New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp

Abstract

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Keywords: Rothia; bacteriolytic; bacteriophages; endolysin; phageome; stomach.

Figure 1

Approximate maximum likelihood (ML) tree of proteins similar to PolaR retrieved from the RefSeq database. The dendrogram was calculated using FastTree 2.1.11 with the Jones–Taylor–Thornton (JTT) model. Branch labels represent support from the Shimodaira–Hasegawa test. Only the two clades closest to PolaR are shown. The complete tree is available as newick/nexus in Supplementary Materials File S1.

Figure 2

Structural characteristics of PolaR endolysin. (a) The domain composition of the lysin was predicted using the InterProScan tool and elements of the secondary structures were assigned based on the AlphaFold2 structure of the monomer using the DSSP method, (b) MMseqs2 MSA coverage plot that shows similarity to potential templates located in the ColabFold databases, (c,d) three-dimensional models of the monomer and putative dimer of PolaR were predicted by AlphaFold2 and visualized using ChimeraX. The ribbon representations are colored according to the polypeptide chain. Residues on the surface representations are colored based on their charge, ranging from red (positive) to blue (negative). Letter “N” and “C” on panels (a,c,d), represents amino-terminus and carboxyl-terminus of the protein respectively.

Figure 3

Antibacterial activity of PolaR against Rothia spp. over time. (a) Rothia dentocariosa PCM 2348 (ATCC 14189), (b) Rothia dentocariosa PCM 2349 (ATCC 17931), (c) Rothia mucilaginosa PCM 2403 and (d) Rothia mucilaginosa PCM 2415 (ATCC 25296) were tested in the Sytox Green fluorometric assay with three different PolaR concentrations: 1, 25 and 100 µg/mL. Increasing fluorescence represents an increasing number of killed bacterial cells. All samples were tested in triplicate. The presented data represent one of three independently conducted experiments on three independently produced PolaR preparations.

Figure 4

The effect of PolaR on Rothia mucilaginosa PCM 2415 (ATCC 25296) aggregates. (a) LIVE/DEAD staining of Rothia mucilaginosa PCM 2415 (ATCC 25296) after 2–4 h treatment with PBS or 150 µg/mL of PolaR. (b) Ratio of aggregate size in 2–4 h after PBS or 150 µg/mL PolaR treatment. PBS fold change is marked as a red dashed line (* p = 0.02).

Figure 5

Antibacterial activity of 150 µg/mL PolaR against sonicated and non-sonicated Rothia mucilaginosa PCM 2415 (ATCC 25296) was tested over time in a fluorometric assay. Increasing fluorescence represents an increasing number of killed bacterial cells.

Figure 6

Cytotoxicity of PolaR testing on mammalian cells in vitro. FaDu (ATCC HTB-43), RAW 264.7 (ATCC TIB-71) and A549 (ATCC CCL-185) cell lines were treated with 0.1, 1, 10 or 100 µg/mL of PolaR. PBS of the same volume served as a control. Cells were incubated with PolaR for 48 h (ns p > 0.2).