Investigating the mechanism of action of aggregation-inducing antimicrobial Pept-ins

•Aggregation-inducing antimicrobial Pept-ins are less prone to inducing resistance•Pept-in resistance mechanism is dominated by limiting Pept-in uptake•Pept-in uptake involves electrostatic attraction and likely many transporters Aggregation can be selectively induced aggregation-prone regions (APRs) contained in the target proteins. Aggregation-inducing peptides (Pept-ins) contain sequences homologous APRs of proteins exert their bactericidal effect causing aggregation a large number To better understand action mechanisms, we analyzed phenotypic, lipidomic, transcriptomic as well genotypic changes laboratory-derived Pept-in-resistant E. coli mutator cells. The analysis showed that decreased uptake, both cells clinical isolates. Our data indicate an between bacterial membrane follows complex potentially involving transporters. Furthermore, it seems more challenging for bacteria become resistant toward dependent on suggesting future should selected this property. Antibiotic emerging global health issue. It cause failure treatment prevention infectious diseases, which could further increase risk performing medical procedures raise cost care (Sommer et al., 2017Sommer M.O.A. Munck C. Toft-Kehler R.V. Andersson D.I. Prediction antibiotic resistance: time new preclinical paradigm?.Nat. Rev. Microbiol. 2017; 15: 689-696Crossref PubMed Scopus (132) Google Scholar). Development classes with unique mode urgently needed address increasingly pressing We have shown promising antibacterial strategy disrupts homeostasis widespread protein using synthetic amyloidogenic (Bednarska 2016Bednarska N.G. van Eldere J. Gallardo R. Ganesan A. Ramakers M. Vogel I. Baatsen P. Staes Goethals Hammarström al.Protein design strategy.Mol. 2016; 99: 849-865Crossref (29) Scholar; Khodaparast 2018Khodaparast L. Louros N.N. Michiels Ramakrishnan Claes F. Young Shahrooei al.Aggregating occur constitute weak spots proteostasis.Nat. Commun. 2018; 9: 866Crossref Protein sequence-specific self-interaction whereby misfolded polymerize into either amorphous aggregates or amyloid fibrils typically composed single (Yoshimura 2012Yoshimura Y. Lin Yagi H. Lee Y.H. Kitayama Sakurai K. So Ogi Naiki Goto Distinguishing crystal-like glass-like from kinetics formation.Proc. Natl. Acad. Sci. U S 2012; 109: 14446-14451Crossref (192) During initial nucleation phase aggregation, slow thermodynamically unfavorable process, stable seeds formed rearranging structures series ? strands. Upon formation seeds, enters elongation phase, during preformed rapidly grow incorporating additional units polymers (Soto Pritzkow, 2018Soto Pritzkow S. misfolding, conformational strains neurodegenerative diseases.Nat. Neurosci. 21: 1332-1340Crossref (313) Thus, accelerated addition seeds; seeding efficiency depends critically sequence homology (Eisenberg Jucker, 2012Eisenberg D. Jucker state human diseases.Cell. 148: 1188-1203Abstract Full Text PDF (1127) mediated short (APRs), average at least once every 100 amino acids almost all (Rousseau 2006Rousseau Serrano Schymkowitz J.W.H. How evolutionary pressure against shaped chaperone specificity.J. Mol. Biol. 2006; 355: 1037-1047Crossref (188) Most given proteome, thus, possible specifically induce peptide (also called Pept-in) encoding APR (Betti 2016Betti Vanhoutte Coutuer De Rycke R.M. Mishev Vuylsteke Aesaert Rombaut Smet al.Sequence-specific generates defined knockdowns plants.Plant Physiol. 171: 773-787PubMed 2016Gallardo Ladan Laleh Couceiro J.R. Langenberg T. Siemons Nyström al.De novo biologically active amyloid.Science. 354: aah4949Crossref (45) However, minority redundant (which same sequence) found several sometimes different (Khodaparast This offers approach induces via these results bacteria, turn lead disruption proteostasis thus eventually cell death Owing action, hundreds proteins, observed no development subculturing experiments Escherichia Staphylococcus aureus Taken together, disrupting targeting shows great therapeutic potential. Although main aggregation-inducing achieved massive amount remains investigated, crucial successful development, efficacy improvement, toxicity minimization mammalian potential mechanisms need investigated well, valuable optimizing delaying occurrence (Martínez 2007Martínez J.L. Baquero Predicting resistance.Nat. 2007; 5: 958-965Crossref (233) generated through laboratory evolution XL1-Red hypermutator strain (Greener 1997Greener Callahan Jerpseth B. An efficient random mutagenesis technique strain.Mol. Biotechnol. 1997; 7: 189-195Crossref (82) Barrick 2009Barrick J.E. Yu D.S. Yoon S.H. Jeong Oh T.K. Schneider Lenski R.E. Kim J.F. Genome adaptation long-term experiment coli.Nature. 2009; 461: 1243-1247Crossref (792) reasons were wild-type buildup allow rapid acquisition Hypermutable been frequently identified settings suitable backgrounds studying acquired genes (Woodford Ellington, 2007Woodford N. Ellington M.J. emergence mutation.Clin. Infect. 13: 5-18Abstract 5,000-fold higher mutation rate than wild type generate mutations genomic DNA P2 very cross-resistance was observed. Phenotypic studies mostly caused reduced accumulation within resulting mainly cellular uptake. P2-resistant show negative charge enriched coding transporters, appear collectively contribute Further isolates also associated P2-resistance These exhibited confirming importance across strains. study cells, performed classic adaptive following conditions: vehicle (PBS), ampicillin (AMP), P33, P2. P33 previously each encodes distinct fragment (Table 1) employ tandem repeat design, includes two identical flanked three solubilizing arginine residues gatekeepers) linked proline Three independent populations evolved 27 days parallel condition daily 1/3 minimum inhibitory concentration (MIC). most significant MIC AMP: 3.25 400 ?g/mL (Figure 1A). slower compared AMP; while increased 6.25 200 1B), rose only 3.12 1C). AMP, P2, did not change vehicle-treated (Figures 1A–1C), pressure, high cells.Table 1Pept-in cellsPeptideSequenceaGLGLALV, LGIAVAL, LLTTLL P5, respectively.MIC (?g/mL)P2RGLGLALVRRPRGLGLALVRR6.5P33RLGIAVALRRPRLGIAVALRR3.25P5RALLTTLLRRPRALLTTLLRR3.25a GLGLALV, respectively. Open table tab Increased elevates rates (Pal 2007Pal Maciá M.D. Oliver Schachar Buckling Coevolution viruses drives rates.Nature. 450: 1079-1081Crossref (209) Scholar), accelerate acquisition. Correspondingly, became antibiotics faster when 2/3 1D–1F). worth noting still occurred much AMP. 12.5 even after continuous 1E). confirm difficult, especially P33. 1G) 1H) led growth longer lag lower density stationary indicating fitness burden antibiotic-resistant and/or (Lee 2010Lee H.H. Molla M.N. Cantor C.R. Collins J.J. Bacterial charity work leads population-wide resistance.Nature. 2010; 467: 82-85Crossref (402) examined whether Pept-in-resistance collateral sensitivity other antibiotics, phenomenon (Jahn 2017Jahn L.J. Ellabaan M.M.H. Sommer Adaptive selection regimes similar phenotypes genotypes.Front. 8: 1-14Crossref (34) Lázár 2018Lázár V. Martins Spohn Daruka Grézal G. Fekete Számel Jangir P.K. Kintses Csörgo al.Antibiotic-resistant peptides.Nat. 3: 718-731Crossref (166) Nichol 2019Nichol Rutter Bryant Hujer A.M. Lek Adams Jeavons Anderson A.R.A. Bonomo R.A. Scott J.G. contingent repeatability evolution.Nat. 2019; 10: 334Crossref (60) For purpose, determined susceptibility 14 six categories derived end S2). RNA synthesis inhibitor, inhibitors, fatty acid division inhibitors 2C 2D ). they developed positively charged 3- 5-fold membrane-disrupting cationic (CAMPs) 2-fold 2D). Notably, resistance-associated profiles vehicle- 2A) AMP- 2B) suggests play role mechanism. verify hypothesis, surface measuring binding fluorescein isothiocyanate (FITC)-labeled poly-L-lysine (FITC-PLL) membrane, often used quantify (Spohn 2019Spohn Vidovics Méhi O. al.Antimicrobial limited 1-13Crossref (90) Compared ancestors, AMP-evolved levels FITC median fluorescence intensity (MFI), whereas significantly MFI 2E 2F). P33-resistant demonstrated level 2E), histogram FITC-fluorescence distribution subpopulation P33-derived FITC-PLL. result confirms evident Preventing access drug targets common antibiotic-resistance (Blair 2015Blair J.M.A. Webber M.A. Baylay A.J. Ogbolu D.O. Piddock L.J.V. Molecular 2015; 42-51Crossref (1748) As exerts its co-translationally cytoplasm next FITC-labeled ancestor strains, under treatments day 13 27. Whereas P2-derived 30-fold increase, rest had ancestors. 3A) 3B) accumulation, time-dependent killing 3C) unaltered MIC, those ancestors; however, decrease (P33- 13, respectively). reduction FITC-accumulation 3A–3C). enhance continuously evolving strengthening ability intracellular Pept-in. FITC-P2 accumulated intracellularly polar faint signal around region 2 h 3D). preventing due efflux conjugating diacetate (FITC-2DA). FITC-2DA non-fluorescent hydrolyzed fluorescent live esterases, but dead diminished esterase activity (Wang 2011Wang Williams E.T. Bourgea Wong Y.N. Patten C.J. Characterization recombinant carboxylesterases: probe substrate carboxylesterase 2.Drug Metab. Dispos. 2011; 39: 1329-1333Crossref (88) Mao 2020Mao Ma Wei Zhang Liu Guan X. Yi ‘A sensitive “off-on” detection application evaluating status discrimination living cells’.Analyst. 2020; 145: 1408-1413Crossref supernatant first FITC-2DA-P2 treatment, since ancestors receiving maintained low 3B). No difference regarding 3E). contributed 6 vehicle-, P33-, P2-evolved lysis subsequent leakage already form FITC-2DA-P2. abundant misfolding accumulate inclusion bodies (IBs) IBs stained amyloid-specific dye pFTAA, binds amyloid-like (Åslund 2009Åslund Sigurdson Klingstedt Grathwohl Bolmont Dickstein D.L. Glimsdal Prokop Lindgren Konradsson al.Novel pentameric thiophene derivatives vitro vivo optical imaging plethora cerebral amyloidoses.ACS Chem. 4: 673-684Crossref (225) 4 ?g/mL, IB few 3F). upon 3F) 6.5 3G). demonstrates able taken up interfering does important may Scholar) evolutionarily conserved Lipid-peptide interaction plays AMP-mediated internalization; pathogens modify lipidomic profile (Zhang Rock, 2008Zhang Y.M. Rock C.O. Membrane lipid bacteria.Nat. 2008; 6: 222-233Crossref (740) modified lipidome gain In general, percentage class underwent 3H). some cases, positive AMPs clustering anionic lipids subsequently promotes pore (Epand 2010Epand R.F. Maloy W.L. Ramamoorthy Epand ‘Probing “charge cluster mechanism” amphipathic helical peptides’.Biochemistry. 49: 4076-4084Crossref (120) observe (PG CL) 3I). Bacteria adopt rigid decreasing unsaturated class, consequently makes harder penetrate (Khondker 2019Khondker Dhaliwal A.K. Saem Mahmood Fradin Moran-Mirabal Rheinstädter M.C. packing determine polymyxin-induced damage.Commun. 2: 1-11Crossref (30) alteration 3J). suggest translocation P2-mediated lipid-peptide interactions. Having sought investigate molecular comparing expression 462 286 downregulated upregulated, affected ClueGO plug-in Cytoscape identify statistically biological pathways (Bindea 2009Bindea Mlecnik Hackl Charoentong Tosolini Kirilovsky Fridman W.H. Pagès Trajanoski Z. Galon ClueGO: decipher functionally grouped gene ontology pathway annotation networks.Bioinformatics. 25: 1091-1093Crossref (3208) revealed metabolic 4A). Since overlapping P2-induced resistance, rather 35 involved translation 4A), peptide-induced IBs2,3. efficacy, CH184 mutant (with per second, respectively) (Proshkin 2010Proshkin Rahmouni Mironov Nudler Polymerase transcription elongation.Science. 328: 504-508Crossref (351) S1A S1B) S1C S1D) type. prolonged exposure unfolded translation, rendering presenc