Au cours des dernières décennies, une augmentation non négligeable du phénomène de résistance aux antibiotiques des bactéries a été observée. Ces bactéries possèdent plusieurs mécanismes de résistance parmi lesquels l’utilisation de transporteurs de type MDR (MultiDrug Resistance) dont certains appartiennent à la famille des transporteurs ABC (ATP-Binding Cassette). Les transporteurs ABC sont des protéines membranaires et ubiquitaires qui possèdent une topologie commune avec deux domaines transmembranaires et deux domaines cytoplasmiques. Les transporteurs ABC de type exportateur permettent le transport de molécules à l’extérieur de la cellule en utilisant l’énergie fournie par l’hydrolyse de l’ATP. PatA-PatB est un transporteur ABC de Streptococcus pneumoniae, un pathogène humain responsable de pneumonies et de méningites. Cette protéine est impliquée dans la résistance de ce pathogène à des antibiotiques de types fluoroquinolones. Pour étudier son mécanisme moléculaire, nous avons optimisé la surexpression fonctionnelle de ce transporteur chez Escherichia coli. Ainsi, nous avons pu caractériser son activité de transport de drogues et son activité d’hydrolyse de nucléotides. Ces expériences ont révélé que PatA-PatB a la particularité d’utiliser préférentiellement le GTP comme source d’énergie, contrairement aux autres membres de cette famille. Afin d’identifier l’origine de cette propriété au niveau moléculaire, des expériences de mutagénèse dirigée ont été effectuées et nous avons ainsi identifié deux simples mutants qui transportent les drogues aussi bien avec du GTP que de l’ATP / The excessive use of antibiotics during the past decades led to the amplification of multidrug resistance in pathogenic bacteria. Bacteria have developed several mechanisms of antibiotic resistance. One of them involves the antibiotic efflux by MDR (MultiDrug Resistance) transporters, some of which belong to the ABC (ATP-Binding Cassette) transporter family. ABC transporter are ubiquitous membrane proteins with a conserved topology comprising four domains : two «TransMembrane Domain» and two cytoplasmic domains named « Nucleotide-Binding Domain ». ABC exporters expel drugs outside the bacteria using the energy of ATP hydrolysis. PatA-PatB is an ABC transporter from Streptococcus pneumoniae, a human pathogen bacterium responsible for pneumonia and meningitis. This protein is involved in S. pneumoniae resistance against fluoroquinolone antibiotics. To study the molecular mechanism, we optimized the functional expression of this transporter in Escherichia coli. Then, we characterized its drug transport activity and its nucleotide hydrolysis activity. These experiments showed that PatA-PatB, in contrast to other members of the ABC superfamily, preferentially uses GTP as energy supply. To identify the origin of this property at a molecular level, mutagenesis experiments were performed and we identified two mutants capable of an even drug transport with ATP and GTP

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport.

In Streptococcus pyogenes, the protein SiaA (HtsA) is part of a heme uptake pathway system and involved in heme transfer from Shp to the ABC transporter. SiaA mutants, in which alanine replaces the axial histidine (H229) and methionine (M79) ligands, as well as a lysine (K61) and cysteine (C58) located near the heme propionates, are reported. Studies on a mutant of a cysteine expected to be at a distance from the propionates (C47A) are also reported. The coordination state and spin state of the selected mutants were determined via Resonance Raman studies. The pKa values of mutants ranged from 9.0 to 9.4, which were close to the pKa of the WT SiaA (9.7). The midpoint reduction potential of lysine (K61A) mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the WT protein (68 ± 3 mV). The addition of guanidinium hydrochloride resulted in protein denaturation that could show more than one process and occurred over days. The ease of protein unfolding was directly related to the extent of interaction of the residues with the heme: changes in the axial ligands resulted in far greater changes in heme protein stability than changes in the residues near the heme propionates. The causative agent of diphtheriae, Corynebacterium diphtheriae, imports heme via an ABC uptake transporter. In this research, two of the five proteins in the heme uptake pathway of C. diphtheriae were studied. These proteins were HmuT, lipoprotein component of the ABC transporter, and HtaA, the heme receptor. UV-visible spectroscopy and fluorescence spectroscopy showed that HmuT protein as isolated bound a porphyrin, rather than heme. Electrospray ionization mass spectrometry (ESI-MS) studies suggested that two tetrapyrroles were bound. To assess stability of this protein towards heme release, thermal denaturation studies were performed. For HtaA, UV-visible and fluorescence spectroscopy also showed the protein as isolated was also bound a porphyrin, rather than heme. Homology studies showed that HtaA protein is quiet distant from homologous heme uptake proteins and could be a member of novel heme binding domain family.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.

Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

ABCD2 (D2) has been proposed as a peroxisomal long-chain acyl-CoA transporter that is essential for very long chain fatty acid metabolism. In the livers of mice, D2 is highly induced by fenofibrate, a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of dietinduced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes. Although proposed as a peroxisomal transporter, the intracellular localization of D2, especially in adipose tissue, has not been validated with direct experimental evidence. Sequential centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but lacked well-known peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with evidence of a dense matrix and a diameter of ~200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment from adipose tissue confirmed the scarcity of PEX19. Proteomic profiling of the D2 compartment revealed the presence of proteins associated peroxisome, endoplasmic reticulum (ER), and mitochondria. We conclude that D2 is localized to a distinct subclass of peroxisomes that lack many peroxisome proteins and may physically associate with mitochondria and the ER.

Zinc is a metal involved in structure of many enzymes, in the growth and differentiation of plants. Wheat is one of the most consumed cereals. Some wheat cultivars can&amp / #8217 / t deal with zinc deficiency and this situation not only reduces grain yield but also weakens the resistance of cereals to diseases and impairs the nutritional quality of the grain. Some wheat cultivars are not affected by zinc deficiency. In this study, &amp / #8216 / differential display&amp / #8217 / , used for determination differentially expressed genes between two samples, was performed. The most zinc efficient bread wheat cultivar Kira&ccedil / -66 was grown in hydroponics medium and samples were taken at different time periods. RNA isolations were done and differential display technique was performed. After examining the results, differentially expressed bands were selected and sequenced. DNA sequence analysis were done in available databases which showed that three of the bands were fragments of putative zinc transporters. In this study we have found threee putative gene fragments using differential display technique on zinc efficient plants grown under differeing zinc concentrations. These fragments showed homology with zinc transporter, ABC transporter and ADH (Alcohol Dehydrogenase). It is known that all of these three genes are involved in zinc efficiency mechanism. Further studies will be conducted on these gene fragments.

Several Fusarium species can cause Fusarium head blight (FHB) and Fusarium crown rot (FCR) diseases in wheat and these are of economic importance in wheat production systems globally. Fusarium graminearum represents a model pathogen species to study these diseases because it has a sequenced genome, commercially available gene expression arrays and an growing collection of mutants impaired in pathogenicity and virulence, at least for FHB. FCR occurs at the stem base of the wheat plant, causing major reductions in grain yield. FCR has been much less intensively researched than FHB and the infection process of F. graminearum during crown rot disease in wheat has not been studied previously at the molecular level. Fungal biomass estimations by real-time quantitative PCR analysis of DNA from inoculated plants identified three distinct phases of infection during FCR, an initial increase in fungal mass in phase 1 up to 2 days post inoculation (dpi), then a reduction during phase 2 until 14 dpi followed by a large increase thereon in phase 3 that corresponded to symptom development. Histological characterisation of F. graminearum colonisation during these three phases of infection showed that initially the spores germinated on the stem surface at the point of inoculation forming a superficial hyphal mat. This occurred within the first two days of infection. The second phase was characterised by a period of low amounts of fungal tissue present in the infected plants and 14 days following infection hyphae were only observed below the point of inoculation at the stem base of the wheat seedling and had penetrated and colonised the adaxial epidermis of the outer leaf sheath. Following this, the third phase was characterised by a major colonisation of the internal tissues of the crown which corresponded to visible symptom development around 35 days after inoculation. Fungal gene expression during all three phases of infection were examined using the Affymetrix GeneChip system comprised of 22,000 F. graminearum gene probe sets. This analysis showed 1,839 genes were significantly up regulated in planta compared to axenic vegetative mycelia, including some known FHB virulence genes (e.g. those involved in the biosynthesis of trichothecene toxins). Fungal genes differentially regulated between the phases were identified indicating that FCR disease development requires a coordinated process involving distinct fungal gene expression programs. A bioinformatic comparison of global F. graminearum gene expression during FCR of wheat with published data for FHB of barley indicated similarities at very early stages of infection but divergence thereafter. It was decided to functionally test whether F. graminearum utilises the same virulence genes in FCR and FHB diseases. Because no virulence genes have been previously identified from FCR studies a small group of genes were initially selected from the FCR gene expression studies for further functional analysis using gene knock-out technology. Only two of these genes showed a changed phenotype during Fusarium infection of wheat plants and they encoded a probable ABC transporter (FgABC1) and a probable superoxide dismutase (FgSOD1). It was interesting to note that even though both FgABC1 and FgSOD1 exhibited similar transcription profiles during both FCR of wheat and FHB of barley it was found that FgABC1 was specifically required for full FCR disease development on the wheat cultivar Kennedy whereas FgSOD1 was specifically required for FHB disease on the same cultivar. This indicated that F. graminearum virulence genes can show specificity to the infection of different plant tissues and that these types of genes cannot be predicted based only on their transcription profiles. It is suggested that F. graminearum induces a global set of virulence factors but only some of these may be effective in particular tissues. To test further whether there was tissue specialisation for specific tissues and FCR & FHB diseases, a group of F. graminearum genes that were known virulence factors during FHB were tested to see if they were also virulence factors for FCR. This analysis showed that two genes displayed specificity only for FHB and five were virulence factors for both FHB and FCR. One of the genes that was a virulence factor for both diseases was the Tri5 gene that is necessary for the biosynthesis of trichothecene mycotoxins. This gene and these toxins did not appear to be necessary for symptom development and the induction of host defence responses but were necessary for fungal colonisation of the crown and stem in later stages of infection. Interestingly there were parallels in the role played by the Tri5 gene in FCR and that reported for FHB where it is necessary for colonisation for the spike. This study is the first molecular analysis of any Fusarium species during crown rot of wheat. Importantly, it shows that there may be specialisation towards host tissues for some virulence genes but also suggests that some factors may be non-specifically required for infection and it is these factors that will represent attractive targets for future control measures of both diseases.

Universiẗat, Diss., 2006--Frankfurt (Main).