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Accueil > Départements > Biologie des Génomes > Yoshiharu YAMAICHI : Intégrité du génome et de la polarité cellulaire chez la bactérie

Publications de l’équipe

2017


  • A. Dostálová, S. Rommelaere, M. Poidevin, et B. Lemaitre, « Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps », BMC biology, vol. 15, nᵒ 1, p. 79, 2017.
    Résumé : BACKGROUND: Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq (Δ) , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. RESULTS: Survival analyses with TEPq (Δ) flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq (Δ) flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. CONCLUSIONS: Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.
    Mots-clés : Beauveria, Complement, DBG, Drosophila, Entomopathogenic fungus, EQYY, innate immunity, Insect, Parasitoid wasp, Phagocytosis.

  • M. Poidevin, E. Galli, Y. Yamaichi, et F. - X. Barre, « WGADseq: Whole Genome Affinity Determination of Protein-DNA Binding Sites », Methods in Molecular Biology (Clifton, N.J.), vol. 1624, p. 53-60, 2017.
    Résumé : We present a method through which one may monitor the relative binding affinity of a given protein to DNA motifs on the scale of a whole genome. Briefly, the protein of interest is incubated with fragmented genomic DNA and then affixed to a column. Washes with buffers containing low salt concentrations will remove nonbound DNA fragments, while stepwise washes with increasing salt concentrations will elute more specifically bound fragments. Massive sequencing is used to identify eluted DNA fragments and map them on the genome, which permits us to classify the different binding sites according to their affinity and determine corresponding consensus motifs (if any).
    Mots-clés : DBG, EMC2, EQYY, Genomics, High-throughput sequencing, Next-generation sequencing (NGS), Site-specific DNA binding protein.

  • Y. Yamaichi et T. Dörr, « Transposon Insertion Site Sequencing for Synthetic Lethal Screening », Methods in Molecular Biology (Clifton, N.J.), vol. 1624, p. 39-49, 2017.
    Résumé : Transposon insertion site sequencing (TIS) permits genome-wide, quantitative fitness assessment of individual genomic loci. In addition to the identification of essential genes in given growth conditions, TIS enables the elucidation of genetic networks such as synthetic lethal or suppressor gene combinations. Therefore, TIS becomes an exceptionally powerful tool for the high-throughput determination of genotype-phenotype relationships in bacteria. Here, we describe a protocol for the generation of high-density transposon insertion libraries and subsequent preparation of DNA samples for Illumina sequencing using the Gram-negative bacterium Vibrio cholerae as an example.
    Mots-clés : DBG, EQYY, Fitness, Genomics, High-throughput sequencing, Mariner transposon, TIS, TnSeq, Transposon mutagenesis.

2016

2015


  • Y. Yamaichi, M. C. Chao, J. Sasabe, L. Clark, B. M. Davis, N. Yamamoto, H. Mori, K. Kurokawa, et M. K. Waldor, « High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4 », Nucleic Acids Research, vol. 43, nᵒ 1, p. 348-360, 2015.
    Résumé : Horizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid's host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.
    Mots-clés : DBG, DNA Transposable Elements, EQYY, Escherichia coli, Gene Library, Gene Transfer, Horizontal, Genes, Bacterial, High-Throughput Nucleotide Sequencing, Plasmids, Sequence Analysis, DNA.
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