Protocols for studying bacteriophage interactions with in vitro epithelial cell layers

Abstract

Summary Interactions between bacteriophages and mammalian cells are poorly understood. Establishing common methodologies investigating these interactions is important for advancing our understanding in this area. The protocols presented here provide an overview of key approaches investigating interactions between bacteriophages and eukaryotic cells using a variety of techniques, including transwells, microscopy, and whole-cell analysis. These techniques allow for the direct measurement of phage-cellular interactions and characterization of how the presence of phages affects cellular pathways, cell biology, immunology, and the microbiome. For complete details on the use and execution of this protocol, please refer to Nguyen et al. (2017) and Bichet et al. (2021) .

Mammalian cells internalize bacteriophages and utilize them as a food source to enhance cellular growth and survival

“…Importantly, the MDCK-I samples were analyzed by the KAM-1325 antibody array, while the A549 samples were analyzed using the improved KAM- 2000 antibody array, which includes most of the antibodies from the prior array, along with 675 additional antibodies for improved detection of cellular changes. We expanded our initial characterization of A549 lung epithelial cells and included a second cell line, the MDCK-I dog kidney epithelial cells, which are known to form strong tight junctions and rapidly internalize and traffic high numbers of T4 phages (Figure 1B) (13,33). Cells were incubated with either purified T4 phages or the Filter control sample for eight hours, followed by cell lysis, chemical cleavage, and protein quantification.…”

“…The endotoxin removal protocol followed the Phage on Tap (PoT) protocol (32). The phages lysate was cleaned four times with 1-Octanol to remove endotoxins from the lysate, reducing endotoxins from 5734 EU/mL to 167 EU/mL in the final phage stock solution (10 11 phages/mL) (see also (33). In all experiments, unless otherwise stated, phages were diluted in endotoxin-free buffers to a final concentration of 10 8 PFU/mL (unless otherwise stated), resulting in an endotoxin concentration below 1 EU/mL.…”

“…This phage was selected as it was previously demonstrated to be internalized by mammalian cells, accumulating intracellularly within macropinosomes over time (13,14). Phage lysates were purified and concentrated via ultrafiltration following the Phage on Tap protocol to produce a single, high-titer phage stock that was used for all subsequent assays (32,33). Phage stocks were treated with DNase and RNase to remove extracellular nucleic acids followed by endotoxin removal using 1-Octanol washes.…”

“…KAM-2000 Kinexus antibody microarray. Following the protocol described in (33), confluent A549 cells were incubated with T4 phages or Filter control samples for eight hours at 37 ºC and 5% CO2. After incubation with the phages or control sample, the cells were scraped in lysis buffer before sonication.…”

“…We demonstrate that phages were internalized by mammalian cells via macropinocytosis, with functional phages continually accumulating within macropinosomes ((13,14). All phage preparations were highly purified and confirmed to be free of bacterial endotoxins (32,33). To further ensure the cellular responses detected were elicited by the phages themselves, we used an additional comparative control that consisted of the highly purified phage lysate filtered four times through a 0.02 μm filter to remove phage particles and obtain a phage-free lysate composed of the background supernatant.…”