Unknown,Transcriptomics,Genomics,Proteomics

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A procedure to identify peptides with -AMP activity

ABSTRACT: We showed previously that insertion of Synechocystis 12-desaturase in Salmonellas membrane alters membrane physical state (MPS) followed by the expression of stress genes causing inability to survive within murine macrophages . Recently, we showed that expression of one membrane lipid domain (MLD) of 12-desaturase (ORF200) interferes with Salmonella MPS causing loss of virulence in mice and immunoprotection. We postulated that a putative -AMP intercalates specifically within phospholipids but, depending on its amino acid sequence, does so within particular key sensors of MLD. In this study we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg2+ concentration during murine macrophages infection. We synthesized a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in Salmonella LT2 strain. We show that the pattern of gene expression controlled by PhoP/PhoQ and other pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens are dysregulated. We also present RNAseq of strain expressing ORF200 and showed that the pattern of the same genes is altered. Accumulation of NUF conferred temporary immunoprotection suggesting that it is possible to address a synthetic peptide to a specific MLD. This represents a powerful procedure to synthesize -AMPs and generate live non virulent strains for vaccination.

INSTRUMENT(S): Illumina HiSeq 1500

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

SUBMITTER: Domenico Memoli

PROVIDER: E-MTAB-4544 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.

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A procedure to identify peptides with -AMP activity

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