High-throughput discovery of human antimicrobial peptide activators of Salmonella Typhimurium PhoPQ
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ABSTRACT:
ORGANISM(S): Escherichia coli K-12
PROVIDER: GSE174191 | GEO | 2022/11/09
REPOSITORIES: GEO
Similar Datasets
Project description:The cross compatibility of functional PhoQ-PhoP mutants with altered specificity residues was analyzed by sort seq. We cloned and combinatorially combined 79 PhoQ* and 71 PhoP* variants, producing a library with a theoretical diversity of 5,609. The library was 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 (Fig. 2b). 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.
2019-07-27 | GSE120786 | GEO
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.
2019-09-20 | GSE120780 | GEO
Project description:The Yersinia pestis PhoPQ gene regulatory system is induced during infection of the flea digestive tract and is required to produce adherent biofilm in the foregut, which greatly enhances bacterial transmission during a flea bite. To understand the in vivo context of PhoPQ induction and to determine PhoP-regulated targets in the flea, we undertook whole genome comparative transcriptional profiling of Y. pestis wild-type and ΔphoP strains isolated from infected fleas and from temperature-matched in vitro planktonic and flowcell biofilm cultures. In the absence of PhoP regulation, the gene expression program indicated that the bacteria experience diverse physiological stresses and are in a metabolically less active state. Multiple stress response genes, including several toxin-antitoxin loci and YhcN family genes responsible for increased acid tolerance, were upregulated in the phoP mutant during flea infection. The data imply that PhoPQ is induced by low pH in the flea gut, and that PhoP modulates physiologic adaptation to acid and other stresses encountered during infection of the flea. This adaptive response, together with PhoP-dependent modification of the bacterial outer surface that includes repression of pH 6 antigen fimbriae, supports stable biofilm development in the flea foregut.
2017-02-09 | GSE61558 | GEO
Project description: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.
2016-07-15 | E-MTAB-4544 | biostudies-arrayexpress
Project description:To test the global insulation of selected PhoQ*-PhoP* variants, we used RNA-Seq to examine gene expression in strains carrying one of six different PhoQ-PhoP variant pairs. In each case, cells were grown in medium with either excess or limiting extracellular Mg2+ to repress or stimulate PhoQ, respectively, before harvesting RNA. Each system produced a similar induction of known PhoP-dependent genes. To assess whether a variant PhoQ cross-phosphorylated other response regulators, we took advantage of the fact that, when active, most response regulators autoregulate and promote expression of themselves and their cognate histidine kinase2. Notably, none of the six strains tested showed significant induction of other two-component systems relative to a wild-type control. Similar results are seen for the chimeric pathway AQ4-PhoP4, which was induced with its ligand: trans-zeatin.
2019-09-20 | GSE128611 | GEO
Project description:To survive during colonization or infection of the human body, microorganisms must defeat antimicrobial peptides, which represent a key component of innate host defense in phagocytes and on epithelia. However, is not known how the clinically important group of Gram-positive bacteria sense antimicrobial peptides to coordinate a directed defensive response. By determining the genome-wide gene regulatory response to human beta defensin 3 in the nosocomial pathogen Staphylococcus epidermidis, we discovered an antimicrobial peptide sensor system that controls major specific resistance mechanisms to antimicrobial peptides and is unrelated to the Gram-negative PhoP/PhoQ system. Keywords: Wild type control vs treated vs mutant
2008-02-26 | GSE7163 | GEO
Project description:To survive during colonization or infection of the human body, microorganisms must defeat antimicrobial peptides, which represent a key component of innate host defense in phagocytes and on epithelia. However, is not known how the clinically important group of Gram-positive bacteria sense antimicrobial peptides to coordinate a directed defensive response. By determining the genome-wide gene regulatory response to human beta defensin 3 in the nosocomial pathogen Staphylococcus epidermidis, we discovered an antimicrobial peptide sensor system that controls major specific resistance mechanisms to antimicrobial peptides and is unrelated to the Gram-negative PhoP/PhoQ system. Wild type untreated in triplicate is compared to wild type treated in triplicate along with three mutants in triplicate with and without treatment of human beta defensin 3, totalling 30 samples
2010-05-26 | E-GEOD-7163 | biostudies-arrayexpress
Project description:In bacteria, two-component regulatory system (TCSs) is generally characterized by a simple phosphotransfer scheme, composed of a sensor domain (a histidine kinase) and a response domain (a response regulator), which is responsible for detection of external stimuli. PhoP-PhoQ TCS of Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight disease in rice, was previously shown to be negatively regulated by RaxR-RaxH, another TCS that senses population cell density as well as modulates the activity of AvrXA21, a bacterial effector, recognized by a bacterial blight resistance gene, Xa21. In this work, whole-genome microarray was performed to analyze transcription profiling and identify member of PhoP regulon under Mg2+ and Ca2+ limited condition. This analysis revealed that PhoP governs broad cellular pathways including stress defense response, cation transportation, general metabolism, broad regulatory system, bacterial motility, and bacterial virulence. Array results provide a set of candidate genes, further biochemical pathway analysis, and signaling pathway crosstalks that need to be characterized to understand PhoP-dependent mechanisms and also suggest a putative regulatory loop between two phoP-phoQ and raxR-raxH TCSs. Implication of this analysis suggested that Xoo adopt PhoP-PhoQ system to perceive extracellular signals from certain environment such as low concentration of metal ions, and to regulate intracellular signals of other regulatory systems. Keywords: Comparative transcription profiling under limited Mg2+ Ca2+ condition
2008-07-16 | GSE12099 | GEO
Project description:In bacteria, two-component regulatory system (TCSs) is generally characterized by a simple phosphotransfer scheme, composed of a sensor domain (a histidine kinase) and a response domain (a response regulator), which is responsible for detection of external stimuli. PhoP-PhoQ TCS of Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight disease in rice, was previously shown to be negatively regulated by RaxR-RaxH, another TCS that senses population cell density as well as modulates the activity of AvrXA21, a bacterial effector, recognized by a bacterial blight resistance gene, Xa21. In this work, whole-genome microarray was performed to analyze transcription profiling and identify member of PhoP regulon under Mg2+ and Ca2+ limited condition. This analysis revealed that PhoP governs broad cellular pathways including stress defense response, cation transportation, general metabolism, broad regulatory system, bacterial motility, and bacterial virulence. Array results provide a set of candidate genes, further biochemical pathway analysis, and signaling pathway crosstalks that need to be characterized to understand PhoP-dependent mechanisms and also suggest a putative regulatory loop between two phoP-phoQ and raxR-raxH TCSs. Implication of this analysis suggested that Xoo adopt PhoP-PhoQ system to perceive extracellular signals from certain environment such as low concentration of metal ions, and to regulate intracellular signals of other regulatory systems. Keywords: Comparative transcription profiling under limited Mg2+ Ca2+ condition Three biological and dye-swap replicates, total six samples for each dataset. Three datasets contained; (i) phoP knockout mutant vs. wildtype PXO99A in low concentration (10 µM) of MgCl2 and CaCl2, (ii) phoP knockout mutant in low (10 µM) vs. high (10mM) concentration of MgCl2 and CaCl2, and (iii) PXO99A in low (10 µM) vs. high (10mM) concentration of MgCl2 and CaCl2.
2008-07-16 | E-GEOD-12099 | biostudies-arrayexpress
Project description:The clinical development of antimicrobial peptides (AMPs) is currently under evaluation to combat the rapid increase in multi-drug resistant bacterial pathogens. However, many AMPs closely resemble components of the human innate immune system, and the ramifications of prolonged bacterial exposure to AMPs are not fully understood. Here we show that in vitro serial passage of a clinical USA300 methicillin-resistant Staphylococcus aureus strain in a host-mimicking environment containing host-derived AMPs results in the selection of stable AMP-resistance. AMP-resistant S. aureus mutants often displayed little to no fitness cost and caused invasive disease in mice. Further, this phenotype coincided with diminished susceptibility to both clinically prescribed antibiotics and human defense peptides. These findings suggest that therapeutic use of AMPs could select for virulent mutants with cross-resistance to human innate immunity as well as antibiotic therapy. Thus, therapeutic use of AMPs and the implications of cross-resistance need to be carefully monitored and evaluated.
2016-09-26 | PXD004036 | Pride