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• W2275361191 abstract "Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs. Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs. Natural antimicrobial peptides (AMPs) 1The abbreviations used are:AMPsAntimicrobial peptidesBac 7Bactenecin 7Lfcin BLactoferricin BP-DerHybrid of pleurocidin and dermaseptinPR-39Proline-arginine-rice peptide. are an evolutionarily conserved defense system of organisms against invading microorganisms. AMPs are effective against a wide range of microorganisms including bacteria, fungi, parasites, and some viruses (1.Zasloff M. Antimicrobial peptides of multicellular organisms.Nature. 2002; 415: 389-395Crossref PubMed Scopus (6744) Google Scholar). In general, AMPs are cationic peptides with fewer than 50 amino acid residues. To date, two main mechanisms of action have been identified for AMPs acting as antimicrobial agents (2.Nicolas P. Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides.FEBS J. 2009; 276: 6483-6496Crossref PubMed Scopus (271) Google Scholar): (1) membrane integrity disruption and (2) intracellular activity inhibition. Although the basic properties of all AMPs are similar, each AMP has a unique structure and microbial intracellular activity inhibition mechanism. For example, indolicidin inhibits DNA synthesis (3.Hsu C.H. Chen C. Jou M.L. Lee A.Y. Lin Y.C. Yu Y.P. Huang W.T. Wu S.H. Structural and DNA-binding studies on the bovine antimicrobial peptide, indolicidin: evidence for multiple conformations involved in binding to membranes and DNA.Nucleic Acids Res. 2005; 33: 4053-4064Crossref PubMed Scopus (230) Google Scholar), whereas buforin II binds to DNA and RNA (4.Park C.B. Kim H.S. Kim S.C. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions.Biochem. Biophys. Res. Commun. 1998; 244: 253-257Crossref PubMed Scopus (689) Google Scholar), mersacidin (a lantibiotic) binds to Lipid II and blocks peptidoglycan metabolism (5.Brotz H. Bierbaum G. Leopold K. Reynolds P.E. Sahl H.G. The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II.Antimicrob. Agents Chemother. 1998; 42: 154-160Crossref PubMed Google Scholar), tachyplesin I binds to the minor groove of DNA duplexes (6.Yonezawa A. Kuwahara J. Fujii N. Sugiura Y. Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action.Biochemistry. 1992; 31: 2998-3004Crossref PubMed Scopus (124) Google Scholar), microcin B17 inhibits DNA gyrase (thus influencing DNA replication) (7.Parks W.M. Bottrill A.R. Pierrat O.A. Durrant M.C. Maxwell A. The action of the bacterial toxin, microcin B17, on DNA gyrase.Biochimie. 2007; 89: 500-507Crossref PubMed Scopus (53) Google Scholar, 8.Heddle J.G. Blance S.J. Zamble D.B. Hollfelder F. Miller D.A. Wentzell L.M. Walsh C.T. Maxwell A. The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition.J. Mol. Biol. 2001; 307: 1223-1234Crossref PubMed Scopus (107) Google Scholar), microcin J25 recognizes the secondary channel of RNA polymerase (inhibiting transcription) (9.Mukhopadhyay J. Sineva E. Knight J. Levy R.M. Ebright R.H. Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel.Mol Cell. 2004; 14: 739-751Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and pyrrhocoricin inhibits the biological function of the heat shock protein, DnaK (10.Kragol G. Lovas S. Varadi G. Condie B.A. Hoffmann R. Otvos Jr., L. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.Biochemistry. 2001; 40: 3016-3026Crossref PubMed Scopus (377) Google Scholar). However, because no systematic study has been reported, the intracellular targets of numerous AMPs, such as bactenecin 7 (Bac7), hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39), remain unclear (11.Podda E. Benincasa M. Pacor S. Micali F. Mattiuzzo M. Gennaro R. Scocchi M. Dual mode of action of Bac7, a proline-rich antibacterial peptide.Biochim. Biophys. Acta. 2006; 1760: 1732-1740Crossref PubMed Scopus (112) Google Scholar, 12.Boman H.G. Agerberth B. Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine.Infect. Immun. 1993; 61: 2978-2984Crossref PubMed Google Scholar, 13.Patrzykat A. Friedrich C.L. Zhang L. Mendoza V. Hancock R.E. Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli.Antimicrob. Agents Chemother. 2002; 46: 605-614Crossref PubMed Scopus (275) Google Scholar). The N-terminal fragment of Bac7 (1–35) inhibits bacterial growth through a nonlytic mechanism at low concentrations (11.Podda E. Benincasa M. Pacor S. Micali F. Mattiuzzo M. Gennaro R. Scocchi M. Dual mode of action of Bac7, a proline-rich antibacterial peptide.Biochim. Biophys. Acta. 2006; 1760: 1732-1740Crossref PubMed Scopus (112) Google Scholar), and P-Der inhibits macromolecular synthesis in bacteria at the lowest inhibition concentration (13.Patrzykat A. Friedrich C.L. Zhang L. Mendoza V. Hancock R.E. Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli.Antimicrob. Agents Chemother. 2002; 46: 605-614Crossref PubMed Scopus (275) Google Scholar). PR-39 inhibits protein and DNA synthesis without cell membrane lysis (12.Boman H.G. Agerberth B. Boman A. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine.Infect. Immun. 1993; 61: 2978-2984Crossref PubMed Google Scholar). These reports suggest that Bac7, P-Der, and PR-39 have intracellular activities against microorganisms. However, the exact mechanism of action and the intracellular targets remain unclear. Thus, we used an Escherichia coli proteome microarray to systematically identify the protein targets. The E. coli proteome microarray contained the entire proteome of E. coli K12 and provided a high-throughput rapid platform for protein interactome identification (14.Chen C.S. Korobkova E. Chen H. Zhu J. Jian X. Tao S.C. He C. Zhu H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.Na.t Methods. 2008; 5: 69-74Crossref PubMed Scopus (101) Google Scholar). The E. coli proteome microarray has been used in many studies such as research on protein-DNA (14.Chen C.S. Korobkova E. Chen H. Zhu J. Jian X. Tao S.C. He C. Zhu H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.Na.t Methods. 2008; 5: 69-74Crossref PubMed Scopus (101) Google Scholar) and protein-peptide interaction (15.Ho Y.H. Sung T.C. Chen C.S. Lactoferricin B inhibits the phosphorylation of the two-component system response regulators BasR and CreB.Mol. Cell. Proteomics. 2012; 11 (M111.014720)Abstract Full Text Full Text PDF Scopus (39) Google Scholar, 16.Tu Y.H. Ho Y.H. Chuang Y.C. Chen P.C. Chen C.S. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.PLoS ONE. 2011; 6: e28197Crossref PubMed Scopus (34) Google Scholar) and biomarker identification (17.Chen C.S. Sullivan S. Anderson T. Tan A.C. Alex P.J. Brant S.R. Cuffari C. Bayless T.M. Talor M.V. Burek C.L. Wang H. Li R. Datta L.W. Wu Y. Winslow R.L. Zhu H. Li X. Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip.Mol. Cell. Proteomics. 2009; 8: 1765-1776Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We previously applied the E. coli proteome microarray as a high-throughput platform to identify the intracellular targets of lactoferricin B (LfcinB). Our results indicated that LfcinB reduces the ability of E. coli to respond to irregular environments by inhibiting the phosphorylation of two response regulators (basR and creB) and by influencing the metabolic process through multiple protein targets (15.Ho Y.H. Sung T.C. Chen C.S. Lactoferricin B inhibits the phosphorylation of the two-component system response regulators BasR and CreB.Mol. Cell. Proteomics. 2012; 11 (M111.014720)Abstract Full Text Full Text PDF Scopus (39) Google Scholar, 16.Tu Y.H. Ho Y.H. Chuang Y.C. Chen P.C. Chen C.S. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.PLoS ONE. 2011; 6: e28197Crossref PubMed Scopus (34) Google Scholar). Antimicrobial peptides Bactenecin 7 Lactoferricin B Hybrid of pleurocidin and dermaseptin Proline-arginine-rice peptide. In the present study, we identified the protein targets of Bac7, P-Der, and PR-39 and also included the LfcinB data to systematically elucidate the association between AMPs and their specific or common target proteins. Clusters of Orthologous Groups (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam analysis were used to characterize the unique and common target proteins of all four AMPs. KEGG and GO analysis revealed that LfcinB had synergistic effects in combination with Bac7 or PR-39 and that all four AMPs targeted arginine decarboxylase. We further validated these findings through antibacterial assays. The detailed protocol was described in our previous study (14.Chen C.S. Korobkova E. Chen H. Zhu J. Jian X. Tao S.C. He C. Zhu H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli.Na.t Methods. 2008; 5: 69-74Crossref PubMed Scopus (101) Google Scholar). Briefly, each E. coli K12 ASKA library clone (18.Kitagawa M. Ara T. Arifuzzaman M. Ioka-Nakamichi T. Inamoto E. Toyonaga H. Mori H. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research.DNA Res. 2005; 12: 291-299Crossref PubMed Scopus (1042) Google Scholar) was incubated in LB medium containing 30 μg/ml chloramphenicol at 37 °C. The overnight cultures were diluted in fresh LB medium and further grown until the optical density at 595 nm (OD595) reached ∼0.8. Isopropyl β-d-thiogalactoside was then added to induce protein expression by further incubation for 2 h. After induction, cell pellets were collected through centrifugation at 4 °C and stored at −80 °C before protein purification. For purification, the cell pellets were resuspended in a lysis buffer containing benzonase, CelLytic B, imidazole, lysozyme, proteinase inhibitor mixture, and Ni-NTA superflow resins. Subsequently, the resuspended cultures were transferred into 96-well filter plates and then washed with wash buffer I (50 mm NaH2PO4, 300 mm NaCl, 20% glycerol, 20 mm imidazole, and 0.1% Tween 20) and wash buffer II (50 mm NaH2PO4, 150 mm NaCl, 30% glycerol, 30 mm imidazole, and 0.1% Tween 20, pH 8). Finally, the proteins were eluted using an elution buffer (50 mm NaH2PO4, 150 mm NaCl, 30% glycerol, 300 mm imidazole, and 0.1% Tween 20, pH 7.5). To fabricate the chips, the purified proteins were transferred to 384-well plates and individual proteins were printed (in duplicate) on aldehyde slides at 4 °C by using a high throughput microarray spotter with 48 pins (SmartArrayerTM 136, Capitalbio Corporation, Beijing, China). The chips were first blocked using 1% BSA and then probed with N-terminal-biotinylated Bac7, P-Der, and PR-39 in Tris-buffered saline and Tween 20 (TBS-T; 0.05% Tween 20) with 1% BSA individually in a hybridization chamber with shaking at room temperature for 1 h. After washing, the chips were probed with DyLightTM 549-labeled anti-His antibody (Abcam®, Cambridge, UK) and DyLightTM 649-labeled streptavidin (Thermo Fisher Scientific, Waltham, MA) at room temperature for 30 min. Finally, the chips were washed three times with TBS-T. After the final wash with distilled water, the chips were dried through brief centrifugation and then scanned using a microarray scanner (LuxScan 10K, CapitalBio). The following amino acid sequences of Bac7, P-Der, and PR-39 were used: LfcinB: FARRVCTISPAGLKKMRWQWRRCKFBac7: PFPLPRPIPRPGPRPFPLPRPRPRPLRPPRPRIRRP-Der: LYHTLAAKGVHKGVHAAKKLMTKWLAPR-39: PFRPPFRPPFGPPIRPPLRPPFFPPPRPRPLYPPRPRRR For Bac7, only the 1–35-amino acid residue fragment was used because its antibacterial and intracellular activities are similar to those of intact Bac7 (11.Podda E. Benincasa M. Pacor S. Micali F. Mattiuzzo M. Gennaro R. Scocchi M. Dual mode of action of Bac7, a proline-rich antibacterial peptide.Biochim. Biophys. Acta. 2006; 1760: 1732-1740Crossref PubMed Scopus (112) Google Scholar). GenePix Pro 6.0 was used to align each protein spot and export all chip assay image results as text files. ProCAT (19.Zhu X. Gerstein M. Snyder M. ProCAT: a data analysis approach for protein microarrays.Genome Biol. 2006; 7: R110Crossref PubMed Google Scholar) was applied to normalize the signals of the four AMPs and the anti-His antibody. Subsequently, the relative binding ability of each AMP to each protein was estimated using the ratio of the fluorescence intensity of each AMP to that of the anti-His antibody. The signals were considered positive only when they fulfilled the following cutoffs: (1) the local cutoff, defined as two standard deviations above the signal mean for each spot, and (2) the fold change of each AMP signal to anti-His antibody is at least 0.5. For each AMP, three chips were used to conduct triplicate assays and each protein was printed duplicately in one chip. The protein was determined as a hit only if all the 6 spots on 3 chips were defined as positive signals. All bioinformatics analyses were performed using Perl 5.0. COGs (20.Natale D.A. Shankavaram U.T. Galperin M.Y. Wolf Y.I. Aravind L. Koonin E.V. Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs).Genome Biol. 2000; 1 (RESEARCH0009)Crossref PubMed Google Scholar), GO (21.Blake J.A. Harris M.A. The Gene Ontology (GO) project: structured vocabularies for molecular biology and their application to genome and expression analysis.Curr. Protoc. Bioinformatics. 2002; (Chapter 7, Unit 72)PubMed Google Scholar), KEGG (22.Kanehisa M. Goto S. Sato Y. Furumichi M. Tanabe M. KEGG for integration and interpretation of large-scale molecular data sets.Nucleic Acids Res. 2012; 40: D109-114Crossref PubMed Scopus (3371) Google Scholar), and Pfam (23.Punta M. Coggill P.C. Eberhardt R.Y. Mistry J. Tate J. Boursnell C. Pang N. Forslund K. Ceric G. Clements J. Heger A. Holm L. Sonnhammer E.L. Eddy S.R. Bateman A. Finn R.D. The Pfam protein families database.Nucleic Acids Res. 2012; 40: D290-301Crossref PubMed Scopus (2901) Google Scholar) were used to analyze the protein hits. To determine significant enrichment, for the statistical analysis, Fisher's exact test was performed using the free software R. GO analysis was performed on BiNGO 2.44. E. coli MG1655 was grown for 8–9 h in LB medium and then diluted 500-fold by using fresh LB medium (∼1.0 × 106 cells/ml). Culture growth was observed with or without AMPs in NuncTM F 96-well plates by using fresh LB medium as a blank. The working concentrations of LfcinB, PR-39, and Bac7 were 40, 25, and 100 μg/ml respectively. A 96-well plate was incubated at 37 °C in an automated Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT) with OD600 readings obtained at 20-min intervals for 7 h (shaking for 15 s before each reading). Data were collected automatically by using Gen5TM reader control and data analysis software (BioTek Instruments). We calculated the expected value at a given interval (EXY) as follows (24.Hegreness M. Shoresh N. Damian D. Hartl D. Kishony R. Accelerated evolution of resistance in multidrug environments.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13977-13981Crossref PubMed Scopus (196) Google Scholar): EXY=OD0×(ODX/OD0)×(ODY/OD0) Here OD0 is the OD of E. coli MG1655 in the absence of AMPs and ODX and ODY are the ODs of E. coli MG1655 in the presence of LfcinB and PR-39 or Bac7 at that interval, respectively. We performed this assay as described previously (25.Iyer R. Williams C. Miller C. Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.J. Bacteriol. 2003; 185: 6556-6561Crossref PubMed Scopus (128) Google Scholar, 26.Gong S. Richard H. Foster J.W. YjdE (AdiC) is the arginine:agmatine antiporter essential for arginine-dependent acid resistance in Escherichia coli.J. Bacteriol. 2003; 185: 4402-4409Crossref PubMed Scopus (100) Google Scholar). E. coli MG1655 was cultured overnight at 37 °C in M9 minimal medium (6 g/L of Na2HPO4, 4 g/L of glucose, 3 g/L of KH2PO4, 1 g/L of NH4Cl, 0.5 g/L of NaCl, 0.25 g/L of MgSO47H2O, 15 mg/L of CaCl22H2O, and 1 mg/L of thiamine-HCl). To test the cell survival rate after acid shock and various AMP treatments, overnight E. coli cultures were diluted 100-fold by using EG medium (73 mm K2HPO4, 17 mm NaNH4HPO4, 0.8 mm MgSO4, 10 mm citrate, 15 mm arginine, and 0.4% glucose, and then adjusted to pH 2.5); in addition, the AMPs (Bac7, LfcinB, P-Der, and PR-39) as well as the negative control cecropin P1 were individually added. After 1-h incubation at 37 °C, the cells were plated on LB agar and incubated overnight and then subjected to colony counting. To elucidate the association between each AMP and their specific or common target proteins, the E. coli proteome microarray was applied to provide a global profile of each AMP. The overall schematic of this study is depicted in Fig. 1. The chips were used to probe the biotinylated AMPs individually and then to probe the DyLightTM 649-labeled streptavidin and DyLightTM 549-labeled anti-His antibody. Because all proteins on the chips have His tags, the signal of DyLightTM 549-labeled anti-His antibody represents the relative protein amount. We applied ProCAT for data normalization and positive hit selection (19.Zhu X. Gerstein M. Snyder M. ProCAT: a data analysis approach for protein microarrays.Genome Biol. 2006; 7: R110Crossref PubMed Google Scholar); because the proteins were printed randomly on the chips, the signal distribution was adjusted to fit a normal distribution. Spot signals were identified as positive if their signals were larger than the local cutoff, which was mean plus two standard deviations. In addition, the signal fold change of AMP to anti-His antibody should be at least 0.5. For each AMP, three chips were used to conduct triplicate assays and each protein was printed duplicately in one chip. The protein was determined as a hit only if all the six spots on three chips were defined as positive signals. By analyzing these protein hits, we elucidated the unique and common target proteins of the four AMPs. Under identical experimental procedures, each AMP displayed a unique image pattern. This phenomenon may be because of individual characteristics of the four AMPs such as charge and hydrophobicity. To show the reproducibility of the chip assays, the triplicate enlarged chip images of PR-39 (as an example) are shown in Fig. 2. Moreover, to show the reproducibility of duplicates within a chip, we also used PR-39 as an example to plot a correlation figure (supplemental Fig. S1). After data normalization and positive hit selection, the protein targets for each AMP were generated and ranked according to their relative binding affinity, defined as the signal of the AMP divided by the signal from the anti-His antibody. The total number of protein hits for Bac7, LfcinB, P-Der, and PR-39 were 323, 303, 252, and 434, respectively. The protein hit list is displayed in supplementary Information.Fig. 2.Representative chip images. Triplicate enlarges chip images of PR-39 to show the reproducibility of the chip assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To provide a phylogenetic classification of the protein hits of the four AMPs, COG analysis was performed. The results of COG analysis in Fig. 3 indicate the function enrichment of the AMP protein hits. Bac7 showed enrichment in the nucleotide transport and metabolism, amino acid transport and metabolism, post-translational modification protein turnover chaperones, and coenzyme transport and metabolism. Renato et al. (27.Mardirossian M. Grzela R. Giglione C. Meinnel T. Gennaro R. Mergaert P. Scocchi M. The host antimicrobial peptide Bac71–35 binds to bacterial ribosomal proteins and inhibits protein synthesis.Chem. Biol. 2014; 21: 1639-1647Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) reported that Bac7 binds to bacterial proteins and inhibits protein synthesis. Therefore, we anticipated enrichment in ribosomal proteins. Although this was not observed in Bac7, we observed 11 ribosomal proteins in the Bac7-binding protein list (supplementary Information). Renato et al. (28.Scocchi M. Lüthy C. Decarli P. Mignogna G. Christen P. Gennaro R. The proline-rich antibacterial peptide Bac7 binds to and inhibits in vitro the molecular chaperone DnaK.Int. J. Peptide Res.Therapeutics. 2009; 15: 147-155Crossref Scopus (49) Google Scholar) also showed that Bac7 binds to the heat shock protein DnaK. Although DnaK was not in the protein list because of our strict cutoff, we also observed that Bac7 can bind to DnaK. In addition to Bac7, P-Der showed enrichment in carbohydrate transport and metabolism, whereas PR-39 showed enrichment in nucleotide transport and metabolism, secondary metabolites biosynthesis transport and catabolism, translation, and coenzyme transport and metabolism (Fig. 3). The category inorganic ion transport and metabolism was underrepresented for Bac7, P-Der, and PR-39, indicating that these intracellular targeting AMPs may have low preference toward recognizing proteins in this category. Different AMPs have different antimicrobial mechanisms, possibly because of their unique protein targets. Thus, in addition to the COG analysis, we also used other bioinformatics tools (GO and KEGG) to analyze the unique protein targets of each AMP (i.e. the protein hits shared by more than one AMP were not included in this analysis). The Venn diagram in Fig. 4 shows the protein hit number distribution of all four AMPs. The four ellipses show the total number of hits for the four AMPs (Bac7, yellow; LfcinB, red; P-Der, cyan; and PR-39, green). The nonoverlapping parts of the four ellipses represent the unique protein hits for each AMP (Bac7, 55; LfcinB, 231; P-Der, 47; and PR-39, 157). To analyze the unique protein targets of each AMP, we used GO analysis and characterized the unique hits for each AMP (Table I). For Bac7, enrichment was observed in nucleobase, nucleoside, and nucleotide interconversion and kinase activity. DNA synthesis is crucial for cell survival; in addition, kinase performs essential signal transduction functions. For LfcinB, enrichments were observed in transcriptional activator activity and biosynthetic processes of many carbohydrates (both p < 0.01) including lipopolysaccharide (LPS). LPS biosynthesis is particularly crucial because LPS is the major component of the outer membrane of Gram-negative bacteria. Several studies have designed specific compounds against LPS biosynthesis (29.Yethon J.A. Whitfield C. Lipopolysaccharide as a target for the development of novel therapeutics in gram-negative bacteria.Curr. Drug Targets Infect. Disord. 2001; 1: 91-106Crossref PubMed Scopus (71) Google Scholar, 30.Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (924) Google Scholar). Here, we observed that LfcinB might interrupt LPS biosynthesis through intracellular inhibitory activity (rfaG, rfaI, wcaC, yibB, yfbF, wcaL, yefE, rfaZ, rfaY, yefG, htrB, rfaQ, and rfaS). For P-Der, half of the GO analysis results were associated with the catabolic processes, including those of small molecules (e.g. pentose and l-arabinose catabolism), suggesting that the major antibacterial mechanism of P-Der is the inhibition of the bacterial catabolic processes. For PR-39, half of the GO analysis results were associated with RNA (RNA binding, ribonucleoprotein complex and ribosome biogenesis, RNA metabolic process, RNA modification, and ncRNA processing). Thus, PR-39 attacks proteins involved in RNA-related processes; therefore, this AMP may interrupt several cellular RNA processes.Table IGO analysis of the unique hits of each AMPDescriptionp valueHit in this CategoryTotal gene in this categoryBac 7Nucleobase, nucleoside and nucleotide interconversion0.0221419Kinase activity0.04059177Lfcin BTranscription activator activity0.00111694Carbohydrate biosynthetic process0.001323181Polysaccharide biosynthetic process0.002021162Cellular polysaccharide biosynthetic process0.003216109Cellular carbohydrate biosynthetic process0.006117128Lipopolysaccharide biosynthetic process0.00951386Sequence-specific DNA binding0.011276Transcription repressor activity0.01714104Pigment metabolic process0.024516Purine base biosynthetic process0.0335Transferase activity, transferring glycosyl groups0.031065P-DerWide pore channel activity and porin activity0.027427Pentose catabolic process0.034313L-arabinose catabolic process0.03424Small molecule catabolic process0.0348189Pore complex0.034317Isomerase activity0.0476113PR-39RNA binding0.005216145Cytosol0.018315148Ribonucleoprotein complex and ribsome biogenesis0.0183847RNA metabolic process0.018316171RNA modification0.0261966ncRNA processing0.02851085One-carbon metabolic process0.0322745Tetrahydrofolate metabolic and biosynthetic process0.032236Dihydrofolate reductase activity0.049822 Open table in a new tab" @default.
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• W2275361191 title "Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays" @default.
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