Bioinformatics-driven discovery of novel Clostridioides difficile lysins and experimental comparison with highly active benchmarks

Abstract

Clostridioides difficile is the single most deadly bacterial pathogen in the United States, and its global prevalence and outsized health impacts underscore the need for more effective therapeutic options. Towards this goal, a novel group of modified peptidoglycan hydrolases with significant in vitro bactericidal activity have emerged as potential candidates for treating C. difficile infections (CDI). To date, discovery and development efforts directed at these CDI-specific lysins have been limited, and in particular there has been no systematic comparison of known or newly discovered lysin candidates. Here, we detail bioinformatics-driven discovery of six new anti-C. difficile lysins belonging to the amidase-3 family of enzymes, and we describe experimental comparison of their respective catalytic domains (CATs) with highly active CATs from the literature. Our quantitative analyses include metrics for expression level, inherent antibacterial activity, breadth of strain selectivity, killing of germinating spores, and structural and functional measures of thermal stability. Importantly, prior studies have not examined stability as a performance metric, and our results show that the panel of eight enzymes possess widely variable thermal denaturation temperatures and resistance to heat inactivation, including some enzymes that exhibit marginal stability at body temperature. Ultimately, no single enzyme dominated with respect to all performance measures, suggesting the need for a balanced assessment of lysin properties during efforts to find, engineer, and develop candidates with true clinical potential.

Keywords: C. difficile; antibiotic; autolysin; peptidoglycan hydrolase; thermostability.

Figure 1.

Sequence analysis of C. difficile CAT candidates. (A) A circular cladogram of lysin candidates identified by PSI-BLAST of the PlyCD and CD27L seed lysins. The homology search yielded 2,109 total sequences. Shown is a cladogram of representative sequences clustered at 95% identity. The search templates are highlighted in cyan, and novel lysin candidates from this study are highlighted in orange. (B) Multiple sequence alignment of catalytic domains (CATs) from the eight lysins in this study. Key functional residues based on prior structural analysis of CD27L are marked with arrows: catalytic glutamate (pink), zinc ion coordination (purple), conserved hydrogen bonding network (orange), and Clostridia selectivity determinants (blue).

Figure 2.

Purity analysis of CATs. SDS-PAGE analysis following 1-step IMAC purification of CATs. Lanes 1, 6, and 8=protein ladder; lane 2=CDC192; lane 3=CDHM11; lane 4=CD27L; lane 5=PlyCD; lane 7=CDW0003; lane 9=CDBJ082; lane 10=CD6356; lane 11=CDS9P1.

Figure 3.

Specific lytic rates of CATs towards panel of C. difficile isolates. Strains are (A) BAA-1801, (B) BAA-1382, (C) BAA-1814, and (D) BAA-1812. (E) Average normalized activity across the entire panel of 10 C. difficile isolates, where the remaining isolates are provided in Fig. S2. Shown are mean and standard deviation of assay replicates (n=5).

Figure 4.

Stability analysis of CATs. (A) T_m as determined through DSF. (B and C) Incubation time until loss of 50% activity was determined for CATs stored at (B) 22 °C and (C) 37 °C. CATs that failed to drop below 50% residual activity over the testing period are reported as >168 hours, shown as a hashed line in panels B and C. Note panel A temperatures are on a linear scale axis and panel B and C times are on log scale axes.

Figure 5.

Bactericidal activity of CATs towards actively germinating spores. CFUs from quantitative cultures following germination of C. difficile strain BAA-1801 spores and CAT treatment. Shown are mean and standard deviation of assay replicates (n=4).