Structural and Functional Characterization of Aerobactin Synthetase IucA from a Hypervirulent Pathotype ofKlebsiella pneumoniae

Abstract

Iron is a vital mineral nutrient required by virtually all life forms to prosper; pathogenic bacteria are no exception. Despite the abundance of iron within the human host, highly regulated iron physiology can result in exceedingly low levels of iron bioavailable to prospective invading bacteria. To combat this scarcity of iron, many pathogenic bacteria have acquired specific and efficient iron acquisition systems, which allow them to thrive in iron-deficient host environments. One of the more prominent bacterial iron acquisition systems involves the synthesis, secretion, and reuptake of small-molecule iron chelators known as siderophores. Aerobactin, a citrate-hydroxamate siderophore originally isolated nearly 50 years ago, is produced by a number of pathogenic Gram-negative bacteria. Aerobactin has recently been demonstrated to play a pivotal role in mediating the enhanced virulence of a particularly invasive pathotype of Klebsiella pneumoniae (hvKP). Toward further understanding of this key virulence factor, we report the structural and functional characterization of aerobactin synthetase IucA from a strain of hvKP. The X-ray crystal structures of unliganded and ATP-bound forms of IucA were solved, forming the foundation of our structural analysis. Small angle X-ray scattering (SAXS) data suggest that, unlike its closest structurally characterized homologues, IucA adopts a tetrameric assembly in solution. Finally, we employed activity assays to investigate the substrate specificity and determine the apparent steady-state kinetic parameters of IucA.

Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae

“…Functional characterization of the Type A NIS synthetase AcsD described a highly stereoselective mechanism for the condensation of citrate and L-serine (38). Furthermore, structural models of the AcsD and IucA active sites predicted a mechanism in which only one of the two pro-chiral carboxymethyl groups of citrate is activated by ATP to form a citryladenylate intermediate (36,38). Experiments presented herein confirmed the structural predictions by functionally demonstrating the stereoselective mechanisms of IucA and IucC.…”

“…Finally, we report the steady-state kinetics, and structural characterization of IucC by X-ray crystallography and small-angle solution scattering. Together with our previous studies of IucA (36), hvKP IucA and IucC represent the first pair of Type A and Type C NIS synthetases from a single pathway to be structurally and functionally characterized. This work furthers our understanding of NIS biosynthesis and provides the biochemical and structural foundation to further explore this potential antivirulence target.…”

“…Furthermore, no one has described the in vitro reconstitution of aerobactin biosynthesis to functionally demonstrate the proposed pathway. Recent structural and functional studies of NIS synthetases, including hvKP IucA (36), achromobactin synthetase AcsD (37,38), as well as petrobactin synthetases AsbA (39) and AsbB (40), have all advanced our understanding of the catalytic function of these closely-related enzymes.…”

Emergence of a novel mobile colistin resistance gene, mcr-8 , in NDM-producing Klebsiella pneumoniae

“…Whole-genome sequencing indicated that KP91 belonged to the ST42 group and contained multiple antimicrobial resistance genes, including the β-lactam resistance genes bla SHV-1 and bla CTX-M-14 , aminoglycoside resistance genes strA , strB , armA , and aph(4)-Ia , macrolide resistance genes mph (E) and msr (E), quinolone resistance genes oqxA and qnrB4 , sulfonamide resistance genes sul1 , sul2 , and sul3 , tetracycline resistance genes tet (A), tet (B), and tet (34), and trimethoprim resistance gene dfrA12 (Table 1 ). KP91 also carried multiple virulence genes, including kfuC , mrkABCDF , irp2 , iucA and iucB , which have been associated with urinary tract infections, septicemia, and pneumonia 19 – 21 .…”

Discovery of anti-mucoviscous activity of rifampicin and its potential as a candidate antivirulence agent against hypervirulent Klebsiella pneumoniae

“…Strains of hvKP typically possess biosynthetic and transport genes for aerobactin (iucABCD and iutA) and salmochelin (iroBCDN) on the virulence plasmid [12]. Aerobactin has been shown to be a critical virulence factor under some circumstances [25], leading to aerobactin synthetase (IucA) being thoroughly investigated [26]. Russo et al recently demonstrated that aerobactin is a key marker that distinguishes hvKP from non-hvKP strains [27].…”