In Rhizobium leguminosarum the ABC transporter responsible for rhamnose transport is dependent on RhaK, a sugar kinase that is necessary for the catabolism of rhamnose. It was hypothesized that RhaK has two separate functions; phosphorylation of rhamnose, and an unknown interaction with the rhamnose ABC transporter. To address this hypothesis a linker-scanning mutagenesis of rhaK was carried out. Two generated variants (RhaK72 and RhaK73) were found to maintain kinase activity, but were severely impaired in rhamnose transport function. Structural modelling suggested that both RhaK72 and RhaK73 affect surface exposed residues in two distinct regions localized to one face of the protein. This suggests that this proteins face may play a role in a protein-protein interaction that affects rhamnose transport. Using a two-hybrid system, an N-terminal and a C-teminal fragment of RhaK were both shown to interact with the N-terminal fragment of RhaT. These fragments span the regions that contain the rhaK73 and rhaK72 inserts respectively. When the rhaK72 and rhaK73 insert alleles were cloned and assayed using the two-hybrid system, these they were unable to interact with the RhaT fragment, suggesting these inserts abolish transport by interfering with a physical interaction between RhaT and RhaK. A phylogeny was generated based on the amino acid sequence of RhaK like proteins found in syntenous opereons. To gain insight into what residues may constitute a binding domain a PRALINE alignment of the orthologous kinases was combined with secondary structure analysis, known informative mutations, and functional residue predictions. A putative 12 amino acid binding site was identified using this method. An alanine scanning mutagenesis and subsequent two-hybrid analysis was carried out on this region. The substitution of any of these residues greatly affected the interaction between RhaT and RhaK. Although heterologous complementation of RhaK is possible, cosmid complementation anomalies and phylogenetic analysis of RhaK indicates the R. leguminosarum and S. meliloti kinases are different. Through a series of heterologous complementation experiments, enzyme assays, gene fusions, and transport experiments we show that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the Sinorhizobium meliloti kinase does not have rhamnose kinase activity. / May 2015

Göttingen, Univ., Diss., 2004. / Computerdatei im Fernzugriff.

Göttingen, Universiẗat, Diss., 2004.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection.

Universiẗat, Diss.--Heidelberg, 2006. / Zsfassung in dt. u. engl. Sprache.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection.

Campylobacter jejuni is a human pathogen that causes severe diarrhea! disease. However, our understanding of C. jejuni virulence mechanisms and survival during disease and transmission remains limited. Amino acid ATP Binding Cassette (AA-ABC) transporters in C. jejuni have been proposed as being important for bacterial physiology and pathogenesis. We have investigated a novel AA-ABC transporter system, encoded by cj0467-9, by generating targeted deletions of cj0467 (membrane transport component) and cj0469 (ATPase component) in C. jejuni 81-176. Analyses described herein have led us to designate these genes paqP and paqQ, respectively [pathogenesis-ssociated glutamine (q) ABC transporter permease () and ATPase (Q)]. We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Both ΔpaqP and ΔpaqQ mutants also exhibited a surprising but significant increase in short-term intracellular survival in macrophages and epithelial cells. Levels of resistance to a series of environmental and in vivo stresses were examined. Both mutants were hyper-resistant to aerobic and oxidative stress, and while ΔpaqP was also hyper-resistant to heat and osmotic shock, ΔpaqQ was more susceptible than wild-type to the latter two stresses. Annexin-V staining coupled with fluorescence microscopy revealed that macrophages infected with the ΔpaqP and ΔpaqQ mutants underwent a lower level of apoptosis than cells infected with wild-type bacteria. Macrophages infected with the mutant strains exhibited a transient decrease in ERK activation compared to wild type-infected macrophages, potentially explaining the reduced apoptosis phenotype. The ΔpaqP mutant did not exhibit a defect for short or longer term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation between an AA-ABC transporter with bacterial stress tolerance, intracellular survival, host cell damage, and host signal transduction in response to pathogen infection. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Cancer remains the most cause death disease in Taiwan at least ten years. Liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the most common cause of cancer mortality in men and the second most in women. Not only liver section and liver transplantation are used in HCC therapy but also local ablation therapy and transarterial therapy. Transarterial chemoembolization (TACE) is one of the local therapies that inject chemotherapeutic drugs directly into liver tumor. However, drug resistance is the mainly restriction in patient after chemotherapy. Moreover, it is known that drug resistance was associated to over-expression of certain ABC transporter genes, especially ABCB1, ABCG2, and ABCC family in cancer cell and those ABC transporters were also expressed in liver. Base on clinical study, they use 5-fluororuacil, cisplatin and mitomycin-C for liver cancer treatment. In this study, we hypothesized that cancer therapies may be augmented through blocked the drug efflux ABC channels with the ABC transporter inhibitors such as verapamil. The associations among drug treatments, inhibitor incorporation and the expression of ABC transporters were evaluated in HepG2 and Hep3B cells. MTT assay demonstrated that the cell viability was considerable decreased by treating triple drugs with verapamil. RT-PCR data showed that ABC transporters mRNA expression has no significantly change. However, membrane ABCB1 and ABCG2 were induced after drugs and inhibitors treatment either 1 or 24 hours by flow cytometry analysis. P-glycoprotein functional assay also showed p-glycoprotein was inhibited by verapamil, and hence Rhodamine 123 retention was increased. Taken together, there are different response of ABC transporters in HepG2 and Hep3B after drugs and inhibitors treatment. Membrane ABCB1 and ABCG2 were induced by drugs and inhibitors treatment. However, p-glycoprotein¡¦s function was restrained simultaneously by inhibitors treatment. Therefore, verapamil can enhance cell death by inhibiting ABC transporters and its cytotoxic effect rather than the increased expression of ABC transporters. This finding might provide a better way in liver cancer therapy.

University, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

Regensburg, Univ., Diss., 2009.