Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers

Abstract

Bacterial viruses are among the most numerous biological entities within the human body. These viruses are found within regions of the body that have conventionally been considered sterile, including the blood, lymph, and organs. However, the primary mechanism that bacterial viruses use to bypass epithelial cell layers and access the body remains unknown. Here, we used in vitro studies to demonstrate the rapid and directional transcytosis of diverse bacteriophages across confluent cell layers originating from the gut, lung, liver, kidney, and brain. Bacteriophage transcytosis across cell layers had a significant preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total bacteriophages applied being transcytosed over a 2-h period. Bacteriophages were capable of crossing the epithelial cell layer within 10 min with transport not significantly affected by the presence of bacterial endotoxins. Microscopy and cellular assays revealed that bacteriophages accessed both the vesicular and cytosolic compartments of the eukaryotic cell, with phage transcytosis suggested to traffic through the Golgi apparatus via the endomembrane system. Extrapolating from these results, we estimated that 31 billion bacteriophage particles are transcytosed across the epithelial cell layers of the gut into the average human body each day. The transcytosis of bacteriophages is a natural and ubiquitous process that provides a mechanistic explanation for the occurrence of phages within the body.IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria. They cannot infect eukaryotic cells but can penetrate epithelial cell layers and spread throughout sterile regions of our bodies, including the blood, lymph, organs, and even the brain. Yet how phages cross these eukaryotic cell layers and gain access to the body remains unknown. In this work, epithelial cells were observed to take up and transport phages across the cell, releasing active phages on the opposite cell surface. Based on these results, we posit that the human body is continually absorbing phages from the gut and transporting them throughout the cell structure and subsequently the body. These results reveal that phages interact directly with the cells and organs of our bodies, likely contributing to human health and immunity.

Keywords: bacteriophages; endocytosis; phage-eukaryotic interaction; symbiosis; transcytosis.

FIG 1

Transcytosis of bacteriophages occurs in a preferential apical-to-basal direction across diverse cell layers. (A) Experimental system to investigate phage transcytosis. Phage T4 was applied to either the apical or basolateral (basal) cell chambers and incubated for 2 h at 37°C, and transcytosed phages were sampled and quantified in the contralateral chamber by bacterial plating. (B) Transcytosis of T4 phages across Madin-Darby canine kidney (MDCK) cells in either an apical-to-basal or basal-to-apical direction. (C) Transcytosis of T4 phages across T84 cells (colon epithelial), CaCo2 cells (colon epithelial), A549 cells (lung epithelial), Huh7 cells (hepatocyte epithelial cell-like), and hBMec cells (brain endothelial). Scatter plots show medians; error bars represent 95% confidence intervals; each point represents the average from three technical replicates.

FIG 2

Rate of apical-to-basal phage transcytosis across diverse phages. (A) Rate of T4 phage transcytosis across MDCK cells over a 2 h period. (B) Transcytosis of T4 phage applied to MDCK cells at sequentially decreasing log₁₀ concentrations. (C) Transcytosis of unprocessed (4 × 10⁴ endotoxin units [EU] ml⁻¹) and cleaned (1.4 EU ml⁻¹) T4 phages across MDCK cells. (D) Transcytosis of diverse phage types (T3, T5, T7, SP01, SPP1, and P22 phages) across MDCK cells. Bar plot shows mean; error bars show minimum and maximum values. Scatter plots show medians; error bars represent 95% confidence intervals; each point represents the average from two technical replicates.

FIG 3

MDCK cells were grown on gridded dishes, incubated with SYBR gold-labeled T4 phages (green), and imaged under differential interference contrast and confocal microscopy. Red arrows indicate cells containing discrete fluorescent signal; blue arrows indicate cells with diffuse fluorescent signal spread across the cytoplasm.

FIG 4

Visualization of intracellular phages. (A to I) Representative correlative micrographs of an MDCK cell stained with Hoechst stain (blue) and CellMask (red) after application of T4 phages stained with SYBR gold (green). (A and B) SYBR gold-positive target cell was selected during confocal microscopy (A) and then processed and aligned for inspection of the same structures by transmission electron microscopy (TEM) (B). (C to I) Spatially aligned electron micrographs showing SYBR gold-positive endomembrane structures (G and H), adjacent to SYBR gold-negative virus particles (F and I). (J and K) Representative electron micrographs of extracellular (J) and intracellular (K) virus particles found in CLEM samples. Data in panel A are maximum projection between the 37th and 43rd optical sections (3.0 µm to 4.2 µm above coverslip surface). Data in panel B are a distortion-corrected TEM montage from the 47th resin section (3,670-nm sample depth, 85 nm thick) acquired at 25 kx. Arrows indicate virus-like particles within membrane-bound vesicles. Data used for spatial alignment are shown in Fig. S2. Bars, 10 µm (A and B), 500 nm (C to E), and 100 nm (F to K).

FIG 5

MDCK cells were treated with T4 phage labeled with both the DNA-complexing SYBR gold stain (green) and capsid-conjugated Cy3 stain (red) for either 30 min (A) or 2 h (F) and imaged under confocal microscopy. Cells were stained with Hoechst stain (blue) and CellMask (white) after application of T4 phage. The 30-min treatment sample showed correlation of DNA and capsid fluorescence signals, compared with the 2 h treatment, where there was a disassociation of fluorescence. Bar, 10 μm.

FIG 6

Subcellular fractionation of phage-treated MDCK and A549 cells. (A) Fractionation of MDCK cells treated with T4 phages (lysate) for 18 h. Cells were washed and lysed using the lysosome enrichment kit, and the total number of phages in cells (Total Cell Lysate) was determined. Total cell lysates were fractionated, and 10 cellular fractions were collected and split for either phage quantification by bacterial plating or Western blot analysis of Golgi apparatus and endoplasmic reticulum (ER). (B) Fractionation of A549 cells treated with T4 phages for 18 h. Scatter plots show medians; error bars represent 95% confidence intervals. Bar plot shows mean; error bars show standard deviations.

FIG 7

Inhibition of phage transcytosis. Percent transcytosis of T4 phages across MDCK cells pretreated with chemical inhibitors for 18 h compared to a solvent control. Bar plot shows mean; error bars show standard deviations.