Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation

The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum.

Growing awareness of cerebellar involvement in addiction is based on the cerebellum's intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that lead to addiction.

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance.

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5' regulatory sequence variation in the corresponding genes is indeed increased. However, approximately 42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.

Cross-species and cross-platform gene expression studies with the Bioconductor-compliant R package 'annotationTools'.

Background: The variety of DNA microarray formats and datasets presently available offers an unprecedented opportunity to perform insightful comparisons of heterogeneous data. Cross-species studies, in particular, have the power of identifying conserved, functionally important molecular processes. Validation of discoveries can now often be performed in readily available public data which frequently requires cross-platform studies.Cross-platform and cross-species analyses require matching probes on different microarray formats. This can be achieved using the information in microarray annotations and additional molecular biology databases, such as orthology databases. Although annotations and other biological information are stored using modern database models ( e. g. relational), they are very often distributed and shared as tables in text files, i.e. flat file databases. This common flat database format thus provides a simple and robust solution to flexibly integrate various sources of information and a basis for the combined analysis of heterogeneous gene expression profiles.Results: We provide annotationTools, a Bioconductor-compliant R package to annotate microarray experiments and integrate heterogeneous gene expression profiles using annotation and other molecular biology information available as flat file databases. First, annotationTools contains a specialized set of functions for mining this widely used database format in a systematic manner. It thus offers a straightforward solution for annotating microarray experiments. Second, building on these basic functions and relying on the combination of information from several databases, it provides tools to easily perform cross-species analyses of gene expression data.Here, we present two example applications of annotationTools that are of direct relevance for the analysis of heterogeneous gene expression profiles, namely a cross-platform mapping of probes and a cross-species mapping of orthologous probes using different orthology databases. We also show how to perform an explorative comparison of disease-related transcriptional changes in human patients and in a genetic mouse model.Conclusion: The R package annotationTools provides a simple solution to handle microarray annotation and orthology tables, as well as other flat molecular biology databases. Thereby, it allows easy integration and analysis of heterogeneous microarray experiments across different technological platforms or species.

Estimation of electronic coupling in π -stacked donor-bridge-acceptor systems: correction of the two-state model

Comparison of donor-acceptor electronic couplings calculated within two-state and three-state models suggests that the two-state treatment can provide unreliable estimates of Vda because of neglecting the multistate effects. We show that in most cases accurate values of the electronic coupling in a π stack, where donor and acceptor are separated by a bridging unit, can be obtained as Ṽ da = (E2 - E1) μ12 Rda + (2 E3 - E1 - E2) 2 μ13 μ23 Rda2, where E1, E2, and E3 are adiabatic energies of the ground, charge-transfer, and bridge states, respectively, μij is the transition dipole moments between the states i and j, and Rda is the distance between the planes of donor and acceptor. In this expression based on the generalized Mulliken-Hush approach, the first term corresponds to the coupling derived within a two-state model, whereas the second term is the superexchange correction accounting for the bridge effect. The formula is extended to bridges consisting of several subunits. The influence of the donor-acceptor energy mismatch on the excess charge distribution, adiabatic dipole and transition moments, and electronic couplings is examined. A diagnostic is developed to determine whether the two-state approach can be applied. Based on numerical results, we showed that the superexchange correction considerably improves estimates of the donor-acceptor coupling derived within a two-state approach. In most cases when the two-state scheme fails, the formula gives reliable results which are in good agreement (within 5%) with the data of the three-state generalized Mulliken-Hush model

Bristle pattern formation in the thorax formation of Drosophila: a systems level approach

A fundamental question in developmental biology is how tissues are patterned to give rise to differentiated body structures with distinct morphologies. The Drosophila wing disc offers an accessible model to understand epithelial spatial patterning. It has been studied extensively using genetic and molecular approaches. Bristle patterns on the thorax, which arise from the medial part of the wing disc, are a classical model of pattern formation, dependent on a pre-pattern of trans-activators and –repressors. Despite of decades of molecular studies, we still only know a subset of the factors that determine the pre-pattern. We are applying a novel and interdisciplinary approach to predict regulatory interactions in this system. It is based on the description of expression patterns by simple logical relations (addition, subtraction, intersection and union) between simple shapes (graphical primitives). Similarities and relations between primitives have been shown to be predictive of regulatory relationships between the corresponding regulatory factors in other Systems, such as the Drosophila egg. Furthermore, they provide the basis for dynamical models of the bristle-patterning network, which enable us to make even more detailed predictions on gene regulation and expression dynamics. We have obtained a data-set of wing disc expression patterns which we are now processing to obtain average expression patterns for each gene. Through triangulation of the images we can transform the expression patterns into vectors which can easily be analysed by Standard clustering methods. These analyses will allow us to identify primitives and regulatory interactions. We expect to identify new regulatory interactions and to understand the basic Dynamics of the regulatory network responsible for thorax patterning. These results will provide us with a better understanding of the rules governing gene regulatory networks in general, and provide the basis for future studies of the evolution of the thorax-patterning network in particular.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

VARIABILITY OF PHENOTYPIC, PROTEOMIC AND TRANSCRIPTOMIC EXPRESSION OF STAPHYLOCOCCUS AUREUS SURFACE ADHESINS

Staphylococcus aureus is a highly successful pathogen responsible of a wide variety of diseases, from minor skin infection to life-threatening sepsis or infective endocarditis, as well as food poisoning and toxic shock syndrome. This heterogeneity of infections and the ability of S. aureus to develop antibiotic-resistance to virtually any available drugs reflect its extraordinary capacity to adapt and survive in a great variety of environments. The pathogenesis of S. aureus infection involves a wide range of cell wall-associated adhesins and extracellular toxins that promote host colonization and invasion. In addition, S. aureus is extremely well equipped with regulatory systems that sense environmental conditions and respond by fine tuning the expression of metabolic and virulence determinants. Surface adhesins referred to MSCRAMMs - for Microbial Surface Component Recognizing Adherence Matrix Molecules - mediate binding to the host extracellular matrix or serum components, including fibrinogen, fibronectin, collagen and elastin, and promote tissue colonization and invasion. Major MSCRAMMs include a family of surface-attached proteins covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif. Genomic analyses indicate that S. aureus contain up to 22 LPXTG surface proteins, which could potentially act individually or in synergy to promote infection. In the first part of this study we determined the range of adherence phenotypes to fibrinogen and fibronectin among 30 carriage isolates of S. aureus and compared it to the adherence phenotypes of 30 infective endocarditis and 30 blood culture isolates. Overall there were great variations in in vitro adherence, but no differences were observed between carriage and infection strains. We further determined the relation between in vitro adherence and in vivo infectivity in a rat model of experimental endocarditis, using 4 isolates that displayed either extremely low or high adherence phenotypes. Unexpectedly, no differences were observed between the in vivo infectivity of isolates that were poorly and highly adherent in vitro. We concluded that the natural variability of in vitro adherence to fibrinogen and fibronectin did not correlate with in vivo infectivity, and thus that pathogenic differences between various strains might only be expressed in in vivo conditions, but not in vitro. Therefore, considering the importance of adhesins expression for infection, direct measurement of those adhesins present on the bacterial surface were made by proteomic approach. 5 In the second series of experiments we assessed the physical presence of the LPXTG species at the staphylococcal surface, as measured at various time points during growth in different culture media. S. aureus Newman was grown in either tryptic soy broth (TSB) or in Roswell Park Memorial Institute (RPMI) culture medium, and samples were removed from early exponential growth phase to late stationary phase. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa) and clumping factor A (ClfA). Peptides of surface proteins were recovered by "trypsin-shaving" of live bacteria, and semi-quantitative proteomic analysis was performed by tandem liquid-chromatography and mass-spectrometry (LC-MS). We also determined in parallel the mRNA expression by microarrays analysis, as well as the phenotypic adherence of the bacteria to fibrinogen in vitro. The surface proteome was highly complex and contained numerous proteins theoretically not belonging to the bacterial envelope, including ribosomal proteins and metabolic enzymes. Sixteen of the 21 known LPXTG species were detected, but were differentially expressed. As expected, 9 known agr-regulated proteins (e.g. including Spa, FnBPA, ClfA, IsdA, IsdB, SasH, SasD, SasG and FmtB) increased up to the late exponential growth phase, and were abrogated in agr-negative mutants. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr negative mutant, while all other LPXTG proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in in vitro fibrinogen adherence tests during late growth (24h), whereas it remained poorly detected by proteomics. Differential expression was also detected in iron-rich TSB versus iron-poor RPMI. Proteins from the iron-regulated surface determinant (isd) system, including IsdA, IsdB and IsdH were barely expressed in iron-rich TSB, whereas they increased their expression by >10 time in iron-poor RPMI. We conclude that semi-quantitative proteomic analysis of specific protein species is feasible in S. aureus and that proteomic, transcriptomic and adherence phenotypes demonstrated differential profiles in S. aureus. Furthermore, peptide signatures released by trypsin shaving suggested differential protein domain exposures in various environments, which might be relevant for antiadhesins vaccines. A comprehensive understanding of the S. aureus physiology should integrate all these approaches.

Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.

Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

Evolving olfactory systems on the fly.

The detection of odour stimuli in the environment is universally important for primal behaviours such as feeding, mating, kin interactions and escape responses. Given the ubiquity of many airborne chemical signals and the similar organisation of animal olfactory circuits, a fundamental question in our understanding of the sense of smell is how species-specific behavioural responses to odorants can evolve. Recent comparative genomic, developmental and physiological studies are shedding light on this problem by providing insights into the genetic mechanisms that underlie anatomical and functional evolution of the olfactory system. Here we synthesise these data, with a particular focus on insect olfaction, to address how new olfactory receptors and circuits might arise and diverge, offering glimpses into how odour-evoked behaviours could adapt to an ever-changing chemosensory world.

The murine interleukin-2 receptor gamma chain gene: organization, chromosomal localization and expression in the adult thymus.

Defects in the interleukin-2 receptor gamma (IL-2R gamma) chain in the man result in an X-linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T-cell differentiation. This phenotype may result from pertubations in IL-2, IL-4-, IL-7- or IL-15-mediated signaling, as the IL-2R gamma chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL-2R gamma. The gene (Il2rg) is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well. Il2rg maps to mouse X chromosome region 40, in a region of synteny with human Xq12-13.1. We have also explored the expression of the IL-2R gamma during thymocyte development. IL-2R gamma transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL-2R gamma will allow for further studies on the regulation and function of this gene in vivo.

MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.

Gene expression often cycles between active and inactive states in eukaryotes, yielding variable or noisy gene expression in the short-term, while slow epigenetic changes may lead to silencing or variegated expression. Understanding how cells control these effects will be of paramount importance to construct biological systems with predictable behaviours. Here we find that a human matrix attachment region (MAR) genetic element controls the stability and heritability of gene expression in cell populations. Mathematical modeling indicated that the MAR controls the probability of long-term transitions between active and inactive expression, thus reducing silencing effects and increasing the reactivation of silent genes. Single-cell short-terms assays revealed persistent expression and reduced expression noise in MAR-driven genes, while stochastic burst of expression occurred without this genetic element. The MAR thus confers a more deterministic behavior to an otherwise stochastic process, providing a means towards more reliable expression of engineered genetic systems.

Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.