Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Volcano plot highlighting significant elevations in N -acetylputrescine and 4-acetamidobutanoate in humans with gram-negative septic shock (B) N -acetylpturescine and 4-acetomidobutanoate levels correlate with worse clinical outcomes as measured by APACHE II scores (C) Putrescine metabolic pathway outlining production of N -acetylputrescine and 4-acetamidobutanoate (D) Putrescine metabolites are elevated in the mouse cecal slurry model of septic shock/BSI; HK = heat killed, CS = cecal slurry; n = 8 PBS, n = 7 HK CS, n = 4 Live CS; p-values were determined by One-way ANOVA followed by Tukey’s multiple comparisons test (E)Septic shock/BSI with heat-killed cecal slurry rescued with E. coli BW25113 results in increased plasma putrescine metabolite levels; n = 5 HK CS, n = 9 HK CS + E. coli BW25113; Two-tailed p-values were determined by unpaired t test (F) Klebsiella pneumoniae pneumonia results in increased bronchial alveolar lavage (BAL) fluid levels of N - acetylputrescine; n = 4 PBS, n = 6 K. pneumoniae (KP9); Two-tailed p-values were determined by Mann Whitney test to include the outlier. (G) Klebsiella pneumoniae pneumonia results in increased plasma levels of N -acetylputrescine; n = 4 PBS, n = 6 K. pneumoniae (KP9); Two-tailed p-values were determined by Mann Whitney test to include the outlier. (H) Bacterial and mouse production of N -acetylputrescine + 4-acetamidobutanoate from putrescine; n = 3 for bacteria, representative data from 1-3 independent repeats; n = 6 mice; Blue = E. coli , Green = K. pneumoniae , Red = P. aeruginosa For all panels, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Clinical Proteomics, Two Tailed Test, MANN-WHITNEY, Bacteria

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Complementation in E. coli BW25113 demonstrates SpeG can produce N -acetylputrescine; n = 3 per condition, representative data from 3 independent experiments (B) 1 hour production of N -acetylputrescine from putrescine by recombinant purified enzymes; n = 3 per condition, representative data from 3 independent experiments; NE = no enzyme, GFP = green fluorescent protein (C) Kinetics of putrescine acetylation by SpeG is consistent with previously demonstrated cooperative mechanism on spermidine, n = 2-3 per substrate concentration, representative data from 4 independent experiments; summary parameters includes all experiments (D) Maximum-likelihood phylogenetic tree of a representative member of each group of protein sequences sharing >80% amino acid ID (RepNode80); Blue = E. coli SpeG clade, Purple = mammalian SAT1 clade, Turquoise = B. subtilis BltD clade (E) Complementation in E. coli BW25113 demonstrates PA1472 can produce N -acetylputrescine; n = 3 per condition, representative data from 2-3 independent experiments (F) 1 hour production of N -acetylputrescine from putrescine by recombinant purified enzymes; n = 3 per condition, representative data from 2-3 independent experiments; NE = no enzyme, GFP = green fluorescent protein (G) Kinetics of PA1472 on putrescine; n = 2 per concentration, representative data from 3 independent experiments; summary parameters includes all experiments (H) SpeG (PDB: 3WR7) and PA1472 (AlphaFold2) dimers For all panels, data presented are means ± SEM

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Recombinant, Purification, Concentration Assay

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Suppression of speG expression with inducible CRISPRi in E. coli patient bloodstream isolate E23; n = 3 per condition, representative data from 2 independent experiments (B) Decreased levels of extracellular N -acetylputrescine with inducible CRISPRi of speG in E23, concentrations normalized to OD600 = 1.0; n = 3 per condition, representative data from 2 independent experiments (C) Increased relative intracellular putrescine levels (concentrations normalized to OD600 = 1.0 and then normalized to concentration of control condition) and decreased ratio of N -acetylputrescine/putrescine with inducible CRISPRi of speG in E23; n = 3 per condition, representative data from 2 independent experiments (D) Inducible CRISPRi of speG suppresses E23 growth in culture; n = 3 per condition, representative data from 3 independent experiments (E) Inducible CRISPRi of speG delays mortality in a cecal slurry model of sepsis with E23; n = 10 mice per group for all groups except n = 9 for RFP – aTC; p-value determined by Log-rank (Mantel-Cox) test For panels A-D, data presented are means ± SEM; Two-tailed p-values were determined by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Expressing, Concentration Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Checkerboard assays demonstrate synergy between diminazene and vancomycin in E. coli E23; representative data from 3 independent experiments (B) Inducible CRISPRi of speG reduces MIC of vancomycin in E. coli E23; mean growth of n = 3 per condition, representative data from 3 independent experiments (C) Inducible CRISPRi of speG enhances membrane permeability in E23; n = 3 per condition, representative data from 2 independent experiments; Two-tailed p-values were determined by unpaired t test (D) Diminazene treatment for 6 hours enhances membrane permeability in E. coli E23; n = 3 per condition, representative data from 2 independent experiments; p-values were determined by One-way ANOVA followed by Dunnett’s multiple comparisons test with all comparisons made against no drug control For panels B-D, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Membrane, Permeability, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Identification and targeting of microbial putrescine acetylation in bloodstream infections

doi: 10.1101/2023.09.21.558834

Figure Lengend Snippet: (A) Checkerboard assays demonstrate synergy between diminazene and antibiotics to which the assayed MDR strains are clinically resistant; representative data from 3-4 independent experiments per strain (B) Diminazene treatment enhances uptake of antibiotics to which MDR strains are resistant; n = 3 per condition, representative data from 1-3 independent experiments; Two-tailed p-values were determined by unpaired t test (C) Combination of diminazene and tetracycline treatment reduces mortality in a cecal slurry model of sepsis with the tetracycline resistant clinical isolate E. coli E1; n = 10 mice per group; p-value determined by Log-rank (Mantel-Cox) test For panels B, data presented are means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p<0.0001 aTC = anhydrotetracycline

Article Snippet: SpeG was amplified from E. coli K12 MG1655 genomic DNA and SAT1 , PA4114, PA1472, and PA1377 were ordered as E. coli codon optimized sequences (ThermoFisher); all were cloned cloned into pET-28A-inducible expression vectors using Gibson assembly (including an in-frame either N-terminal or C-terminal polyhistidine sequence) (primers in Table S6 ).

Techniques: Two Tailed Test