Cellular adaptation of Clostridioides difficile to high salinity encompasses a compatible solute-responsive change in cell morphology

Abstract

Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacte rial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environ mental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation path ways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthe sis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addi tion of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC transporter imported most of the used compatible sol utes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of L-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.