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

Abstract

Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive-and PAMP-dependent LC3assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or ifnβ. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally.

Engineered Bacteriophage Therapeutics: Rationale, Challenges and Future

“…Møller-Olsen et al [ 260 ] used CRISPR-Cas-based selection to obtain a GE derivative of a T7-like phage, K1F, equipped with a gene for green fluorescence protein (GFP). The GFP-labeled phage showed some delay in infection as compared to wild-type K1F [ 261 ]. However, tracing K1F-GFP by fluorescence microscopy demonstrated its ability to penetrate human urinary bladder epithelial cells and human cerebral microvascular cells.…”

“…However, tracing K1F-GFP by fluorescence microscopy demonstrated its ability to penetrate human urinary bladder epithelial cells and human cerebral microvascular cells. Moreover, the phage was able to kill inside human cells a hybrid between E. coli strains K12 and K1, the latter being a nosocomial pathogen responsible for urinary tract infections, meningitis, and sepsis [ 260 , 261 ].…”

Preclinical data and safety assessment of phage therapy in humans

“…Further phage therapy testing on appropriate cell lines to mimic infection sites provides extended information on uptake, and phage/bacterial, phage/cell lines and on bacterial/cell lines interactions. Such studies provide valuable data on immune responses, safety, efficacy and toxicity to support phage treatment of pathogenic bacteria in clinical settings [ 52 , 53 , 54 ]. Cell lines that have been used in phage studies include several human cerebral microvascular endothelial cells, epithelial cells most commonly HeLa, A549 and HT29 from the cervix, lung and colon, respectively [ 52 , 53 , 55 ].…”

“…Such studies provide valuable data on immune responses, safety, efficacy and toxicity to support phage treatment of pathogenic bacteria in clinical settings [ 52 , 53 , 54 ]. Cell lines that have been used in phage studies include several human cerebral microvascular endothelial cells, epithelial cells most commonly HeLa, A549 and HT29 from the cervix, lung and colon, respectively [ 52 , 53 , 55 ]. Useful fibroblast cell lines used include BJ from human skin, the endothelial cell line HUVEC from umbilical veins, and monocyte-induced macrophages-THP-1 cells [ 55 ].…”

“…To illustrate the power of these studies, we discuss phage K1F that infects Escherichia coli EV36 displaying the K1 capsule in the presence of human cerebral microvascular endothelial cells (hCMECs) in an in vitro bacterial neonatal meningitis model [ 53 ]. Data from flow cytometry, confocal and live microscopy showed that the bacterium infected hCMECs in a time-dependent and concentration-dependent manner, and that the phage successfully entered the human cells, but was degraded by constitutive-dependent and Pathogen Associated Molecular Patterns (PAMP)-dependent LC3-assisted phagocytosis.…”