Protein sequences for beta-lactamase TEM, SHV, and NDM bacterial variants were obtained from UniProt46. We employed the software algorithm TANGO25 using default settings to identify APRs across this work, using a score of 5 per residue as the lower threshold and a parameter configuration of temperature at 298 K, pH at 7.5, and ionic strength at 0.05 M. Known TEM variants were retrieved from the Beta-Lactamase DataBase (BLDB)4. The mutations found in these variants were cross-referenced with literature to classify them according to their observed effects: offering resistance to an extended spectrum of B-lactams, offering resistance to inhibitors, stabilizing the TEM structure or other9,47,48. The effect of each mutant on protein stability was predicted through the FoldX forcefield49. To this end, PDB-structure 1xpb50 was first energy-minimized using the FoldX RepairPDB command, and subsequently, the effect on the stability of individual mutations was assessed using the BuildModel command, with default settings. As stated above, the TEM sequence was further analyzed using the TANGO aggregation prediction software, using default settings. The mutated residues were visualized in the TEM structure using YASARA51.
Peptides design and synthesis
Peptide hits were ordered from Genscript at >90% purity and were also produced in-house using the Intavis Multipep RSi automated synthesizer using solid-phase peptide synthesis. After synthesis, crude peptides were stored as dry ether precipitates at –20 °C. Stock solutions of each peptide were either prepared in 100% DMSO (only for initial screening assays) or following the optimized protocol: peptides were dissolved in 1 M NH4OH, allowed to dissolve for ~5 min, and dried in 1.0 mL glass vials with a N2 stream to form a peptide film. This film was dissolved in a buffer containing 50 mM Tris (pH 8.0) and 20 mM guanidine thiocyanate. Peptides were N-terminally acetylated and C-terminally amidated. Sample peptide QC data are shown in Supplementary Fig. 16.
A DynaPro DLS plate reader instrument (Wyatt, Santa Barbara, CA, USA) equipped with an 830 nm laser source was used to determine the hydrodynamic radius (RH) of the peptide particles. Two hundred microliters of each sample (at 100 or 10 μM, unless stated otherwise) were placed into a flat-bottom 96-well microclear plate (Greiner, Frickenhausen, Germany). The autocorrelation of scattered light intensity at a 32° angle was recorded for 5 s and averaged over 20 recordings to obtain a single data point. The Wyatt Dynamics v7.1 software was used to calculate the hydrodynamic radius by assuming linear particles. The amyloid-specific dye Thioflavin-T (Th-T, Sigma-Aldrich, CAS number 2390-54-7) was used to study the aggregation state of peptides. Two hundred microliters of each peptide sample (at 100 μM, unless stated otherwise) was placed into a flat-bottom 96-well microclear plate (Greiner, Frickenhausen, Germany), and the dye was added to a final concentration of 25 μM. A ClarioStar plate reader (BMG Labtech, Germany) was used to measure fluorescence by exciting the samples at 440-10 nm, and fluorescence emission was observed at 480-10 nm (or a complete spectrum ranging from 470 to 600 nm). Aggregation kinetics were obtained by placing 200 μL of the peptide solution with a final concentration of 25 μM thioflavin-T (Th-T) into a flat-bottom 96-well microclear plate. Fluorescence emission was monitored at 480-10 nm after excitation at 440-10 nm. Every 5 min Th-T fluorescence was measured.
Bacterial collection and growth conditions
Beta-lactamase clinical samples were collected from University Hospitals Leuven and tested for ESBL production using the disk diffusion method52. The beta-lactamase reference isolates were purchased from International Health Management Associates (IHMA). Bacterial strains were cultivated in Mueller–Hinton Broth (MHB, Difco) at 37 °C. Whenever required, growth media were supplemented with appropriate antibiotics to the medium or plates (kanamycin 30 µg/mL, l-arabinose 0.5 mg/mL, and IPTG 1 mM/mL). Escherichia coli BL21 (ThermoFisher Scientific, Belgium) was used for cloning and plasmid amplification. For the selection of antibiotic resistant colonies, E. coli carrying plasmids was grown in LB agar plates supplemented with the relevant antibiotic. Bacterial CFU counting was done on blood agar plates (BD Biosciences, Belgium) or MHA agar plates. Species identification and antibiograms for all clinical isolates were performed using MALDI-Tof and VITEK® 2 automated system (BioMérieux, France). All strains and their resistance profiles are listed in Suppl Table 2.
In vitro toxicity on the mammalian cells
The Cell Titer Blue assay was performed to evaluate the cell viability according to the instructions of the manufacturer (Promega, USA). The peptide treatments were done in DMEM medium without serum. Briefly, cells were seeded to approximately 20,000 mammalian cells per well in a 96-well flat-bottom plate (BD Biosciences 353075) and incubated at 37 °C with 5% CO2 and 90% humidity. Peptides were diluted in cell medium, and cells were treated for 24 h. 20 μL of the CellTiter Blue reagent was added to each well and the plate was incubated for 1 h at 37 °C. The fluorescence was measured at 590 nm by exciting at 560 nm with a ClarioStar plate reader (BMG Labtech, Germany).
Hemolytic activity was evaluated by measuring the amount of released hemoglobin. Fresh blood was pooled from healthy volunteers (collected from Rode Kruis Vlaanderen, Mechelen, Belgium). EDTA was used as the anticoagulant. Briefly, erythrocytes were collected by centrifugation 3000 × g for 10 min. The cells were washed with phosphate-buffered saline (PBS) several times and diluted to a concentration of 8% in PBS. 100 microliters of 8% red blood cells solution were mixed with 100 μL of serial dilutions of peptides in PBS buffer in 96-well plates (BD Biosciences, Belgium). The reaction mixtures were incubated for at least 1 h at 37 °C after which plates were centrifuged for 10 min at 3000 × g. The release of hemoglobin was determined by measuring the absorbance of the supernatant at 495 nm. Erythrocytes lysed in 1% Triton were used as control for 100% hemolysis.
Antibody and antibiotic product codes
The antibodies and antibiotic product codes used are as follows: monoclonal anti-TEM (Abcam, UK ab12251-8A5A10) 0.5 µg/mL, polyclonal rabbit anti-SHV (custom-made by Eurogentec, Belgium) 1 µg/mL, chicken polyclonal anti-beta Galactosidase (Abcam, ab145634 antibody (ab9361) 2 µg/mL. Goat Anti-Mouse IgG HRP secondary antibodies (ab97040); Rabbit Anti-Mouse IgG HRP (ab6728); Goat Anti-Chicken HRP (ab97135).
The antibiotics used for this study: Penicillin G sodium (Benzylpenicillin sodium, Abcam, catalog # ab145634) 1 µg/mL, Ampicillin (Duchefa Biochemie, Netherlands, A0104.0025), tazobactam sodium salt (Sigma-Aldrich, catalog # T2820-10MG), erythromycin, CAS number 114-07-8 (Sigma-Aldrich, catalog # E5389), chloramphenicol, CAS number 56-75-7 (Duchefa Biochemie), and kanamycin CAS number 56-75-7 (Duchefa Biochemie).
Determination of MIC values was performed using the broth microdilution method according to the EUCAST guideline, which was performed in 96-well polystyrene flat-bottom microtiter plates (BD Biosciences). Briefly, a single colony was inoculated into 5 mL Difco™ Mueller–Hinton Broth (BD Biosciences Ref 275730) and grown to the end-exponential growth phase in a shaking incubator at 37 °C. Cultures were subsequently diluted to a MacFarland (0.5 optical density) to reach 106 CFU/mL in fresh MHB medium. 50 µl of different concentrations of peptides ranging from 128 to 2 µg/mL were serially diluted to the sterile 96-well plate in MHB. 50 µL of the diluted bacteria in MHB were next pipetted into 96-well plates to reach the final volume of 100 µL. The bacteria grown with the maximum concentration of carrier and medium were considered positive and negative controls, respectively. The plates were statically incubated overnight at 37 °C to allow bacterial growth. OD was measured at 590 nm using a multipurpose ultraviolet–visible plate reader, and the absorbance of the bacterial growth was measured using an absorbance reader. Bacterial growth was also visually inspected which agreed well with the OD reading.
β-lactamase activity assays
The beta-lactamase assay procedure is based on the hydrolysis of the substrate Nitrocefin, a chromogenic cephalosporin, which produces a colored product (detectable at OD = 490 nm) that is directly proportional to the quantity of beta-lactamase activity. This experiment was carried out in 96-well black polystyrene flat-bottom microtiter plates (BD Biosciences). In brief, 50 μl of different concentrations of peptides or tazobactam in PBS were added to each well, followed by 50 μl of beta-lactamase protein at a final concentration of 12 ng. The plate was incubated at 37 °C for 1 h. The control heat was heated for 1 h at 95 degrees Celsius. Each well received 5 μl of Nitrocefin at a stock concentration of 0.5 mg/mL Nitrocefin. The hydrolyzed Nitrocefin was identified by absorption at 490 nm, which is proportional to the amount of beta-lactamase activity.
For the analysis of synergy between the peptides and other antibiotics, a checkerboard assay was performed. Based on the MICs of the selected peptides, a checkerboard assay was designed to define their FICIs (Fractional Inhibitory Concentration Index) in combinations against different clinical isolates53,54. Briefly, a total volume of 100 μL of Mueller–Hinton broth was distributed into each well of the 96-well plates. The first compound (peptide) of the combination was serially diluted vertically (128, 64, 32, 16, 8, 4, 2, 0 μg/mL) while the other drug (Beta-lactam or Kanamycin) was diluted horizontally in a 96-well plate (from 3200 to 3 μg/ mL). The total volume of each microtiter well was inoculated with 100 μL of MHB containing 1 × 106 CFU/mL bacteria. The plates were incubated at 37 °C for 24 h under aerobic conditions without shaking. Calculation of the FICI is used to analyze the results of the checkerboard assay by estimating the degree of synergistic effect. FICI is calculated as the sum of the individual fractional inhibitory concentrations (FICs) for each drug (where MIC A and MIC B denote the MIC of each drug alone, and MIC AA+B and MIC BA+B denote the concentrations of A and B in the drug combination). FICI = (MIC AA+B/MIC A) + (MIC BA+B/MIC B). With FICI ≤ 0.5, the combination of antibiotics is considered as a synergistic effect, 0.5 < FICI ≤ 1 indicates additivity, FICI > 1 indicates indifference.
Flow cytometry analysis
Bacterial cells in cleaned suspensions were stained with both propidium iodide (PI) and FITC-labeled peptides to evaluate the killing rate and peptide uptake in a two-dimensional analysis. Briefly, end-exponential growth phase E. coli cells (106 CFU/mL) were washed with PBS and treated with peptides at sub-MIC (0.25 x MIC) and sub-MIC of Penicillin for several hours at 37 °C. Treated bacteria were washed with PBS buffer two times. One microliter of PI (Invitrogen) was added to the bacteria and incubated for 5 min. The bacteria were counted by FACS to reach 40000 events. To correlate the activity of the peptides with cell death, the fluorescence intensity was measured in two channels using the GalliosTM Flow Cytometer (Beckman Coulter, USA), PI: excitation 536 nm and emission 617 nm, FITC: excitation 490 nm and emission 525 nm. Heated bacteria at 90 °C for 10 min were used as PI-positive control.
Staining with luminescent conjugated oligothiophenes
The bacterial cultures were washed with PBS, and the number of bacteria was adjusted to 108–109 cells/mL. Bacteria were then treated with peptides (at sub-MIC or MIC concentration based on the aim of the study) or buffer for 2 h at 37 °C. Then, cells were treated with LCO dyes (pFTAA; AmytackerTM680 or AmytackerTM545: final concentration of 0.5 µM; Ebba Biotech, Sweden) for at least 90 min. The absorption, emission, and excitation spectra for each dye were measured based on the standard Ebbabiotec advice (ebbabiotech.com).
Inclusion body (IB) purification
Overnight cultures of bacteria were centrifuged for 30 min at 4000 × g and cells were washed with physiological water (NaCl 0.9%). Bacterial cells were treated by peptide at the appropriate concentration for at least 2 h at 37 C. The bacterial pellets were washed with 10 mL buffer A (50 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM β-mercaptoethanol, 1.0 mM EDTA) and centrifuged at 4 °C for 30 min at 4000 × g. The supernatant was discarded and 20 mL of buffer B (buffer A plus 1 tablet of the protease and phosphatase Inhibitor Cocktail (ab201119, Abcam, UK) was added to the bacterial pellet. In order to break the cells, a High-Pressure Homogenizer (Glen Creston Ltd) with the pressure set to 20,000–25,000 psi was used on ice, and in addition, the suspensions were sonicated (Branson Digital sonifier 50/60 Hz) on ice with alternating 2 min cycle (15 pulses at 50% power with 30 s pauses on ice, until completing 2 min total sonication time). The lysed cells were centrifuged at 4 °C for 30 min at 11,000 × g. The precipitated fraction was afterward resuspended with 10 mL buffer D (buffer A plus 0.8% (V/V) Triton X-100, 0.1% sodium deoxycholate), and the suspension was sonicated to ensure the pellet was completely dissolved. This step was repeated three times. Centrifugation was performed at 4 °C for 30 min at 11,000 × g. Finally, to solubilize IBs, the pellet was suspended in 500 μl of buffer F (50 mM HEPES, pH 7.5, 8.0 M urea).
SHV and TEM protein purification
Plasmids were obtained from Genscript (USA) vector construction services. TEM (870 bp) and SHV (894 bp) were each sub-cloned into a PUC57 vector cloning site NdeI/ XhoI, with an N-terminal HIS-tag followed by the TEV cleavage site. The proteins were expressed in E. coli BL21 (DE3) by inducing with 1 mM IPTG overnight at 20 °C. Cells were harvested by centrifugation (15 min at 5000 rpm (2800 x g) at 4 °C), resuspended in buffer (500 mM Sucrose, 200 mM Tris pH 8.5 plus protease inhibitors (mini ETDA free (Sigma-Aldrich), one tablet per 25 mL of buffer) and lysed using a high-pressure homogenizer (EmulsiFlex C5, Avestin, Canada). The cell debris was removed by centrifugation (30 min, 18 x g), and the soluble lysate was loaded on a size-exclusion chromatography (SEC) column 26/600 75 pg column (column vol 320 mL, GE Healthcare, USA). The protein was equilibrated with buffer 50 mM Tris pH 8.5, 300 mM NaCl.
GFP fusion protein construction
TEM- and SHV-GFP fusion proteins were sub-cloned into the Invitrogen pBAD myc/his A vector. To this end, a vector expressing GFP with a linker (sequence KPAGAAKGG) at its C-term, designed in a previous study55, was modified. A multiple cloning site containing EcoRI and SpeI restriction sites was introduced C-terminally of the linker sequence through site-directed mutagenesis (using the New England Biolabs Q5® Site-Directed Mutagenesis Kit). Next, SHV and TEM sequences with EcoRI and SpeI restriction sites at their N- and C-terminus, respectively, were produced through PCR amplification from the expression constructs used for purification (discussed above). Finally, both the vector and PCR inserts were digested with SpeI-HF® and EcoRI-HF® (New England Biolabs) and ligated according to the manufacturer’s instructions.
Expression of TEM or SHV fusion GFP in E. coli
For protein expression and solubility analysis, bacterial strains were grown overnight in Lysogeny Broth (LB DifcoTM) supplemented with Ampicillin for GFP expression and both Ampicillin and chloramphenicol for co-expression of the GFP constructs with pKJE7. The overnight cultures were diluted 1:100 in fresh LB supplemented with the appropriate antibiotics and grown to an OD of about 0.6, after which expression was induced with 0.2 % arabinose. Expression was allowed to proceed for 3 h after which cells were lysed in B-PER™ reagent (ThermoFisher, USA) supplemented with 0.1 mg/mL lysozyme (Sigma-Aldrich), Complete™ Protease Inhibitor Cocktail (Sigma-Aldrich) and Pierce™ universal nuclease for cell lysis (ThermoFisher). Cells were lysed on ice for 30 min, after which soluble and insoluble fractions were separated through centrifugation at 17,100 x g for 30 min at 4 °C. The supernatant was removed, and the insoluble fraction dissolved in an equal volume of 8 M urea. GFP in soluble and insoluble fractions was then quantified through SDS-PAGE followed by Western blotting. Blots were developed using chemiluminescence after incubation with an anti-GFP antibody (Antibody 2555 S, Cell Signaling Technologies) or anti-DnaK antibody (D8076, USBio USA) and an HRP-conjugated secondary antibody. Blots were quantified using Bio-Rad’s Image LabTM Software. Soluble GFP fractions were determined by calculating the ratio of soluble over total (soluble + insoluble) protein.
Female C57BL/6Jax mice of 6 to 8 weeks with uniform weight (between 20 and 23 g) were used in this study (Harlan, The Netherlands). Mice were housed in plastic cages, four mice per cage on softwood granules as bedding. The room was kept between 21 °C and 25 °C with 12/12 h light–dark cycles. The animals had free access to water and pelleted rodent food. To avoid stress-induced confounding factors, the mice were transferred to the lab one week before experimental manipulation.
Efficacy of TEM3.2 in the treatment of urinary tract infection in mouse
To test the efficacy of the peptides, a urinary tract infection model was performed as described previously56. Briefly, female C57BL/6Jax mice female mice were deprived of water for at least 1 h. Then, they were anesthetized by IP administration of the mixture of ketamine (Nimatek)/xylazine (XYL-M 2% BE-V170581). The bladder of the mouse was massaged with fingers and pushed down gently to expel the remaining urine. Mice were slowly inoculated urethrally with 50 µL of a bacterial suspension slowly over 5 s to avoid vesicoureteral reflux (108 CFU/ mouse) using a sterile catheter (pediatric intravenous-access cannula (GS391350)). The catheter was removed directly after inoculation. After surgery, the animals were visually monitored for full recovery. After 1 h post inoculation, all mice received Ampicillin (30 mg/kg_PO-orally) and at the same time 3 groups of animals received the peptides via different administration routes (10 mg/kg_IV—intravenous; IP—intraperitoneal or SC—sub-cutaneous) and the positive control groups received tazobactam (10 mg/kg, PO). The negative control groups received vehicle or saline (IV administration). 2 h post inoculation, all mice received a second injection with the same concentration of each treatment as explained above. Twenty-four hours post-infection, mice were sacrificed and organs (kidney, bladder, ureter) were washed with PBS and were homogenized (Thermo Savant FastPrep FP120 Homogenizer/24 s). The homogenized tissues were serially diluted and cultured on blood agar plates. The plates were incubated overnight at 37 °C, and the number of bacteria was measured by CFU value.
Fluorescence microscopy of co-cultures of bacteria and mammalian cells
Human HeLa cells were grown to create a confluent monolayer on a small-cell-view cellular plate with a glass bottom (Greiner Bio-One GmbH/35 mm Ref: 627860) for imaging purposes. Next, cells were inoculated for 24 h with 200 μL of a mixture of overnight culture of TEM1 E. coli strain and FITC-Peptides (3xMIC). Cells were stained for 30 min with CellMask Deep Red plasma membrane dye (ThermoFisher catalog # C10046) and 1 L of NucBlue reagent (Invitrogen), after which the medium was removed, and 2 mL paraformaldehyde 4% was added to the dish for fixation. The dish was kept at room temperature for 6 h. Prior to imaging, the co-cultured cells were rinsed at least three times with 1 mL PBS.
Structured illumination microscopy (SIM)
Bacteria were fixed by adding 2.5% paraformaldehyde and 0.04 % glutaraldehyde (final concentrations) to the culture media, followed by incubation at room temperature for 15 min and 30 min on ice. Bacteria were then washed in PBS and resuspended in GTE buffer (50 mM glucose, 25 mM Tris, and 10 mM EDTA, pH 8.0). Directly preceding microscopic analysis, cells were transferred to a glass slide and covered with a coverslip. Imaging was performed using a Zeiss Elyra S.1 system in the VIB BioImaging Core at KU Leuven.
Statistical analysis was performed with Prism or R, using unpaired student’s t-tests, one-sample t-tests, and ANOVA to determine the statistical significance of differences between samples unless otherwise indicated. Significance levels: * for P < 0.05; ** for P < 0.01; *** for P < 0.001; **** for P < 0.0001. Non-significant differences are not separately labeled, unless stated otherwise.
All mouse experiments were conducted according to the national (Belgian Law 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. All protocols were approved by the KU Leuven Institutional ethics committee on animal experimentation. All relevant animal characteristics and housing conditions are specified in the materials and methods.
All blood samples were obtained from healthy volunteers from the biobank of the Red Cross Flanders in accordance with all relevant national legislation, including informed consent. Blood samples were completely anonymized prior to transfer to our facilities. Ethical approval was obtained from the medical ethical committee of the University Hospitals Leuven (study number S60497).
Cell lines were verified by Eurofins Genomics Europe (accredited acc. to DIN EN ISO/IEC 17025:200). Genetic characteristics were determined by PCR-single-locus-technology. 16 independent PCR-systems D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, AMEL, D5S818, FGA, D19S433, vWA, TPOX, and D18S51 were investigated. Results were compared with the online database of the DSMZ (http://www.dsmz.de/de/service/services-human-and-animal-cell) and the Cellosaurus database (https://web.expasy.org/cellosaurus. Only the PCR systems with ANSI/ATCC standard ASN-0002 were aligned in the final comparison.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.