Identification and characterization of a periplasmic trilactone esterase, Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter

Abstract

Summary Ferric enterobactin (FeEnt) acquisition is a highly efficient and conserved iron scavenging system in Gram-negative bacteria. Recently, we have characterized two FeEnt receptors (CfrA and CfrB) in Campylobacter jejuni and C. coli, the enteric human pathogens that do not produce any siderophores. In this study, whole genome sequencing and comparative genomic analysis identified a unique Ent trilactone esterase Cee (Cj1376) in C. jejuni. Genomic analysis and biochemical assay strongly suggested that Cee is the sole trilactone esterase in C. jejuni. Thin layer chromatography and HPLC analyses showed high efficiency of the purified Cee to hydrolyze Ent. Three Cee homologs previously characterized from other bacteria (IroE, IroD, and Fes) were also purified and analyzed together with Cee, indicating that Cee, Fes, and IroD displayed similar hydrolysis dynamics for both apo and ferric forms of Ent while IroE catalyzed Ent inefficiently. Unlike cytoplasmic Fes and IroD, Cee is localized in the periplasm as demonstrated by immunoblotting using Cee-specific antibodies. Genetic manipulation of diverse Campylobacter strains demonstrated that Cee is not only essential for CfrB-dependent FeEnt acquisition but also involved in CfrA-dependent pathway. Together, this study identified and characterized a novel periplasmic trilactone esterase and suggested a new model of FeEnt acquisition in Campylobacter.

Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product

“…As a result, the Fe(III) complex of enterobactin is retarded in the periplasm where it is exposed to esterase-catalyzed hydrolysis to its linear components. Because it has been reported that C. jejuni can use bisDHBS 5− as a siderophore independently of enterobactin (10) and the discovery of the trilactone esterase Cee in the periplasm of C. jejuni (25), it appears that CeuE, previously considered to be a [Fe(ENT)] 3− binding protein, is instead adapted to preferentially bind [Fe(bisDHBS)] 2− . This ability to use the enterobactin hydrolysis product for Fe(III) uptake and hence to avoid the metabolic costs of siderophore production provides C. jejuni with a competitive advantage when it encounters enterobactin-secreting bacteria either within the host or contaminated water.…”

“…The identification of the esterase Cee and in particular the observation that bisDHBS 5− can be used independently of enterobactin (25) raise important questions about the siderophore preference of the PBP CeuE and its role in the iron uptake in C. jejuni. By using siderophore mimics, we previously demonstrated that CeuE can bind the Fe(III) complexes of both hexadentate and tetradentate catecholate ligands (26,27).…”

“…An alternative Campylobacter Fe(III) acquisition model that relies on these linear hydrolysis products was recently proposed based on the finding that Cee, the sole trilactone esterase of C. jejuni and Campylobacter coli, is located in the periplasm, i.e., these bacteria are unable to degrade enterobactin within the cytoplasm (25). The model suggests that, once the Fe(III) complex of enterobactin enters the periplasm, its ester backbone is cleaved by Cee, which is highly efficient in hydrolyzing both the Fe(III) complex and apo-enterobactin.…”

Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4− siderophore analogues of varied linker length

“…1)17. Indeed, the periplasmic enterobactin esterase Cee has been shown to play an important role in the uptake of Fe(III) in C. jejuni , since it hydrolyzes the enterobactin backbone in the periplasm followed by the import of Fe(III)-enterobactin fragment complexes, including bis(2,3-dihydroxybenzoyl-L-Ser), into the cytoplasm18. The observation that there is no known cytoplasmic enterobactin esterase in C. jejuni suggests that hexadentate analogues may first require cleavage to their tetradentate counterparts before transportation into the cytoplasm18.…”

Campylobacter jejuni ferric–enterobactin receptor CfrA is TonB3 dependent and mediates iron acquisition from structurally different catechol siderophores

“…C. jejuni has been shown to acquire iron through enterobactin, hemin, transferrin/lactoferrin and through ferrous ion import [21–26]. Iron acquisition has also been demonstrated to be an important colonization and virulence determinant in C. jejuni [21, 22, 27]. Although C. jejuni is unable to synthesize siderophores such as enterobactin, it is able to utilize exogenous enterobactin as an iron source through the action of the CfrA or CfrB transporters [21, 25–27].…”