Bacterial surface properties influence the activity of the TAT-RasGAP317-326 antimicrobial peptide

•Antimicrobial activity of TAT-RasGAP317-327 is affected by medium composition•Peptide exposure induces metabolic and stress responses•Cell surface modifications influence peptide antimicrobial activity•Resistance to does not lead multidrug resistance Antibiotic an increasing threat for public health, underscoring the need new antibacterial agents. Antimicrobial peptides (AMPs) represent alternative classical antibiotics. TAT-RasGAP317-326 a recently described AMP effective against broad range bacteria, but little known about conditions that may its activity. Using RNA-sequencing screening mutant libraries, we show Escherichia coli Pseudomonas aeruginosa respond regulating response pathways, possibly implicating two-component systems. Our results also indicate bacterial properties, in particular integrity lipopolysaccharide layer, binding entry. Finally, found differences between species with respect their rate emergence this peptide. findings provide basis future investigation on mode action TAT-RasGAP317-326, which help developing treatments based The spread antibiotic many severely limiting benefits antibiotics growing number infections are becoming harder treat (O'Neill, 2016O'Neill J. Tackling drug-resistant globally: final report recommendations review resistance.amr-review.org. 2016; Google Scholar). Therefore, there antimicrobials could be used treatment infections. (AMPs), several already clinical trials promising results, large source agents (Kumar et al., 2018Kumar P. Kizhakkedathu J.N. Straus S.K. peptides: diversity, mechanism strategies improve biocompatibility vivo.Biomolecules. 2018; 8: 4Crossref PubMed Scopus (313) They attractive alternatives due broad-spectrum allows targeting wide variety (Di Somma 2020Di A. Moretta Cane C. 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Resist. 2021; 25: 227-231Crossref (2) composed permeable moiety, TAT HIV 48–57 sequence, 10 acid sequence Src homology 3 domain p120 RasGAP. 22 long, molar mass 2830 g/mol net charge +9 at pH 7.0. comparison, 26 2847 +6 7.0 mixture highly similar non-ribosomal cyclic heptapeptide tripeptide side chain (molar 1302 g/mol) (Figure S1) (Yang 2001Yang L. Harroun T.A. Weiss T.M. Ding Huang H.W. Barrel-stave model toroidal model? A case study pores.Biophys. 2001; 81: 1475-1485Abstract Full Text PDF Velkov 2013Velkov Roberts K.D. Nation R.L. Thompson P.E. Pharmacology polymyxins: insights 'old' class antibiotics.Future 2013; 711-724Crossref (258) Hong was initially compound sensitizes cancer cells genotoxins (Michod 2004Michod J.Y. Chen Bonny RasGAP-derived potently enhances genotoxin-induced cytotoxicity tumor cells.Oncogene. 2004; 23: 8971-8978Crossref (44) Scholar, Michod 2009Michod Annibaldi Schaefer Dapples Rochat Effect RasGAP N2 fragment-derived growth mice.J. Natl. 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TAT-RasGAP317-326-mediated sensitization occur independently Bax Bak.Apoptosis. 719-733Crossref 2016Heulot Chevalier Puyal Margue Michel Kreis Kulms Nahimana anti-cancer kill caspase-, apoptosis-, necroptosis-independent manner.Oncotarget. 7: 64342-64359Crossref We previously shown tryptophan residue position 317 essential both eukaryotic 2014aBarras Zoete Dempsey Lapouge Olayioye M.A. Michielin WXW motif required peptide.J. Biol. Chem. 289: 23701-23711Abstract (15) minimal inhibitory concentrations (MICs) low Acinetobacter baumannii (11 μM 2.9 respectively) higher Staphylococcus aureus (23 46 respectively), indicating levels tolerance toward showed limited protection mouse coli-induced peritonitis Physiological factors contributed poor biodistribution rapid clearance and, subsequently, efficacy setting study, assessed experimental settings better characterize well exposure. important will pave way further properties. grown Mg2+ EDTA, gentamicin, via involves outer (Macfarlane 1999Macfarlane E.L. Kwasnicka Ochs M.M. Hancock R.E. PhoP-PhoQ homologues regulate expression outer-membrane protein OprH resistance.Mol. 1999; 34: 305-316Crossref (166) McPhee 2003McPhee J.B. Lewenza Cationic activate regulatory system, PmrA-PmrB, regulates aeruginosa.Mol. 2003; 50: 205-217Crossref (306) Olaitan 2014Olaitan A.O. Morand Rolain J.M. Mechanisms resistance: acquired intrinsic bacteria.Front. 5: 643Crossref determine whether levels, how modulated MIC concentration inhibiting 50% (IC50) three laboratory strains, MG1655, ATCC 25922, PA14. these experiments summarized Table 1 detail Figures S2–S4. standard Luria-Bertani (LB) determined 8 32 (corresponding 22.63 μg/ml 90.56 μg/ml), respectively. There small difference LB BM2 supplemented 2 mM MgSO4 (BM2 Mghigh) aeruginosa. However, displayed 8-fold decrease containing 20 Mglow) relative Mghigh). Mghigh measured (1.66 0.83 μM), Low magnesium resulted 4-fold increase had no impact coli. agree earlier data increases Altogether, culture renders more susceptible B.Table 1E. ATCC25922, PA14 sensitivity varies depending usedStrainMediumTAT-RasGAP317-326Polymyxin BMIC (μM)IC50 (μM)MIC (μg/mL)IC50 (μg/mL)E. MG1655LB8310.6LB Mghigh3216.320.6BM2 Mglow0.50.321.0BM2 Mghigh4220.8E. 25922LB85.221.8LB Mghigh1285242.7P. PA14LB327.242.1LB Mghigh6420.821.0BM2 Mglow41.741.0BM2 Mghigh3210.110.5The indicated overnight LB, (LB Mghigh), Mglow), Culture then diluted OD600 = 0.1 hr. Bacterial suspension times μL added per 96-well plate 100 μl serial dilutions OD590 16 hr incubation. defined lowest completely proliferation. IC50 inhibit calculated using GraphPad Prism 8. detailed curves presented Open table tab Because bacteriological medium, investigated affects complex LB. compared high increased (Table 1), consistent obtained medium. Moreover, ability hamper clearly inhibited (Figures 1A 1B). decreased result independent used. presence accordance our Would cations than render bacteria less sensitive TAT-RasGAP317-326? Addition Fe2+ Ca2+ 1C 1D). context sulfate chloride counterions 1B 1E). contrast, ammonium did affect susceptibility 1F). Collectively, Fe2+, divalent bacteria. Since characteristics, (LPS) (Hancock, 1997Hancock drug barrier.Trends 1997; 37-42Abstract (285) questioned internalization altered cations. lower extent Mg2+, FITC-labeled bound coli, amount internalized 1G). – finding suggest restricts entry relationship viable (culturable) performed colony unit (CFU) assays. induced 2- 5- fold surviving upon longer incubation 2A). pronounced viability observed ≥15 μM, bactericidal Confocal microscopy studies accumulated intracellularly when exposed leading 2E). Furthermore, led changes morphology seen electron 2F). Mglow 0.5–2 untreated control, while 5–10 strongly reduced numbers 2B). order analyze kinetics early time points, survival These correspond 2.5 1) 2A monitored hours B; latter (2.5 μg/mL aeruginosa). Interestingly, slow time-kill comparison 2C). aeruginosa, however, 2D). RNA sequencing analysis evaluate transcriptome. this, peptide, prevents proliferation dramatic drop Among 4419 transcripts predicted MG1655 genome, 95.6% (n 4223) detected least one condition (Data S1). Figure 3A presents change gene incubated without average level each gene. excluded genes whose below threshold set reads kilobase million (RPKM). Overall, 962 (fold >4): 11.0% total upregulated (red dots 3A) 11.8% downregulated (blue 3A). Detailed lists Tables S1 S2, validated twelve RNA-Seq qRT-PCR extracted same analyses. Five (lpxL, fabF, marA, entB, fepA, depicted red 3B) reported upregulated, five (bssR, frdA, ompF, nuoE, sdhC, blue unchanged according (tolC ompR). One genes, ompR, housekeeping reference normalization. good correlation qRT-PCR, confirming validity 3B). profiles sequencing, associated TAT-RasGAP317-326. accomplish systematic manner, Gene Ontology (GO) Kyoto Encyclopedia Genes Genomes (KEGG) pathway enrichment analyses (Kanehisa Goto, 2000Kanehisa Goto KEGG: kyoto encyclopedia genomes.Nucleic 2000; 28: 27-30Crossref Kanehisa 2019Kanehisa Sato Y. Furumichi Morishima Tanabe New approach understanding genome variations KEGG.Nucleic 47: D590-D595Crossref (821) Kanehisa, Toward origin evolution cellular organisms.Protein 1947-1951Crossref (439) Ashburner 2000Ashburner Ball C.A. Blake J.A. Botstein Butler H. Cherry Davis A.P. Dolinski Dwight S.S. Eppig J.T. al.Gene ontology: unification biology. Consortium.Nat. Genet. 25-29Crossref (23758) Ontology, 2019The ontology resource: years still GOing strong.Nucleic D330-D338Crossref (1436) differentially expressed genes. KEGG revealed information-processing enriched among 3C 3D). seven eight responsible enterobactin synthesis (included “biosynthesis siderophore group nonribosomal peptides” pathway) treatment. Other carbon metabolism (citrate cycle, pyruvate metabolism) oxidative phosphorylation downregulated. Similarly, GO term processes related general S5). From data, distinguish peptide-specific mediating adaptation stress. address question, decided perform comprehensive deletion library Keio collection mutants consists single clones non-essential replaced cassette (Baba 2006Baba Ara Hasegawa Takai Okumura Baba Datsenko Tomita Wanner B.L. Mori Construction K-12 in-frame, single-gene knockout mutants: collection.Mol. Syst. 2006; (2006 0008)Crossref (4789) collection, strain 5 non-bactericidal 2A) measurement specific points (detailed measurements all individual available Data S2). strain, wild-type 6 24 4, panels B). 27 thus having normalized 270 replicates parental +2 deviation 4A S3). 356 hypersensitive (having deletions 279 genes) minus 4B). While grew slowly absence similarly detectable i