1-Deoxy-d-xylulose 5-phosphate reductoisomerase as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterization and biological evaluation of inhibitors.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Šresolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

New insights into the resistance mechanism for the BceAB-type transporter SaNsrFP.

Treatment of bacterial infections is one of the major challenges of our time due to the evolved resistance mechanisms of pathogens against antibiotics. To circumvent this problem, it is necessary to understand the mode of action of the drug and the mechanism of resistance of the pathogen. One of the most potent antibiotic targets is peptidoglycan (PGN) biosynthesis, as this is an exclusively occurring and critical feature of bacteria. Lipid II is an essential PGN precursor synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into nascent PGN. Antimicrobial peptides (AMPs), such as nisin and colistin, targeting PGN synthesis are considered promising weapons against multidrug-resistant bacteria. However, human pathogenic bacteria that were also resistant to these compounds evolved by the expression of an ATP-binding cassette transporter of the bacitracin efflux (BceAB) type localized in the membrane. In the human pathogen Streptococcus agalactiae, the BceAB transporter SaNsrFP is known to confer resistance to the antimicrobial peptide nisin. The exact mechanism of action for SaNsrFP is poorly understood. For a detailed characterization of the resistance mechanism, we heterologously expressed SaNsrFP in Lactococcus lactis. We demonstrated that SaNsrFP conferred resistance not only to nisin but also to a structurally diverse group of antimicrobial PGN-targeting compounds such as ramoplanin, lysobactin, or bacitracin/(Zn)-bacitracin. Growth experiments revealed that SaNsrFP-producing cells exhibited normal behavior when treated with nisin and/or bacitracin, in contrast to the nonproducing cells, for which growth was significantly reduced. We further detected the accumulation of PGN precursors in the cytoplasm after treating the cells with bacitracin. This did not appear when SaNsrFP was produced. Whole-cell proteomic protein experiments verified that the presence of SaNsrFP in L. lactis resulted in higher production of several proteins associated with cell wall modification. These included, for example, the N-acetylmuramic acid-6-phosphate etherase MurQ and UDP-glucose 4-epimerase. Analysis of components of the cell wall of SaNsrFP-producing cells implied that the transporter is involved in cell wall modification. Since we used an ATP-deficient mutant of the transporter as a comparison, we can show that SaNsrFP and its inactive mutant do not show the same phenotype, albeit expressed at similar levels, which demonstrates the ATP dependency of the mediated resistance processes. Taken together, our data agree to a target protection mechanism and imply a direct involvement of SaNsrFP in resistance by shielding the membrane-localized target of these antimicrobial peptides, resulting in modification of the cell wall.

Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H.

Lantibiotics are a growing class of antimicrobial peptides, which possess antimicrobial activity against mainly Gram-positive bacteria including the highly resistant strains such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci. In the last decades numerous lantibiotics were discovered in natural habitats or designed with bioengineering tools. In this study, we present an insight in the antimicrobial potential of the natural occurring lantibiotic nisin H from Streptococcus hyointestinalis as well as the variant nisin H F1I. We determined the yield of the heterologously expressed peptide and quantified the cleavage efficiency employing the nisin protease NisP. Furthermore, we analyzed the effect on the modification via mass spectrometry analysis. With standardized growth inhibition assays we benchmarked the activity of pure nisin H and the variant nisin H F1I, and their influence on the activity of the nisin immunity proteins NisI and NisFEG from Lactococcus lactis and the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1. We further checked the antibacterial activity against clinical isolates of Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis via microdilution method. In summary, nisin H and the nisin H F1I variant possessed better antimicrobial potency than the natural nisin A.

Characterization of the nucleotide-binding domain NsrF from the BceAB-type ABC-transporter NsrFP from the human pathogen Streptococcus agalactiae.

Treatment of bacterial infections is a great challenge of our era due to the various resistance mechanisms against antibiotics. Antimicrobial peptides are considered to be potential novel compound as antibiotic treatment. However, some bacteria, especially many human pathogens, are inherently resistant to these compounds, due to the expression of BceAB-type ABC transporters. This rather new transporter family is not very well studied. Here, we report the first full characterization of the nucleotide binding domain of a BceAB type transporter from Streptococcus agalactiae, namely SaNsrF of the transporter SaNsrFP, which confers resistance against nisin and gallidermin. We determined the NTP hydrolysis kinetics and used molecular modeling and simulations in combination with small angle X-ray scattering to obtain structural models of the SaNsrF monomer and dimer. The fact that the SaNsrFH202A variant displayed no ATPase activity was rationalized in terms of changes of the structural dynamics of the dimeric interface. Kinetic data show a clear preference for ATP as a substrate, and the prediction of binding modes allowed us to explain this selectivity over other NTPs.

EZH2 Loss Drives Resistance to Carboplatin and Paclitaxel in Serous Ovarian Cancers Expressing ATM.

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations.