Investigating Zinc Metalloprotease Protein Mutations in Clostridioides difficile through Multiple Sequence Alignment

Purpose: Antibiotic resistance is a growing health crisis projected to cause more deaths annually than cancer by 2050. Per CDC’s 2019 report, Clostridioides difficile qualifies as an urgent antimicrobial resistance threat. To aid in understanding antibiotic resistance, we studied C. diff protein mutations using all the available sequences on the NCBI website. This approach enabled us to find certain regions of the zinc metalloprotease (Zmp1) protein displaying high rates of mutation, and contributes to our growing understanding of how microorganisms evolve in the context of modern antibiotic use.

Procedure: We inputted the Zmp1 reference sequence into the NCBI BLAST database to search for other similar Zmp1 protein sequences (N=93). Using those sequences, we conducted a multiple sequence alignment via Clustal Omega’s website, then viewed the alignment results in Jalview. Here, we were able to highlight important mutations and count the number of appearances of each amino acid by position using a Python program my mentor, Jacob, had written. Finally, we used ChimeraX, a protein visualization software, to pinpoint the locations of these mutations on a molecular scale.

Data collected & Conclusions: We collected data on the protein’s conservation scores and notable mutations near its active site. The results mostly supported my hypothesis, as the active site in the Zmp1 protein was mostly well conserved. However, a few mutations in the zinc binding site demonstrated significant differences in amino acid structure. For example, the glutamic acid to lysine mutation is very interesting because their side chains have completely different charges.