Searching for a Bacteriophage Lysin to Treat Corynebacterium bovis in Immunocompromised Mice

Abstract

Corynebacterium bovis is the causative agent of Corynebacterium-associated hyperkeratosis in immunocompromised mice. The resulting skin pathology can be profound and can be associated with severe wasting, making the animals unsuitable for research. Although the administration of antibiotics is effective in resolving clinical symptoms, antibiotics do not eradicate the offending bacterium. Furthermore, antibiotic use may be contraindicated as it can affect tumor growth and is associated with Clostridioides difficile enterotoxemia in highly immunocompromised murine strains. Lysins, which are lytic enzymes obtained from bacteriophages, are novel antimicrobial agents for treating bacterial diseases. The advantage of lysins are its target specificity, with minimal off-target complications that could affect the host or the biology of the engrafted tumor. The aim of this study was to identify lysins active against C. bovis. Chemical activation of latent prophages by using mitomycin C in 3 C. bovis isolates did not cause bacteriophage induction as determined through plaque assays and transmission electron microscopy. As an alternative approach, 8 lysins associated with other bacterial species, including those from the closely related species C. falsenii, were tested for their lytic action against C. bovis but were unsuccessful. These findings were congruent with the previously reported genomic analysis of 21 C. bovis isolates, which failed to reveal bacteriophage sequences by using the PHAST and PHASTER web server tools. From these results, we suggest C. bovis is among those rare bacterial species devoid of lysogenic bacteriophages, thus making the identification of C. bovis-specific lysins more challenging. However, C. bovis may be a useful model organism for studying the effects of antiphage systems.

Figure 1.

Growth curve of C. bovis. A turbid culture of C. bovis strain 7894 was diluted 1:100 in fresh BHI medium supplemented with Tween 80 and subsequently incubated at 37 °C with aeration for a total of 48 h. The bacterial concentration was quantitated every 6 h by either (A) measuring the OD₆₀₀ or (B) serially diluting and plating to determine the number of cfu per mL. Error bars, SEM of duplicate experiments.

Figure 2.

Analyzing the lytic activity of several non-Corynebacterium lysins against C. bovis by means of the turbidity reduction assay. (A) To confirm each lysin was enzymatically functional, PlyC (S. pyogenes), PlySs2 (S. aureus), PlyG (B. anthracis), Cpl-1 (S. pneumoniae), PlyCD (C. difficile), LysK (S. aureus) and ClyS (S. aureus) were incubated at 25 µg/mL with a susceptible bacterial species. (B) The bacteriolytic activity of each lysin at 25 µg/mL was determined against C. bovis strain 7894. For each turbidity reduction assay, the OD_600nm was measured every 15 s for 1 h at 37 °C. A decrease in turbidity is indicative of bacteriolytic activity. Bacteria absent lysin treatment were used as a negative control for lytic activity. Each sample was analyzed in duplicate.

Figure 3.

Measuring the concentration of ATP released by C. bovis when treated with various non-Corynebacterium lysins. ATP assays were used to measure the amount of intracellular ATP released, as a function of luminescence, by C. bovis strain 7894 after incubation with PlyC, PlySs2, PlyG, Cpl-1, PlyCD, LysK, and ClyS (final concentration, 25 µg/mL) for 1 h at 37 °C. S. pyogenes treated with PlyC was used as a positive control; ATP-free water, buffer only, and untreated bacteria were used as negative controls. Error bar, SEM of duplicate experiments. RLU, relative light units.

Figure 4.

Evaluating the bacteriolytic activity of the Corynebacterium lysin PlyCf against C. bovis. In a turbidity reduction assay, the C. falsenii lysin PlyCf (final concentration, 125 µg/mL) with either (A) C. falsenii strain ATCC BAA-596 (positive control) or (B) C. bovis strain 7894. OD₆₀₀ was measured every 30 s for 1 h at room temperature. A decrease in turbidity is indicative of bacteriolytic activity. Bacteria absent lysin treatment were used as a negative control for lytic activity. Each sample was analyzed in duplicate.