TSP purification

pET_AV101_TSP1-4 were transformed into electrocompetent E. coli BL21 cells for the expression of the TSPs (Sørensen et al. 2021). A single colony of each of transformants carry one of the four pET_AV101_TSP1-4 was inoculated into LB medium with 50 µg ml⁻¹ kanamycin and incubated overnight at 37°C and 170 rpm. Next day, 10 ml of the starter culture was added to 1 l of LB medium with 50 µg ml⁻¹ kanamycin at 37°C and incubated until an OD₆₀₀ value of 0.6. Then, a final concentration of 0.5 mM of isopropyl-b-d-thiogalactopyranoside was added to induce protein expression. The culture was incubated for an entire night at a reduced temperature of 16°C. The culture was centrifuged the following morning for 10 min at 13 000 g, and the pellet was then resuspended in 9 ml of lysis buffer (0.5 M NaCl, 20 mM Na2HPO4, 50 mM Imidazole, pH 7.4). Sonication was used to disrupt the cells, with a program of nine cycles lasting 30 s each at 80% power. Centrifugation at 9500 g for 30 min at 4°C separated cell debris. Using 0.22 mm filters, the supernatant containing the expressed proteins was filtered. HisGraviTrapTM (GE Healthcare) was then used to purify the proteins using an elution buffer (0.5 M NaCl, 20 mM Na₂HPO₄, 0.5 M Imidazole, pH 7.4). To transfer the TSPs into a new buffer [20 mM HEPES (pH 7.4)], Amicon Ultra-15 Centrifugal Filter Units with a 50 kDa cutoff (Merck Milipore) were utilized. Using a Qubit 2.0 Fluorometer and the QubitTM Protein Assay Kit, protein concentration was determined (Invitrogen).

The TSP spot and inhibition assay was performed as previously described (Sørensen et al. 2021). Shortly, bacterial strains were grown to an OD₆₀₀ value of 0.6. A volume of 100 µl of the bacterial culture was then added to 4 ml top-agar and poured onto an LB agar plate. After the bacterial lawns were solidified, 1.5 µg of the four TSPs were spotted onto the bacterial lawn and left to dry for 30 min. Phage AV101 and the protein buffer [20 mM HEPES (pH 7.4)] were used as a positive and negative control, respectively. The plates were incubated overnight at 37°C and the next day the presence of a translucent zone was evaluated. To further validate the spot assay, the inhibitory effect of the TSPs on the infectivity of the bacterial strains were evaluated. The four strains ESBL-038, -040, -058, and -144 were used as they represented strains that TSP1-4 recognize, respectively. A single colony of the bacterial strains was incubated in LB medium at 37°C at 170 rpm until OD₆₀₀ reached 0.3. The cells were then cooled on ice before 100 µl of the cells were added to 5 mg of the TSPs in individually tubes. The cell-TSP suspension was then preincubated at 37°C for 20 min. Afterwards, the suspension was added to 4 ml top agar and poured onto an LB agar plate and left to solidify. Three times 10 µl phage AV101 dilutions (10⁻¹–10⁻⁸) was spotted on top of the plate and incubated overnight at 37°C. Next day, the inhibitory effect of the TSPs were evaluated by comparing the PFU ml⁻¹ of the phage sensitive strains with the PFU ml⁻¹ with strains incubated with the respective TSPs. The inhibition assay was carried out in triplicates and the results were visualized in Graphpad Prism9 with the mean standard deviations shown in the figure.

Models for the TPSs were predicted in a Nvidia Quadro RTX 8000 using Alphafold2-multimer (version 3.2.1) (Jumper et al. 2021, Evans et al. 2022). We utilized global search for the multiple sequence alignment, five recycling rounds, and the amber relaxation was skipped. The best model was ranked based on the iptm+ptm score and was the one selected for the analysis.