Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis – a rich resource to identify new transcripts, proteins and to study gene regulation

Background: Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. Results: A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file. Conclusions: The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes

Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs

Establishing the precise evolutionary history of a gene improves prediction of disease-causing missense mutations

PURPOSE: Predicting the phenotypic effects of mutations has become an important application in clinical genetic diagnostics. Computational tools evaluate the behavior of the variant over evolutionary time and assume that variations seen during the course of evolution are probably benign in humans. However, current tools do not take into account orthologous/paralogous relationships. Paralogs have dramatically different roles in Mendelian diseases. For example, whereas inactivating mutations in the NPC1 gene cause the neurodegenerative disorder Niemann-Pick C, inactivating mutations in its paralog NPC1L1 are not disease-causing and, moreover, are implicated in protection from coronary heart disease. METHODS: We identified major events in NPC1 evolution and revealed and compared orthologs and paralogs of the human NPC1 gene through phylogenetic and protein sequence analyses. We predicted whether an amino acid substitution affects protein function by reducing the organism’s fitness. RESULTS: Removing the paralogs and distant homologs improved the overall performance of categorizing disease-causing and benign amino acid substitutions. CONCLUSION: The results show that a thorough evolutionary analysis followed by identification of orthologs improves the accuracy in predicting disease-causing missense mutations. We anticipate that this approach will be used as a reference in the interpretation of variants in other genetic diseases as well. Genet Med 18 10, 1029–1036

A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.We analyzed the immunological consequences of the interaction of MHC class II (I-Au) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4+ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation

Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome

The results presented in this paper have not been published previously in whole or in part. The work reported in this paper was supported by grants from the BBSRC awarded to M.R.E (BB/M001644/1) and J.H.S. (BB/M001032/1). L.A.Y.G is supported by a PhD studentship awarded by the Mexican Council of Science and Technology (CONACyT studentship no. 418612) and Queen Mary University of London. We are grateful to Philipp Bauknecht and Gáspár Jékely (Max Planck Institute for Developmental Biology, Tübingen, Germany) for providing the Gα16 plasmid and the CHO-G5A cells, which were originally generated by Baubet et al. (Proc Natl Acad Sci USA 97:7260–7265). We are also grateful to Phil Edwards for his help with collecting starfish, Paul Fletcher for maintaining our seawater aquarium and Maria Eugenia Guerra for creating the silhouettes of animals used in Figure 7

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

International audienceHuman MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K. The structure of MUC1-9 in complex with H-2K has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide's central region, partially characterized as a -turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics

Interaction Pattern of Arg 62 in the A-Pocket of Differentially Disease-Associated HLA-B27 Subtypes Suggests Distinct TCR Binding Modes

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype