CagW, a VirB6 homologue interacts with Cag-type IV secretion system substrate CagA in Helicobacter pylori.

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.

Identification and interplay of sequence specific DNA binding proteins involved in regulation of human Pregnane and Xenobiotic Receptor gene.

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.

Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

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.

C-terminal domain of CagX is responsible for its interaction with CagT protein of Helicobacter pylori type IV secretion system.

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.

Quantitative proteomics and metabolomics approaches to demonstrate N-acetyl-D-glucosamine inducible amino acid deprivation response as morphological switch in Candida albicans.

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.

Ncb2 is involved in activated transcription of CDR1 in azole-resistant clinical isolates of Candida albicans.

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.

Responses of pathogenic and nonpathogenic yeast species to steroids reveal the functioning and evolution of multidrug resistance transcriptional networks.

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.

The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function.

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.

Biochemical characterization of the Helicobacter pylori Cag-type IV secretion system unique component CagU.

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.

Analyzing the role of CagV, a VirB8 homolog of the type IV secretion system of Helicobacter pylori.

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.

The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans.

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.

Pregnane and Xenobiotic Receptor (PXR/SXR) resides predominantly in the nuclear compartment of the interphase cell and associates with the condensed chromosomes during mitosis.

Pregnane and Xenobiotic Receptor (PXR) is a transcription factor that is activated by a diverse range of xenobiotics and endogenous metabolites including steroids, bile acids and about 50% of the prescription drugs. In specific cell types (e.g. liver and intestine) it serves as a 'xenosensor' by regulating expression of a network of genes involved in xenobiotic clearance from the body. PXR expression in several cancerous tissues and its regulated expression of multi-drug resistance proteins highlight its significance in prognosis of malignancies. The view that subcellular localization and ligand induced movements of transcription factors is one of the major phenomena in regulating transcriptional activity, we used a green fluorescent protein tagged PXR chimera to study its dynamic behaviour in living cells. Under all experimental conditions, PXR was observed to be a predominantly nuclear protein maintaining a dynamic equilibrium between the nuclear and cytoplasmic compartments of the interphase cells. Interestingly, for the first time, a member of the nuclear receptor superfamily, PXR, has been observed to be associated with condensed chromosomes during all the mitotic stages of cell division. The significance of PXR association with mitotic chromosomes is discussed.

Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo.

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

Functional characterization of Helicobacter pylori DnaB helicase.

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5' to 3' helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.

Identification and Antifungal Susceptibility Testing of Candida Species: A Comparison of Vitek-2 System with Conventional and Molecular Methods.

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.

Purification of full-length human pregnane and xenobiotic receptor: polyclonal antibody preparation for immunological characterization.

Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

Interactions between bacteria and Candida in the burn wound.

In order to understand whether the presence of some bacterial species affects the growth of Candida sp. in the wounds of burn patients, we have studied the effect of various bacterial species, collected from burn wounds on the growth of Candida sp. on Sabouraud dextrose agar (SDA) slants. A total of 300 burn patient samples were analyzed over a period of 2 years. Results of this analysis revealed that Pseudomonas sp. when present alone or in combination with other bacterial species invariably inhibited Candida sp. growth. Thus, we conclude that the absence of Candida sp. in burn wounds, where Pseudomonas sp. is present, is due to the inhibition of Candida growth by this bacterial species.

Biochemical Analysis of CagE: A VirB4 Homologue of Helicobacter pylori Cag-T4SS.

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.

A approximately 35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP.

BACKGROUND: The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested. RESULTS: Partially purified fractions containing GSTCdc6 or GST showed an ACS binding activity in an ATP dependent manner. However, further purification revealed the presence of a putative 35 kDa insect cell protein (p35) which was found responsible for the DNA binding activity. A close match to the 9/11 bases of the ARS consensus sequence was sufficient for p35 binding activity. A DNA fragment from the human c-myc origin region containing yeast ACS like elements also showed p35 binding activity. CONCLUSIONS: We have identified a Spodoptera frugiperda protein with ATP dependent DNA binding activity to ACS like elements. ACS like elements have been reported to be essential for ORC binding and replication initiation in yeast but their role in higher eukaryotes still remains elusive. Like the ARS consensus sequence elements of yeast, ACS like elements found in c-myc and lamin beta 2 origin regions may play similar roles in replication and indicate a conserved role for this DNA motif among eukaryotes.