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Better vaccines and new therapeutic drugs could be a successful breakthrough against intracellular bacteria. M. tuberculosis ABC transporter ATPase (Rv0986) plays a role in mycobacterial virulence by inhibiting phagosome-lysosome fusion. Thus, it could be a potential vaccine candidate. C. pneumoniae another important intracellular bacterium possesses a protein named CpB0255, which is homologous with the mycobacterial Rv0986. The aim of this study was the cloning, over-expression and purification of CpB0255 ABC transporter ATPase protein to study its biological properties. The immunogenicity and protective effect of recombinant chlamydial ATPase protein combined with Alum adjuvant were investigated in mice. The immunization resulted in the reduction of the number of viable C. pneumoniae in the lungs after challenge. Our results confirm that chlamydial ATPase induces protective immunity in mice.
Pathogenic yeasts from the genus Candida can cause serious infection in humans particularly, in immunocompromised patients and are now recognized as major agents of hospital acquired (nosocomial) infections. In the recent years, there has been a marked increase in the incidence of treatment failures in candidiasis patients receiving long-term antifungal therapy, which has posed a serious problem in its successful use in chemotherapy. Candida cells acquire drug resistance (MDR) during the course of the treatment. The mechanisms of resistance to azole antifungal agents have been elucidated in Candida species and can be mainly categorized as (i) changes in the cell wall or plasma membrane, which lead to impaired drug (azole) uptake; (ii) alterations in the affinity of the drug target Erg11p (lanosterol 14∝-demethylase) especially to azoles or in the cellular content of Erg11p due to target site mutation or overexpression of the ERG11 gene; and (iii) the efflux of drugs mediated by membrane transport proteins belonging to the ATP-binding cassette (ABC) transporters, namely CDR1 and CDR2 or to the major facilitator superfamily (MFS) transporter, CaMDR1 . Many such manifestations are associated with the formation of Candida biofilms including those occurring on devices like indwelling intravascular catheters. Biofilm-associated Candida show uniform resistance to a wide spectrum of antifungal drugs. A combination of different resistance mechanisms is responsible for drug resistance in clinical isolates of Candida species.
Introduction Although most of the ATP-binding cassette (ABC) transporter family members are not linked to multidrug resistance, the probably most prominent one, ABCB1 (P-gp), is mainly known for its role in therapy resistant
resistance-nodulation-division (RND) family, the small multidrug resistance (SMR) family, the ATP-binding cassette (ABC) family and the toxic compound extrusion (MATE) family, with broad substrates and also antibiotics [ 13 ]. Members of ABC transporter
The general properties of ABC transporters, from bacteria to humans, including a brief history of their initial discovery, are considered. ABC transporters, one of the largest protein super families and vital for human health, are in toto responsible for the transport of an enormous range of molecules from ions (CFTR) or anti-tumour drugs (Pgp/MDR) to large polypeptides. Nevertheless, all ABC transporters are powered by a conserved ATPase the ABC or NBD domain, using in all probability the same basic mechanism of action for the hydrolysis of ATP and its coupling to the transport process. Based on recent high resolution structures of several NBDs and an intact transporter, a model of how dimers of these important proteins function will be discussed, with particular attention to HlyB, the ABC transporter from E. coli.
Scald caused by Rhynchosporium secalis, is an economically important disease found worldwide. In order to profile genes and pathways responding to R. seclais infection, leaf transcriptomes before and after fungus inoculation in susceptible barley were compared using cDNA-AFLP technique. Transcriptional changes of 144 expressed sequence tags (ESTs) were observed, of which 18 have no previously described function. Functional annotation of the transcripts revealed a wide range of pathways including cell wall fortification, cytoskeleton construction and metabolic processes at different time points. Furthermore, the results of RT-PCR analysis on candidate genes, ABC transporters and lycine-specific demethylase were consistent with the cDNA-AFLP data in their expression patterns. Taken together, our data suggest that susceptible barley reprograms metabolic and biological processes to initiate a suitable response R. secalis infection.
In this study, we describe the membrane lipid composition of eight clinical isolates (azole resistant and sensitive strains) of Candida albicans isolated from AIDS/ HIV patients. Interestingly, fluorescence polarization measurements of the clinical isolates displayed enhanced membrane fluidity in fluconazole resistant strains as compared to the sensitive ones. The increase in fluidity was reflected in the change of membrane order, which was considerably decreased (decrease in fluorescence polarization “p” value denotes higher membrane fluidity) in the resistant strains. The ergosterol content in azole susceptible isolates was greater, almost twice as compared to the resistant isolates. However, no significant alteration was observed in phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of phosphatidylethanolamine exposed to the membrane’s outer leaflet was higher in the resistant strains as compared to the sensitive strains, indicating increased floppase activity of the two major ABC drug efflux pumps, CDR1 and CDR2 possibly due to their overexpression in resistant strains. The results of the present study suggest that changes in the status of membrane lipid phase especially the ergosterol content and increased activity of drug efflux pumps by overexpression of ABC transporters, CDR1 and CDR2 might contribute to fluconazole resistance in C. albicans isolated from AIDS/HIV patients.
The main target of the thesis was to investigate the drug resistance reversal on prokaryotic and eukaryotic model organisms. Based on DNA and protein complex formation properties of the given compounds the plasmid elimination of bacteria and the modification of the drug transporter proteins various experimental systems have been studied in bacteria and tumor cells.It was found that E. coli cells isolated from clinical specimen were less sensitive for the plasmid elimination than the laboratory strain carrying F prime plasmid, however, there was a complex formation between the antiplasmid compounds and the plasmid DNA isolated from both the clinical and laboratory strains. In addition there was a difference between the curing effect of two phenothiazines – the PMZ and TFP – on some E. coli strains in this study. The mechanism of action of different antiplasmid compounds was investigated on model nucleic acids such as calf thymus DNA and plasmid DNA. The pyrido[3,2-g] quinoline and phenothiazine derivatives seemed to have a complex formation with the model nucleic acids. Some of the compounds modified the activity of membrane efflux proteins. Based on the effect of trifluoromethyl ketones earlier studied my attention was focused on the combination of the trifluoromethyl ketone proton pump inhibitor TF18 with well-known antiplasmid compounds such as promethazine, trifluoperazine and 9-aminoacridine. In checkerboard studies the interaction between proton pump inhibitor and tricyclic compounds has been examined and it turned out that the interaction of proton pump inhibitor and trifluoperazine exerted synergistic antibacterial and plasmid curing effect on E. coli doxycycline resistant clinical strain due to the alteration of activity of membrane transporters. The role of proton pump system of the bacterial membrane was studied on Helicobacter pylori strains. The trifluoroketone proton pump inhibitor was able to block the proton motive forces and the activity of flagellar motor of both clarithromycin sensitive and resistant isolates of Helicobacter pylori . Since swimming was more sensitive to the inhibition than tumbling, I can suppose that TF18 works as an un-coupler in biological motor. The sensitivity of MDR1 type of eukaryotic ABC-transporter to resistance modifiers was studied on cancer cells. The synthetic benzo[b]-1,8-naphthyridine, pyridoquinoline, aza-oxafluorene and pregnane derivatives exerted reversing action of P-glycoprotein. Furthermore natural compounds, like coumarin derivatives and some fractions of persimmon extracts have been found to be potent resistance reversal agents against tumour cells.
in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration. Biochim. Biophys. Acta 1419 , 173–185. Ewart G. D. Mutations in the
Martinoia, E., Klein, M., Geisler, M., Bovet, L., Forestier, C., Kolukisaoglu, C., Mueller-Roeber, B., Schulz, B. 2002: Multifunctionality of plant ABC transporters more than just detoxifiers. Planta 214: 345
