Nickel chelators and urease inhibition in Klebsiella pneumoniae and bacterial nickel transport Original paper

What was studied?

This study investigated nickel chelators and urease inhibition to dissect how different classes of small molecules modulate urease activity and nickel uptake in ureolytic bacteria. Using a standardized high-throughput pH indicator assay, the authors screened 71 commercially available compounds for their capacity to inhibit urease-mediated hydrolysis of urea in Klebsiella pneumoniae and purified jack bean urease, then examined how selected metal chelators alter nickel import through canonical bacterial nickel transporters. By integrating urease activity assays with radiolabeled nickel uptake measurements in recombinant Escherichia coli expressing the NiCoT and Nik(MN)QO transporters, the work links extracellular chelation, transporter-specific nickel flux, and intracellular urease activation as a unified nickel-dependent virulence axis in ureolytic pathobionts.

Who was studied?

The primary microbiological model for ureolytic activity was Klebsiella pneumoniae subsp. pneumoniae, a clinically important, urease positive pathobiont implicated in pneumonia, urinary tract infection, and kidney stone formation. Purified jack bean urease served as a cell-free reference enzyme to separate direct catalytic inhibition from cell-level effects such as membrane permeability, metal competition, or global toxicity. For nickel transport experiments, the authors used E. coli XL1 Blue transformed with plasmids encoding either the NiCoT nickel permease from K. pneumoniae or the ATP-dependent energy coupling factor transporter Nik(MN)QO from Rhodobacter capsulatus, together with a Klebsiella aerogenes urease operon. This composite system allowed precise quantification of radiolabeled nickel uptake and urease activity as a function of individual chelators, thereby modelling how structurally distinct ligands may differentially affect nickel acquisition and urease maturation in gram-negative pathobionts that contribute to dysbiosis, uremic toxin generation, and pH-driven barrier injury in the human microbiome.

Most important findings

The screen identified 30 of 71 compounds that reduced the rate and or final extent of urease-driven pH rise by more than 25 percent in either K. pneumoniae or jack bean urease. Substrate analogues such as acetohydroxamic acid and hydroxyurea were highly effective against purified urease but much less active in whole cells, highlighting the barrier imposed by bacterial membranes and the influence of high ambient urea on competitive inhibitors. Transition state analogues phenyl phosphorodiamidate and N (n butyl)thiophosphoric triamide almost completely blocked ureolysis in K. pneumoniae, yet only phenyl phosphorodiamidate fully inhibited jack bean urease, consistent with the requirement for oxidative conversion of NBTPT to its more potent oxon form in order to achieve full catalytic blockade.

Nickel-targeted mechanisms yielded a much more nuanced picture. EDTA and diethylenetriaminepentaacetic acid eliminated urease activity in K. pneumoniae but paradoxically increased activity of purified urease, likely by stripping inhibitory divalent cations such as zinc while lowering effective nickel availability in vivo. Dimethylglyoxime (DMG), nitrilotriacetic acid, and EDTA significantly decreased radiolabeled nickel uptake through both NiCoT and Nik(MN)QO, whereas oxalic acid and 1,2,4 butanetricarboxylic acid actually increased nickel import, especially via NiCoT. In the recombinant E. coli urease system, EDTA exerted the strongest reduction in urease activity, DMG and NTA showed intermediate effects, and oxalic acid together with 1,2,4 butanetricarboxylic acid enhanced urease activity in NiCoT expressing cells despite their chelating potential.

L cysteine and its ester derivatives emerged as particularly interesting from a microbiome perspective. At 1 mM, they completely abolished urease activity in K. pneumoniae, yet did not reduce nickel uptake through either transporter and instead increased jack bean urease activity. Combined with prior work in Streptococcus salivarius, these data support a regulatory mechanism in which cysteine acts as a signal that suppresses urease expression rather than a classical nickel chelator, implying that sulfur-containing metabolites in the gut or oral environment may downregulate urease-dependent ammonia generation without needing to overcome high metal loads. The study also identified previously unreported anti-ureolytic agents including carbon disulfide, N phenylmaleimide, tannic acid, gallic acid, 1,2,4 butanetricarboxylic acid, sodium pyrrolidinedithiocarbamate, and DTPA, with several compounds preferentially targeting cell free urease, others acting only in bacteria, and some displaying strong antibacterial as well as anti-ureolytic activity.

Key implications

For clinicians interested in microbiome modulation and metallomic targeting of ureolytic pathobionts, this work underscores that not all nickel chelators are functionally equivalent and that nickel chelators and urease inhibition must be evaluated in the context of transporter-specific nickel uptake, competing metal ions, and regulatory effects on urease expression. EDTA-like broad chelators can effectively suppress urease in low-nickel environments, yet may fail where total metal concentrations are high, as in livestock manure or potentially in metal-enriched human gut niches. Oxalic acid and 1,2,4 butanetricarboxylic acid show that some ligands can paradoxically enhance nickel import and urease activation, which is a caution for empiric use of organic acids in patients with urease-mediated disease, such as struvite stone formation, hyperammonemic encephalopathy, or severe dysbiosis in chronic kidney disease. L-cysteine demonstrates that regulatory down-modulation of urease can be decoupled from nickel chelation and may represent a distinct therapeutic strategy for lowering ammonia and pH drift in oral, gastric, and intestinal biofilms without provoking strong selective pressure on metal homeostasis. Conversely, agents such as 1,2,4-butanetricarboxylic acid can act as metallophores that increase nickel uptake and, in some transporter contexts, enhance urease activity, which is a critical consideration when designing microbiome-targeted interventions (MBTIs).  This paper provides a mechanistic framework for selecting or avoiding specific chelators and phenolic compounds when designing microbiome-targeted interventions that seek to remodel nickel-dependent urease activity rather than simply killing bacteria.

Citation

Svane S, Sigurdarson JJ, Finkenwirth F, Eitinger T, Karring H. Inhibition of urease activity by different compounds provides insight into the modulation and association of bacterial nickel import and ureolysis. Sci Rep. 2020;10(1):8503. doi:10.1038/s41598-020-65107-9.