Engineered phage with cell-penetrating peptides for intracellular bacterial infections

ABSTRACT Salmonella infection poses a critical challenge to global public health, and the situation is exacerbated by the increasing prevalence of antibiotic resistance. Bacteriophages (phages) are increasingly being used as antimicrobial agents due to their ability to kill specific bacteria. However, the low cellular uptake of phages has limited their use in treating intracellular bacterial infections. Here, we present a study using engineered phages with cell-penetrating peptides (CPPs) for enhancing the internalization efficiency of phages to inhibit bacterial intracellular infections. Through bioinformatic analysis, we identified a phage-encoded protein harboring an immunoglobulin-like (Ig-like) domain as the potential target for phage display. Using a CRISPR-Cas9-based method, we successfully displayed short peptides on GP94, an Ig-like domain-containing protein, of Salmonella phage selz. We improved phage intracellular uptake in multiple cell types by fusion of various CPPs to GP94. Notably, the phage selzHA-TAT showed promising results in enhancing the intracellular inhibition of Salmonella in different cells. Our research provides a straightforward strategy for displaying CPPs on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. IMPORTANCE Salmonella infection is a significant threat to global public health, and the increasing prevalence of antibiotic resistance exacerbates the situation. Therefore, finding new and effective ways to combat this pathogen is essential. Phages are natural predators of bacteria and can be used as an alternative to antibiotics to kill specific bacteria, including drug-resistant strains. One significant limitation of using phages as antimicrobial agents is their low cellular uptake, which limits their effectiveness against intracellular bacterial infections. Therefore, finding ways to enhance phage uptake is crucial. Our study provides a straightforward strategy for displaying cell-penetrating peptides on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. This approach has the potential to address the global challenge of antibiotic resistance and improve public health outcomes.


Figure S1 .
Figure S1.Predicted Local Distance Difference Test (LDDT) and alignment error per position of the 3D structure of GP94 generated by AlphaFold2.(A) The predicted LDDT scores of the predicted structure by AlphaFold2.These scores are a per-residue measure of how confident AlphaFold2 is about its prediction.(B) The predicted alignment error, which can be used to interpret the relative position of domains.A low predicted alignment error (blue) between the residues of different domains indicates thatAlphaFold2 predicts the relative positions of these domains well.When the predicted alignment error between domains is high (red), the relative position of these domains is uncertain.

Figure S2 .
Figure S2.Structure alignment of the GP94 and its similar protein.Structure alignment of protein GP94 (blue) with tail tube protein (pb6) of phage T5 (A), tail tube protein (gpV) of phage lambda (B), and hoc protein of phage RB49 (C).GP94 structure is colored in blue and the other three protein structures are colored in orange.The no-aligned structure is colored in grey for all proteins.The figure was created via the RCSB.orgweb portal.

Figure S4 .
Figure S4.Characteristics of the WT selz and CPP modified phages.(A) Plaque morphology of WT selz and CPP modified phages on a bacterial lawn of SL1344.(B) The one-step growth curve of WT selz and engineered phages (n=3).Values represent the mean with standard deviation.

Figure S5 .
Figure S5.Cellular uptake of WT and engineered selz phage in different cell lines.Phages were incubated with THP-1 (A) and Raw264.7 cells (80-90% confluence) at 1.5*10 9 PFU for 4 h.Cells were washed with phosphate-buffered saline (PBS) buffer four times, then lysed by ddH2O, then functional phages were quantified using plaque assay.Data are presented as median with interquartile range (IQR) of the results from two independent experiments (n = 6-8, ns, no significance, the comparison was exclusively performed between individual engineered phage and WT selz phage).

Figure S7 .
Figure S7.3D reconstruction of confocal images.A549, Caco-2, and Hela cells were incubated for 4 h with NHS-AF488 labeled selzHA-TAT phages on 35*35 mm glass bottom microscope dishes before live acquisition of a high-resolution z stack to visualize phage dispersion inside of cells.The lysosomal stain LysoTracker (red) and cell nuclei stain Hoechst (blue) were added to the medium 20 min before the end of incubation with phage.The cross in the center of the image shows a cluster of internalized phages with its Z dimension (depth) represented in the side views.Scale bar, 20 μm.

Table S1
. Result of the DALI query on GP94 domain 2 (residue 92-171) and 3 (residue 172-269).Only show results related with phage proteins.rmsd: root-mean-square deviation of C  atoms in the least-squares superimposition of the structurally equivalent C  atoms; lali: number of structurally equivalent residues; % id: percentage of identical amino acids over all structurally equivalent residues.

Table S2 .
Primer used in this study

Table S3 .
Pfam families used to search for Ig-like domains in phage genomes.