Characterization of Putative Oxidoreductase Involved in Resazomycin Resistance in Francisella tularensis

Abstract

Antibiotic resistance poses a critical threat to global public health, emphasizing the need
to identify and characterize novel bacterial resistance mechanisms. Our laboratory
discovered resazurin-based compounds, known as resazomycins, exhibit antimicrobial
activity against Francisella tularensis, the causative agent of tularemia. Previous studies
have shown that overexpression of FTL_0445 in F. tularensis confers resistance to
resazurin. FTL_0445 is annotated to encode for a NADPH-dependent FMN
oxidoreductase that we hypothesize reduces or modifies resazurin, thereby mediating
antibiotic resistance in F. tularensis. To investigate this hypothesis, the FTL_0445 gene
was amplified and cloned into the linearized pET45b (+) expression vector for
heterologous expression in Escherichia coli using In-Fusion cloning. Following
transformation into E. coli, colonies were isolated on LB + ampicillin plates, and plasmid
DNA was extracted using a mini-prep kit. Restriction digest analysis of purified plasmids
revealed DNA fragments of expected sizes: approximately 5300 bp for the pET45b (+)
vector and approximately 600 bp for the FTL_0445 insert, confirming successful cloning
and proper vector-insert assembly. Currently, efforts are underway to induce protein
expression and purify the FTL_0445-encoded protein in E. coli for biochemical
characterization. Subsequent assays will assess its potential oxidoreductase activity
toward resazurin derivatives to further elucidate the gene’s role in resazomycin
resistance. Understanding the function of FTL_0445 could provide valuable insight into
the molecular basis of antibiotic resistance in F. tularensis and may guide the future
development of novel therapeutic strategies to combat antibiotic-resistant pathogens.