion system called the Cag-T4SS. Here, we characterize one of the Cag-T4SS-specific components, CagU (HP0531). We report that CagU is a bacterial inner membrane-associated protein, partially processed at the C terminus, and that it interacts with the VirB6 and VirB8 homologs CagW and CagV, respectively. The level of expression of CagU is partially affected in the absence of cagX, cagW, and cagV. Deletion of cagU aborts surface localization of CagA and affects the expression levels of CagI and CagH, which are involved in the Cag-T4SS pilus formation. Complementation of the cagU null mutation by wild-type cagU restores all these functions, suggesting its importance for Cag-T4SS function.
Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Proteínas de Bactérias/ultraestrutura , Western Blotting , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte ProteicoRESUMO
BACKGROUND: Candida infection is a major cause of morbidity and mortality in immunocompromised patients; an accurate and early identification is a prerequisite need to be taken as an effective measure for the management of patients. The purpose of this study was to compare the conventional identification of Candida species with identification by Vitek-2 system and the antifungal susceptibility testing (AST) by broth microdilution method with Vitek-2 AST system. MATERIALS AND METHODS: A total of 172 Candida isolates were subjected for identification by the conventional methods, Vitek-2 system, restriction fragment length polymorphism, and random amplified polymorphic DNA analysis. AST was carried out as per the Clinical and Laboratory Standards Institute M27-A3 document and by Vitek-2 system. RESULTS: Candida albicans (82.51%) was the most common Candida species followed by Candida tropicalis (6.29%), Candida krusei (4.89%), Candida parapsilosis (3.49%), and Candida glabrata (2.79%). With Vitek-2 system, of the 172 isolates, 155 Candida isolates were correctly identified, 13 were misidentified, and four were with low discrimination. Whereas with conventional methods, 171 Candida isolates were correctly identified and only a single isolate of C. albicans was misidentified as C. tropicalis. The average measurement of agreement between the Vitek-2 system and conventional methods was >94%. Most of the isolates were susceptible to fluconazole (88.95%) and amphotericin B (97.67%). The measurement of agreement between the methods of AST was >94% for fluconazole and >99% for amphotericin B, which was statistically significant (P < 0.01). CONCLUSION: The study confirmed the importance and reliability of conventional and molecular methods, and the acceptable agreements suggest Vitek-2 system an alternative method for speciation and sensitivity testing of Candida species infections.
RESUMO
Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a 'xenosensor' in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug-drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis-elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54kDa. Here, we report the identification of 54kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Receptores de Esteroides/genética , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Receptor de Pregnano X , Regiões Promotoras Genéticas/genética , Receptores de Esteroides/metabolismo , Elementos de Resposta/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMO
Helicobacter pylori are among the most successful human pathogens that harbour a distinct genomic segment called cag Pathogenicity Island (cag-PAI). This genomic segment codes for a type IV secretion system (Cag-T4SS) related to the prototypical VirB/D4 system of Agrobacterium tumefaciens (Ag), a plant pathogen. Some of the components of Cag-T4SS share homology to that of VirB proteins including putative energy providing CagE (HP0544), the largest VirB4 homologue. In Ag, VirB4 is required for the assembly of the system, substrate translocation and pilus formation, however, very little is known about CagE. Here we have characterised the protein biochemically, genetically, and microscopically and report that CagE is an inner membrane associated active NTPase and has multiple interacting partners including the inner membrane proteins CagV and Cagß. Through CagV it is connected to the outer membrane sub-complex proteins. Stability of CagE is not dependent on several of the cag-PAI proteins tested. However, localisation and stability of the pilus associated CagI, CagL and surface associated CagH are affected in its absence. Stability of the inner membrane associated energetic component Cagß, a VirD4 homologue seems to be partially affected in its absence. Additionally, CagA failed to cross the membrane barriers in its absence and no IL-8 induction is observed under infection condition. These results thus suggest the importance of CagE in Cag-T4SS functions. In future it may help in deciphering the mechanism of substrate translocation by the system.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Nucleosídeo-Trifosfatase/metabolismo , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/análise , Linhagem Celular , Infecções por Helicobacter/metabolismo , Humanos , Modelos Moleculares , Nucleosídeo-Trifosfatase/análise , Conformação Proteica , Mapas de Interação de Proteínas , Estabilidade ProteicaRESUMO
Helicobacter pylori are the well known human pathogen associated with gastric cancer and peptic ulcer. Pathogenesis is mainly due to the presence of 40 kb cagPAI (cag Pathogenicity Island) region that encodes the type IV secretion system (TFSS) consisting of a cytoplasmic part, a middle part/core complex (spans from inner membrane to outer membrane), and an outer membrane associated part. CagX and CagT are two important proteins of TFSS that have homology with virB9 and virB7 of Agrobacterium tumefaciens TFSS. In this study, we have shown that the CagX and CagT interact directly by using co-immunoprecipitation of endogenous CagX and CagT and MBP pull down assay. We further authenticate this observation using yeast two-hybrid assay and co-expression of both the protein coding gene in Escherichia coli. We also observed that the C-terminal region of CagX is important for CagT interaction. We reconfirm that CagT depends on CagX for its stabilization. These observations could contribute in overall visualization of assembly and architecture of TFSS because protein-protein interactions among Cag proteins are likely to have an important role in assembly. Thorough understanding about architecture and mechanism of action of cag-TFSS may lead to design controlled drug delivery system.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Proteínas de Transporte/metabolismo , Helicobacter pylori/metabolismo , Antígenos de Bactérias/metabolismo , Clonagem Molecular , Citoplasma/metabolismo , DNA/metabolismo , Sistemas de Liberação de Medicamentos , Ilhas Genômicas , Helicobacter pylori/genética , Imunoprecipitação , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator de Transcrição PAX5/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores de Esteroides/biossíntese , Sítios de Ligação/genética , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Receptor de Pregnano X , Regiões Promotoras Genéticas , Ligação Proteica/genética , Proteína Proto-Oncogênica c-ets-1/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores de Esteroides/genética , Transativadores/metabolismo , Transcrição Gênica , Ativação Transcricional/genéticaRESUMO
Candida albicans causes superficial to systemic infections in immuno-compromised individuals. The concomitant use of fungistatic drugs and the lack of cidal drugs frequently result in strains that could withstand commonly used antifungals, and display multidrug resistance (MDR). In search of novel fungicidals, in this study, we have explored a plant alkaloid berberine (BER) for its antifungal potential. For this, we screened an in-house transcription factor (TF) mutant library of C. albicans strains towards their susceptibility to BER. Our screen of TF mutant strains identified a heat shock factor (HSF1), which has a central role in thermal adaptation, to be most responsive to BER treatment. Interestingly, HSF1 mutant was not only highly susceptible to BER but also displayed collateral susceptibility towards drugs targeting cell wall (CW) and ergosterol biosynthesis. Notably, BER treatment alone could affect the CW integrity as was evident from the growth retardation of MAP kinase and calcineurin pathway null mutant strains and transmission electron microscopy. However, unlike BER, HSF1 effect on CW appeared to be independent of MAP kinase and Calcineurin pathway genes. Additionally, unlike hsf1 null strain, BER treatment of Candida cells resulted in dysfunctional mitochondria, which was evident from its slow growth in non-fermentative carbon source and poor labeling with mitochondrial membrane potential sensitive probe. This phenotype was reinforced with an enhanced ROS levels coinciding with the up-regulated oxidative stress genes in BER-treated cells. Together, our study not only describes the molecular mechanism of BER fungicidal activity but also unravels a new role of evolutionary conserved HSF1, in MDR of Candida.
Assuntos
Antifúngicos/farmacologia , Berberina/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candidíase/microbiologia , Farmacorresistência Fúngica , Resistência a Múltiplos Medicamentos , Proteínas Fúngicas/genética , Proteínas de Choque Térmico/genética , Humanos , Mutação , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world's residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Sistemas de Secreção Bacterianos/imunologia , Helicobacter pylori/imunologia , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Antígenos de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Sítios de Ligação , Desenho de Fármacos , Dados de Sequência Molecular , Ligação Proteica , CoelhosRESUMO
Helicobacter pylori Cag type IV secretion system (Cag-T4SS) is a multi-component transporter of oncoprotein CagA across the bacterial membranes into the host epithelial cells. To understand the role of unique Cag-T4SS component CagI in CagA translocation, we have characterized it by biochemical and microscopic approaches. We observed that CagI is a predominantly membrane attached periplasmic protein partially exposed to the bacterial surface especially on the pili. The association of the protein with membrane appeared to be loose as it could be easily recovered in soluble fraction. We documented that the stability of the protein is dependent on several key components of the secretion system and it has multiple interacting partners including a non-cag-PAI protein HP1489. Translocation of CagA across the bacterial membranes to cell surface is CagI-independent process. The observations made herein are expected to assist in providing an insight into the substrate translocation by the Cag-T4SS system and Helicobacter pylori pathogenesis.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Helicobacter pylori/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Clonagem Molecular , Fímbrias Bacterianas/metabolismo , Mutação , Periplasma/metabolismo , Plasmídeos/metabolismo , Ligação ProteicaRESUMO
Pregnane X Receptor (PXR) is an important ligand-activated nuclear receptor functioning as a 'master regulator' of expression of phase I, phase II drug metabolizing enzymes, and members of the drug transporters. PXR is primarily expressed in hepatic tissues and to lesser extent in other non-hepatic tissues both in human and in mice. Although its expression profile is well studied but little is known about the regulatory mechanisms that govern PXR gene expression in these cells. In the present study, we have cloned and characterized over 5 kb (-4963 to +54) region lying upstream of mouse PXR transcription start site. Promoter-reporter assays revealed that the proximal promoter region of up to 1 kb is sufficient to support the expression of PXR in the mouse liver cell lines. It was evident that the 500 bp proximal promoter region contains active binding sites for Ets, Tcf, Ikarose and nuclear factor families of transcription factors. Electrophoretic mobility shift assays demonstrated that the minimal region of 134 bp PXR promoter was able to bind Ets-1 and ß-catenin proteins. This result was further confirmed by chromatin immunoprecipitation analysis. In summary, the present study identified a promoter region of mouse PXR gene and the transregulatory factors responsible for PXR promoter activity. The results presented herein are expected to provide important cues to gain further insight into the regulatory mechanisms of PXR function.
Assuntos
Regulação da Expressão Gênica , Receptores de Esteroides/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Clonagem Molecular , Biologia Computacional , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Humanos , Luciferases/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Dados de Sequência Molecular , Receptor de Pregnano X , Regiões Promotoras Genéticas/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores de Esteroides/metabolismo , Deleção de Sequência , beta Catenina/metabolismoRESUMO
Candida albicans is a life threatening polymorphic pathogen for immunocompromised patients, causing superficial as well as invasive systemic diseases. The mucosal membranes of the host, which are the primary sites of its infection, are rich in amino sugars like N-acetylglucosamine (GlcNAc). GlcNAc is also one of the potent inducers of morphological transition, an important pathogenic trait of C. albicans. We thus performed proteomic analysis on total soluble proteins to identify the molecules involved in this response. Proteomic analysis using 2-DE demonstrated reproducible upregulation of 36 spots from a total of 585 matched spots. Mass spectroscopy (MS/MS) analyses of upregulated proteins revealed that carbohydrate and amino acid metabolism were the most prominent functional classes. Metabolite profiling using GC-MS allowed a quantitative comparison of 58 metabolites in GlcNAc or glucose grown cells. We observed a significant decrease in the intracellular amino acid pool of GlcNAc grown cells. Moreover, GlcNAc induces both bZIP transcription factor (GCN4) and eIF2α kinase (GCN2) which are responsible for the activation of general amino acid control response in C. albicans. Inactivation of these genes blocks GlcNAc induced morphogenesis. Altogether these results suggest that amino acid starvation is the morphogenetic signal in presence of GlcNAc in C. albicans.
Assuntos
Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Candida albicans/citologia , Candida albicans/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Metaboloma , Proteoma/análise , Candida albicans/genética , Candida albicans/metabolismo , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/análise , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Metabolômica/métodos , Proteômica/métodosRESUMO
We recently demonstrated that CDR1 overexpression in azole-resistant isolates of Candida albicans is due to its enhanced transcriptional activation and increased mRNA stability. In this study, we provide the first evidence of transcriptional regulation of CDR1 by Ncb2, the ß subunit of NC2, a heterodimeric regulator of transcription. Conditional NCB2 null mutants displayed decreased susceptibility toward azole and an enhanced transcription of CDR1. Interestingly, Ncb2 associated with the CDR1 promoter under both repression and activation; however, an increase in recruitment was observed under both transient and constitutive activation states. By chromatin immunoprecipitation (ChIP) assay, we showed the preferential recruitment of Ncb2 to the core TATA region under activation (azole-resistant isolate), while under repression (azole-susceptible isolate) it was present at the TATA upstream region. Further, ChIP analysis revealed that Ncb2 binding was not restricted to the CDR1 gene; instead, it was observed on the promoters of genes coregulated with CDR1 by the transcription activator Tac1. The tac1Δ null mutants, which fail to show the drug-induced transient activation of CDR1, also showed no increase in Ncb2 recruitment at the promoter. Taken together, our results show that Ncb2, in conjunction with Tac1, is involved in the transcriptional activation of CDR1, opening up new therapeutic possibilities to combat multidrug resistance (MDR) in C. albicans.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/metabolismo , Candidíase/microbiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/isolamento & purificação , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Candida albicans and Candida glabrata are the major causes of vulvovaginal candidiasis (VVC) in Indian women with diabetes mellitus. Little information is available regarding the genotyping of Candida species isolated from Indian diabetic women with VVC. METHODS: In this study, a total of 57 Candida species, comprising Candida albicans and Candida glabrata, isolated from Indian women with VVC, were genotyped and tested for fluconazole susceptibility. Arbitrarily primed polymerase chain reaction (AP-PCR) was used to genotype C glabrata isolates, whereas Southern blot hybridization using a Candida albicans repetitive element-2 (CARE-2) probe was used to genotype C albicans. RESULTS: Genotyping showed that all the C albicans isolates were genetically heterogenous. The pattern of DNA bands obtained after AP-PCR for C glabrata strains were predominantly conformed to genotype A. In vitro fluconazole-susceptibility testing of the isolates using the Clinical and Laboratory Standards Institute M27A2 protocol showed that more than 93% of the Candida isolates were susceptible. CONCLUSIONS: Ninety-five percent of the C albicans isolates analyzed were different and genetically unrelated. The analysis of the AP-PCR DNA banding pattern of C glabrata isolates showed that it resembled genotype "A". The Candida isolates were found to be susceptible to fluconazole, with minimum inhibitory concentrations ranging from 0.5 µg/mL to 8 µg/mL. This correlates with the use of fluconazole as a first-choice antifungal for treating VVC in India.
Assuntos
Antifúngicos/uso terapêutico , Candida albicans/genética , Candida glabrata/genética , Candidíase Vulvovaginal/tratamento farmacológico , Complicações do Diabetes/microbiologia , Fluconazol/uso terapêutico , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Candida glabrata/efeitos dos fármacos , Candida glabrata/isolamento & purificação , Candidíase Vulvovaginal/complicações , Candidíase Vulvovaginal/microbiologia , Feminino , Fluconazol/farmacologia , Genótipo , Humanos , Índia , Testes de Sensibilidade Microbiana , Tipagem Molecular , Reação em Cadeia da PolimeraseRESUMO
Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendous potential consequences for human fungal infections. Our analysis of gene expression in Saccharomyces cerevisiae and Candida albicans cells subjected to three different concentrations of progesterone revealed that their pleiotropic drug resistance (PDR) networks were strikingly sensitive to steroids. In S. cerevisiae, 20 of the Pdr1p/Pdr3p target genes, including PDR3 itself, were rapidly induced by progesterone, which mimics the effects of PDR1 gain-of-function alleles. This unique property allowed us to decipher the respective roles of Pdr1p and Pdr3p in PDR induction and to define functional modules among their target genes. Although the expression profiles of the major PDR transporters encoding genes ScPDR5 and CaCDR1 were similar, the S. cerevisiae global PDR response to progesterone was only partly conserved in C. albicans. In particular, the role of Tac1p, the main C. albicans PDR regulator, in the progesterone response was apparently restricted to five genes. These results suggest that the C. albicans and S. cerevisiae PDR networks, although sharing a conserved core regarding the regulation of membrane properties, have different structures and properties. Additionally, our data indicate that other as yet undiscovered regulators may second Tac1p in the C. albicans drug response.
Assuntos
Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla/genética , Proteínas Fúngicas/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Esteroides/farmacologia , Evolução Biológica , Northern Blotting , Candida albicans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Progesterona/farmacologia , Progestinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the absence of steroid receptors and any known mechanism of gene regulation by steroid hormones in Candida albicans, we did a genome-wide analysis of C. albicans cells treated with progesterone using Eurogentec cDNA microarrays to find the complete repertoire of steroid responsive genes. Northern blotting analysis was employed to validate the genes that were differentially regulated by progesterone in the microarray experiments. A total of 99 genes were found to be significantly regulated by progesterone, among them 60 were up-regulated and 39 were down-regulated. It was observed that progesterone considerably enhanced the expression of multi-drug resistance (MDR) genes belonging to ATP Binding Cassette (CDR1 and CDR2) super-family of multidrug transporters, suggesting a possible relationship between steroid stress and MDR genes. Several genes associated with hyphal induction and the establishment of pathogenesis were also found up-regulated. In silico search for various transcription factor (TF) binding sites in the promoter of the affected genes revealed that EFG1, CPH1, NRG1, TUP1, MIG1 and AP-1 regulated genes are responsive to progesterone. The stress responsive elements (STRE; AG(4) or C(4)T) were also found in the promoters of several responsive genes. Our data sheds new light on the regulation of gene expression in C. albicans by human steroids, and its correlation with drug resistance, virulence, morphogenesis and general stress response. A comparison with drug induced stress response has also been discussed.
Assuntos
Candida albicans/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico , Progesterona/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Northern Blotting , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.