The Tyrosine-Autokinase UbK Is Required for Proper Cell Growth and Cell Morphology of Streptococcus pneumoniae

International audienceProtein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

Use of laser capture microdissection to generate a translational map of gene expression patterns in the liver

Animal models are widely used in toxicology studies of new chemical compounds. The metabolism of drugs occurs largely in the liver, but interspecies differences in the molecular mechanisms at the basis of drug metabolism have been widely demonstrated. These differences may limit the translatability of the results to human, and compromise the safety assessment for new compounds. Urge therefore a more in-depth understanding of the molecular mechanisms of liver for the selection of suitable animal models, which are the most representative for the human. In addition an in-depth knowledge of the cells specific transcriptome may drastically improve the identification of the target organ of toxicity. Accordingly, in this experimental work we focused our attention in generating a molecular map of liver gene expression for different animal models, taking advantage from the use of laser capture microdissection for obtaining cell-specific transcriptomes, from rat, dog and monkey liver slices. We developed LCM and immuno-LCM protocols for liver zone I, liver zone III, bile ducts, hepatic arteries and portal veins specimen collection and we profiled the transcriptomes of these samples by oligonucleotide arrays. The analysis confirmed the enrichment of specific cells/tissues and allowed the identification of known and unknown tissue-specific genes. We have then investigated the impact of sexual dimorphism on the expression of zone specific genes. We showed that sexual dimorphism in liver zonation is a phenomenon interesting only rats and we couldn’t observe any significant differentially expressed gene in other species. The cross species comparison of gene signature for liver zone I, III and bile ducts, revealed a surprising low number of common genes. However this data might be dependent from technical limitations more than from actual biological diversity of similar tissues in different species. The specificity of our gene signatures has been proved, at protein level, by IHC, resulting in all the cases valid and revealing additional species specificities unknown so far. We have applied the knowledge generated in the first phase of this project to a retrospective case study on toxic effects of Methapyrilene in rat liver. The histopathology report described minimal histological changes in treated animals, including single cell necrosis, increase of mitotic figures and bile duct hyperplasia. At transcriptomic level, the exposure to Methapyrilene induced a mild impact on the global liver gene expression, and a specific gene expression pattern of toxicity was not detectable. The analysis of differentially expressed genes, revealed 1500 up regulated genes in treated animals vs. controls and the comparison of those genes with the bile duct gene signature revealed a constant number (120-130) of common genes at all the time points. Using two markers belonging to rat bile duct signature (Cldn7 and Krt19), we were able to quantitatively evaluate the bile duct hyperplasia, by measuring the area covered by labeled cells. However the revealed differences were statistically significant at day 7 and 14, but not at day 3. The generation of a liver gene expression map and of tissue specific signatures allowed an “informed” whole liver transcriptome analysis. This uncommon approach revealed subtle drug induced molecular changes and histological alterations, otherwise difficult to identify. Interestingly, the transcriptomic profiling resulted more sensitive and statistically powerful than the histological approach. We can then conclude that, although a mathematical model describing the empirical approach used in this project is still missing, the knowledge generated by our work improved the interpretation of a retrospective case study, allowing the identification of a specific toxicity pattern. Additionally, the possibility to apply the same approach in prospective studies and to different species, represents a powerful contribution for the understanding of liver pathophysiology and DILI

Wzy-dependent bacterial capsules as potential drug targets

The bacterial capsule is a recognized virulence factor in pathogenic bacteria. It likely works as an antiphagocytic barrier by minimizing complement deposition on the bacterial surface. With the continual rise of bacterial pathogens resistant to multiple antibiotics, there is an increasing need for novel drugs. In the Wzy-dependent pathway, the biosynthesis of capsular polysaccharide (CPS) is regulated by a phosphoregulatory system, whose main components consist of bacterial-tyrosine kinases (BY-kinases) and their cognate phosphatases. The ability to regulate capsule biosynthesis has been shown to be vital for pathogenicity, because different stages of infection require a shift in capsule thickness, making the phosphoregulatory proteins suitable as drug targets. Here, we review the role of regulatory proteins focusing on Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli and discuss their suitability as targets in structure-based drug design.Daniel J. Ericsson, Alistair Standish, Bostjan Kobe, and Renato Moron

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium </it>parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of <it>Plasmodium </it>genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (<it>aaRS</it>s) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the <it>Plasmodium falciparum </it>genome to identify and classify <it>aaRSs </it>in this organism.</p> <p>Results</p> <p>Using various computational and bioinformatics tools, we have identified 37 <it>aaRS</it>s in <it>P. falciparum</it>. Our key observations are: (i) fraction of proteome dedicated to <it>aaRS</it>s in <it>P. falciparum </it>is very high compared to many other organisms; (ii) 23 out of 37 <it>Pf-aaRS </it>sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of <it>Pf-aaRSs </it>vary considerably at various life cycle stages of the parasite; (iv) several <it>PfaaRSs </it>posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite <it>aaRS</it>s to bacterial and plants <it>aaRSs</it>; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite <it>aaRSs</it>.</p> <p>Conclusion</p> <p>We have identified 37 <it>Pf-aaRSs </it>based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 <it>Pf-aaRSs </it>based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.</p

Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris

Amino acid residues that are important for metal binding and catalysis in Grampositive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains