Project description:We present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical 6 to 8 residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases 2, 3, 6, 7 and 8, and the ectodomains of the membrane sheddases, ADAMs 10 and 17. The first library (Lib 10AA) was used to determine substrate cleavage motifs. Lib 10AA contains a highly diverse randomized 10-mer substrate peptide sequences (10^9 unique members) that was displayed mono-valently on filamentous phage and bound to magnetic beads via an N-terminal biotin. The protease was allowed to cleave the SPD beads, and the released phage subjected to up to three total rounds of positive selection followed by next generation sequencing (NGS). This allowed us to identify from 10^4 to 10^5 unique cleavage sites over a broad dynamic range of NGS counts (ranging from 3-5000), and produced consensus and optimal cleavage motifs based positional sequencing scoring matrices and state-of the-art machine learning algorithm that closely matched synthetic peptide data. A second SPD-NGS library (Lib hP) was constructed that allowed us to identify candidate human proteome sequences. Lib hP displayed virtually the entire human proteome tiled in contiguous 49AA sequences with 25AA overlaps (nearly 1 million members). After three rounds of positive selection we identified up to 10^4 natural linear cut sites depending on the protease and captured most of the examples previously identified by proteomics (ranging from 30 to 1000) and predicted 10 to 100-fold more.
Project description:The lactococcal phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages in cheese factories worldwide. It infects L. lactis MG1363, a model strain for the study of Gram-positive bacteria. The structural proteins of phage p2 have been thoroughly described. However, most of its non-structural proteins are still uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. We found this small phage protein to have a major impact on the bacterial proteome and to be important to prevent bacterial resistance to phage infection.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.
Project description:Many, if not all, bacteria use quorum sensing (QS) to control gene expression and collective behaviours, and more recently QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or “listen in” on the host’s communication processes, in order to switch between lytic and lysogenic modes of infection. In this project, we studied the interaction of Vibrio cholerae, the causative agent of cholera disease, with the lysogenic vibriophage VP882. The lytic cycle of VP882 is induced by the QS molecule DPO (3,5-dimethylpyrazin-2-ol), however, the global regulatory consequences of DPO-mediated VP882 activation have remained unclear. Using a combination of transcriptomic, genetic, and biochemical approaches, we discovered that induction of VP882 results in binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompete and down-regulate host-derived Hfq-dependent small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs and we demonstrate that one of these sRNAs, named VpdS, modulates the expression of multiple host and phage mRNAs through a base-pairing mechanism and thereby promotes phage replication. We further show that host-derived sRNAs can affect phage replication by interfering with the translation of phage mRNAs and thus might be part of the phage defence arsenal of the host. Taken together, our data draw a complex picture of post-transcriptional interactions occurring between host- and phage-derived transcripts that together determine the phage-mediated lysis program.
Project description:Genomic material isolated from purified phage YerA41 lysate was shown to contain RNA. YerA41 phage lysate was RNase treated to remove phage-external RNA and total RNA was then isolated from the phage preparate using Qiagen Rneasy mini kit. The isolated RNA was sequenced to elucidate its origin. The results suggested that the RNA originated from intact ribosomes of the host bacterium that contaminated the phage lysate.
Project description:hvKP ATCC43816 and its lytic phage H5 were employed as a phage-antibiotic combination model. Based on the comprehensive characterization of phages, including cryo-electron microscopy, we evaluated the synergic effect of H5 on bacterial killing in vitro when combined with multiple antibiotics, and analyzed the advantages of phage-antibiotic combinations from an evolutionary perspective and proposes a novel PAS mechanism by using ceftazidime as an example.