Work

Gain of Function Cytolysin Variant Expressed by Clinically Isolated Urinary Enterococcus faecalis

Year: 2026

Type: preprint

Abstract: ABSTRACT Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it is not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis , Enterococcus raffinosus , Staphylococcus capitis , and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum . Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment. IMPORTANCE Enterococcus faecalis often causes hospital-acquired opportunistic infections, especially urinary tract infections. The virulence factor, cytolysin, is a plasmid-encoded toxin that can lyse red blood cells (RBCs), immune cells, and bacterial cells, and has been implicated in rabbit endocarditis and endophthalmitis models. E. faecalis cytolysin has been reported to lyse human, horse, rabbit, and mouse RBCs, but not sheep. This is thought to be due to the higher levels of phosphatidylcholine (PC) in susceptible RBCs, suggesting cytolysin targets PC. Here, we report a urinary E. faecalis strain C33 that lyses sheep RBCs and does not require PC for membrane lysis, unlike previously reported E. faecalis strains. Our findings suggest a potentially distinct target receptor for the gain of function C33 cytolysin. Additionally, we found enhanced C33 cytolysin activity against human bladder epithelial cells and the urinary microbiota, suggesting potentials roles in competition, colonization, or pathogenesis in the urinary niche.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.02.756073