Hilde De Reuse

Control of protein folding is a conserved function in all domains of life. The ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases) catalyze the cis / trans -isomerization of peptidyl-prolyl bonds, often a rate-limiting step for protein folding. PPIases are involved in central processes in eucaryotes but their functions in bacteria are poorly understood. We applied a large-scale two-hybrid screen to identify novel targets of SlyD, a metallochaperone PPIase, from the model organism Escherichia coli , and the important gastric pathogen Helicobacter pylori . SlyD from both organisms interacts with metal-containing proteins including TCA cycle enzymes and with enzymes driving the biogenesis of iron-sulfur (Fe-S) clusters. In H. pylori , a ∆ slyD mutant contains strongly diminished amounts of MisSU, its sole Fe-S biogenesis proteins as well as diminished activity of the Fe-S aconitase. E. coli and H. pylori ∆slyD mutants are less susceptible to ampicillin and gentamicin antibiotics and under microaerobic conditions to the ROS-producing molecule hypochlorite. In addition, the intracellular ATP content and proton motive force are reduced in the H. pylori ∆ slyD mutant, and its gentamicin and hypochlorite susceptibility phenotypes are rescued upon restoration of MisSU expression. Measurement of malate and fumarate in the H. pylori ∆slyD mutant suggests that the two enzymes before and after fumarase in the TCA cycle are affected. We conclude that SlyD regulates the activity of TCA cycle enzymes by controlling the efficiency of their Fe-S cluster formation. This is the first report of the role of a bacterial PPIase in central metabolism and susceptibility to stress and antibiotics. IMPORTANCE Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis / trans -isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori , measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.