Lytic Bacteriophage EFA1 Modulates HCT116 Colon Cancer Cell Growth and Upregulates ROS Production in an Enterococcus faecalis Co-culture System

Abstract

Enterococcus faecalis is an opportunistic pathogen in the gut microbiota that's associated with a range of difficult to treat nosocomial infections. It is also known to be associated with some colorectal cancers. Its resistance to a range of antibiotics and capacity to form biofilms increase its virulence. Unlike antibiotics, bacteriophages are capable of disrupting biofilms which are key in the pathogenesis of diseases such as UTIs and some cancers. In this study, bacteriophage EFA1, lytic against E. faecalis, was isolated and its genome fully sequenced and analyzed in silico. Electron microscopy images revealed EFA1 to be a Siphovirus. The bacteriophage was functionally assessed and shown to disrupt E. faecalis biofilms as well as modulate the growth stimulatory effects of E. faecalis in a HCT116 colon cancer cell co-culture system, possibly via the effects of ROS. The potential exists for further testing of bacteriophage EFA1 in these systems as well as in vivo models.

Keywords: Enterococcus faecalis; bacteriophage; biofilm; colon cancer proliferation; genomics; reactive oxygen species.

FIGURE 1

EFA1 plaque morphology, TEM image and growth kinetics. Pin prick plaques with diameter approximately 0.3 mm on E. faecalis host (A). TEM revealed Siphoviridae morphology with capsid diameter of 58 ± 3 nm and tail length of 223 ± 7 nm (B). EFA1 OSGC: latent phase of 20 min; burst lasting 10 min; plateau phase occurs after approximately 30 min (C).

FIGURE 2

Bacteriophage EFA1 genome orientated according to bacteriophage termini (PAC site). Putative genes coding for DNA packaging (purple), capsid genes (green), tail genes (blue) and putative endolysin (red) are orientated in the left-to-right direction, while putative DNA manipulation genes (pink) are in the opposite direction. Other putative genes whose functionality could not be predicted were considered hypothetical genes (yellow).

FIGURE 3

Amino acid alignment of the E. faecalis beta-lactamase protein (EPH86145) with EFA1 bacteriophage putative beta-lactamase protein (ORF number 33). Red colored regions highlight areas of identical amino acids [47/272 (17.3%)] between the bacterial and bacteriophage putative beta-lactamase proteins, whereas the non-colored areas define regions where amino acids were non-identical.

FIGURE 4

Proteomic tree showing the genome-wide proteomic diversity between bacteriophage EFA1 (highlighted in bold font) and related bacteriophage genomes. Bacteriophages targeting E. faecalis are highlighted in purple while those targeting E. faecium are highlighted in green. Color coded bar represents the family and subfamily of the bacteriophage (key is at top left).

FIGURE 5

Effects of EFA1 bacteriophage on E. faecalis biofilm viability and mass. (A) Untreated E. faecalis biofilm showing bacteria attached to slide were predominantly membrane intact cells (green), with some bacteria with leaky membranes (red/brown). (B) Bacteriophage EFA1 treated biofilm with scant cells remaining attached to slide. (C) Quantification of biofilm at time 0 h (without EFA1 treatment) is significanlty greater than any other biofilm treated with bacteriophage EFA1. The lowest amount of biofilm mass was seen after 2 h treatment. Biofilm mass steadly increased with exposure to EFA1. (D) 5 days biofilm treated with EFA1 for 24, 2, and 24 h + 2 h, showing similar disruption at 24 + 2 h exposure as that for 2 h.

FIGURE 6

Bacteriophage EFA modulates the effects of E. faecalis on HCT116 cancer cell line by increasing ROS production and attenuating cancer cell proliferation. (A) Cancer cell proliferation was significantly increased in E. faecalis co-cultured HCT116 cells compared to all other cultures. The RPMI growth media and E. faecalis growing in RPMI showed minimal SRB absorbance. HCT116 cells grown with purified bacteriophage EFA1, and the HCT116 cells grown on their own, had similar levels of proliferation. Treatment of the HCT116 cells/E. faecalis co-culture with purified bacteriophage EFA1 resulted in significant reduction of cancer cell growth, to levels below those of the control HCT116 culture. (B) ROS production was significantly increased in the HCT116/E. faecalis co-culture with purified EFA1, and to a lesser extent in the HCT116/E. faecalis co-culture, compared to HCT116 cells on their own. There was no statistical difference in ROS production between untreated HCT116 and those grown with purified bacteriophage EFA1.