Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells

Abstract

Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host and can be genetically modified for different applications. However, whether and how bacteriophages can kill intracellular bacteria in human cells remains elusive. Here, using CRISPR/Cas selection, we have engineered a fluorescent bacteriophage specific for E. coli K1, a nosocomial pathogen responsible for urinary tract infections, neonatal meningitis and sepsis. By confocal and live microscopy, we show that engineered bacteriophages K1F-GFP and E. coli EV36-RFP bacteria displaying the K1 capsule, enter human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP efficiently kills intracellular E. coli EV36-RFP in T24 human urinary bladder epithelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy.

Bacteriophage K1F targets Escherichia coli K1 in cerebral endothelial cells and influences the barrier function

“…n = 3 in each case. (2020) 10:8903 | https://doi.org/10.1038/s41598-020-65867-4 www.nature.com/scientificreports www.nature.com/scientificreports/ Confocal microscopy image analysis also confirmed observations of previous studies performed in T24 human urinary epithelial cells 19 , showing the ability of phage K1F-GFP to locate and infect E. coli EV36-RFP in a human cell environment and the formation of intracellular vacuoles containing phage-aggregates in human cells.…”

“…It is notable that both stains are membrane-impermeable and thus the fluorescent signal is only present in the brief interval from initial lysing to complete dismantlement of the bacterial membrane. The ability and efficiency of phage K1F-GFP to locate and infect intracellular E. coli EV36 in a human cell environment has been confirmed in previous studies 19 , however, the signal stability of GFP is often a point for discussion. We therefore initially assessed the fluorescent signal emitted by phage K1F-GFP during infection in E. coli EV36 using flow cytometry.…”

“…This is considerably higher than the infection rate previously obtained of approx. 20% in human urinary bladder epithelial cells (T24) 19 . This highlighted inherent tissue-specific properties and the necessity for unique tissue-specific model systems to study the mechanism of bacterial infection and phage intervention.…”

“…The internalisation and degradation of phage K1F-GFP and E. coli EV36 in T24 human urinary epithelial cells has previously been studied in the context of in vitro phage therapy 19 . Here we expanded on this study using qualitative confocal microscopy and quantitative flow cytometry to examine the internalization and processing of phage K1F in human cerebral microvascular endothelial cells (hCMECs).…”

Isolation and Characterization of AbTJ, an Acinetobacter baumannii Phage, and Functional Identification of Its Receptor-Binding Modules

“…The adsorption of the phage on the surface of bacteria is the first and most important step in the process of phage infection [31]. In this study, the genes gp52 (Figure 8) indicated that the tail fiber proteins gp52 and gp53 had the ability to recognize and capture A. baumannii.…”