A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

In this study, proteomics and metabolomics were used to study the wheat response to exposure to the SnToxA effector protein secreted by the fungal pathogen Stagonospora nodorum during infection. Ninety-one different acidic and basic proteins and 101 metabolites were differentially abundant when comparing SnToxA- and control-treated wheat leaves during a 72-h time course. Proteins involved in photosynthesis were observed to increase marginally initially after exposure, before decreasing rapidly and significantly. Proteins and metabolites associated with the detoxification of reactive oxygen species in the chloroplast were also differentially abundant during SnToxA exposure, implying that the disruption of photosynthesis causes the rapid accumulation of chloroplastic reactive oxygen species. Metabolite profiling revealed major metabolic perturbations in central carbon metabolism, evidenced by significant increases in tricarboxylic acid (TCA) cycle intermediates, suggestive of an attempt by the plant to generate ATP and reducing equivalents in response to the collapse of photosynthesis caused by SnToxA. This was supported by the observation that the TCA cycle enzyme malate dehydrogenase was up-regulated in response to SnToxA. The infiltration of SnToxA also resulted in a significant increase in abundance of many pathogenicity-related proteins, even in the absence of the pathogen or other pathogen-associated molecular patterns. This approach highlights the complementary nature of proteomics and metabolomics in studying effector-host interactions, and provides further support for the hypothesis that necrotrophic pathogens, such as S. nodorum, appear to exploit existing host cell death mechanisms to promote pathogen growth and cause disease.     

Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of Dictyostelium discoideum

Uridine diphosphoglucose pyrophosphorylase (UTP: -alpha-D-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175-187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997-5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.     

Impact of biased sampling effort and spatial uncertainty of locations on models of plant invasion patterns in Croatia

Very frequently biological databases are used for analysing distribution of different taxa. These databases are usually the result of variable sampling effort and location uncertainty. The aim of this study was to test the influence of geographically biased sampling effort and spatial uncertainty of locations on models of species richness. For this purpose, we assessed the pattern of invasive alien plants in Croatia using the Flora Croatica Database. The procedure applied in testing of the sensitivity of models consisted of sample area sectioning into coherent ecological classes (hereinafter Gower classes). The quadrants were then ranked based on sampling effort per class. This resulted in creation of models using varying numbers of quadrants whose performance was tested with independent validation points. From this the best fitting model was determined, as well as a threshold of sampling effort. The data from quadrants with sampling effort below the threshold were considered too unreliable for modelling. Further, spatial uncertainty was simulated by adding a random term to each location and re-running the models using the simulated locations. Biased sampling effort and spatial uncertainty of locations had similar effects on model performance in terms of the magnitude of the affected area, as in both cases 7% of the quadrants showed statistically significant deviations in alien plant species richness. The model using only on the quandrants with the highest 35% quantile sampling effort best balanced the sampling effort per quadrant and overall geographical coverage. It predicted a mean number of 3.2 invasive alien plant species per quadrant for the Alpine region, 5.2 for the Continental, 6.1 for the Mediterranean and 5.3 for the Pannonian region of Croatia. Thus, the observational databases can be considered as a reliable source for species richness models and, most likely, for other types of species distribution models, given that their limitations are accounted for in the data selection process. In order to obtain precise estimates of species richness it is required to sample the whole range of ecological conditions of the study area.     

The development of Escherichia coli and Listeria monocytogenes variants resistant to high‐pressure carbon dioxide inactivation

Aims: The objective of this study was to investigate whether bacterial cells could develop resistance (as a part of their adaptation strategy) to high‐pressure CO₂ (HPCD) inactivation. Methods and Results: Alternating cycles of exposure to pressurized CO₂ (10·5 MPa, 35°C, 400 min^?1, 70% working volume ratio during 10 min) and re‐growth of the surviving subpopulation were used to investigate possible increases in the resistance of Escherichia coli and Listeria monocytogenes to HPCD. The results show an increased resistance of both pathogens tested after seven cycles of inactivation. Increase in the resistance after 15 cycles resulted in a difference of 2·4 log CFU ml^?1 in log N₀/N_i when parental (N₀) and treated cultures (N_i) of E. coli and L. monocytogenes were compared. Conclusions: Current findings indicate the ability of micro‐organisms to adapt to HPCD preservation technology. Significance and Impact of the Study: The occurrence of HPCD‐resistant micro‐organisms could pose a new hazard to the safety and stability of HPCD‐processed foods.     

Structure-guided design,synthesis and biological evaluation of novel DNA ligase inhibitors with in vitro and in vivo anti-staphylococcal activity

A series of 2-amino-[1,8]-naphthyridine-3-carboxamides (ANCs) with potent inhibition of bacterial NAD⁺-dependent DNA ligases (LigAs) evolved from a 2,4-diaminopteridine derivative discovered by HTS. The design was guided by several highly resolved X-ray structures of our inhibitors in complex with either Streptococcus pneumoniae or Escherichia coli LigA. The structure–activity-relationship based on the ANC scaffold is discussed. The in-depth characterization of 2-amino-6-bromo-7-(trifluoromethyl)-[1,8]-naphthyridine-3-carboxamide, which displayed promising in vitro (MIC Staphylococcus aureus 1 mg/L) and in vivo anti-staphylococcal activity, is presented.     

Discovery of novel benzene 1,3-dicarboxylic acid inhibitors of bacterial MurD and MurE ligases by structure-based virtual screening approach

The peptidoglycan biosynthetic pathway provides an array of potential targets for antibacterial drug design, attractive especially with respect to selective toxicity. Within this pathway, the members of the Mur ligase family are considered as promising emerging targets for novel antibacterial drug design. Based on the available MurD crystal structures co-crystallised with N-sulfonyl glutamic acid inhibitors, a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates—benzene 1,3-dicarboxylic acid derivatives—were identified and compounds 14 and 16 found to possess dual MurD and MurE inhibitory activity.     

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add l-Ala, d-Glu, meso-A₂pm or l-Lys, and d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC₅₀ = 10 μM) and one novel MurF inhibitor (IC₅₀ = 63 μM).