Clostridioides difficile utilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake

Abstract

Clostridioides difficile remains a public health threat commonly observed following antibiotic use. Due to the importance of iron in many cell processes, most bacteria, including C. difficile, have multiple mechanisms of acquiring iron. Previous studies have examined ferrous iron uptake in C. difficile, here we focus on the role of siderophores. In a growth assay, we show that C. difficile can use a variety of siderophores as the sole iron source. In C. difficile, two ABC transporters induced under low iron conditions are predicted siderophore importers: FhuDBGC and YclNOPQ. We hypothesized that these transporters are responsible for the uptake of the siderophores we tested. To investigate the specificity of these transporters, we purified the substrate binding proteins and examined siderophore binding using thermal shift. We demonstrate increased stability between one siderophore binding protein, FhuD, and the siderophore ferrichrome, suggesting a binding interaction. This specificity correlates with the inability of an{Delta} fhuDBGC mutant to grow efficiently under iron limiting conditions in the presence of ferrichrome. While C. difficile used additional siderophores in our growth experiments, we did not observe increased thermal stability between the receptor proteins and any of the other siderophores tested, suggesting these siderophores do not bind these receptors and other siderophore import mechanisms remain to be elucidated. Redundancy in iron acquisition is a microbial survival adaptation to cope with the constant battle for iron within a host. Greater knowledge about how C. difficile acquires iron will provide insight about how C. difficile colonizes and persists in the colon. IMPORTANCEThis study is the first example of C. difficile growing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms which C. difficile uses to compete for iron during an infection.