Investigating the role of EnvZ-OmpR, BaeSR and RamA in the resistance of Klebsiella pneumoniae to Cefiderocol

Abstract

Antimicrobial resistance (AMR) is one of the most serious global public health threats of this century. The modified Cephalosporin Cefiderocol (CFDC) was clinically approved in the EU as of 2020 to attempt to combat multi-drug resistant (MDR) bacteria, including Gram-negative ESKAPES pathogen Klebsiella pneumoniae (Kp). Like all bacteria, Kp requires ferric iron (Fe3+) for viability, due to its involvement in many essential cellular processes. Limited iron stores in an infected host impose a significant challenge for bacterial iron uptake. Hence, Kp must compete with the host for this nutrient. This is achieved by the release and re-uptake of high affinity Fe3+ chelating molecules called siderophores, including the catecholate-type siderophore enterobactin. Enterobactin efflux from the cell has been shown to occur synergistically through resistance-nodulation-division (RND) family efflux pumps MdtABC-TolC, AcrAAcrD-TolC and AcrAB-TolC. The re-uptake of enterobactin into Kp in complex with Fe3+ has been shown to occur through iron regulated outer membrane (OM) proteins (IROMPs) CirA, Fiu, FepA and FecA.

CFDC can mimic enterobactin’s action of Fe3+ binding due to its conjugation with a catechol moiety. This allows CFDC to hijack the IROMPs to gain cellular entry. However, with the catechol moiety conferring an element of structural similarity between CFDC and enterobactin, it has been speculated that CFDC could also be effluxed from the cell by MdtABC-TolC, AcrA-AcrD-TolC and AcrAB-TolC. It is known that the two-component system (TCS) EnvZ-OmpR negatively regulates the IROMPs, the TCS BaeSR positively regulates MdtABC-TolC and AcrA-AcrD-TolC, and the AraC-XylS family regulator RamA positively regulates AcrAB-TolC. Thus, EnvZ-OmpR, BaeSR and RamA have been implicated in reducing Kp susceptibility to CFDC. Therefore, this study’s primary objective was to establish how Kp resists CFDC under iron limitation through EnvZ-OmpR, BaeSR and RamA. Broth microdilution susceptibility testing confirmed cirA deletion to confer a biologically insignificant 2-fold increase to CFDC MIC, with RT-(q)PCR evidencing this to be the consequence of upregulated RNA transcript expression of fiu, fepA and fecA. Deletion of envZompR reduced CFDC MIC by 8-fold. A gain-of-function substitution in histidine kinase EnvZ (L230Q), which rendered EnvZ-OmpR constitutively active, increased CFDC MIC by 4 to 8-fold. Thus, EnvZ-OmpR was confirmed to inhibit CFDC influx into Kp via its repression of cirA, fiu, fepA and fecA transcription. The Chrome Azurol S (CAS) agar assay revealed mdtABC and baeSR deletions to significantly reduce siderophore export from Kp, which was positively correlated with both deletions conferring a 4 to 8-fold decrease in CFDC MIC. Thus, BaeSR was confirmed to induce CFDC efflux from Kp via its activation of mdtABC transcription. The CAS assay revealed no impact on siderophore export due to acrAB deletion or ramA overexpression. Further, acrAB deletion conferred no impact to CFDC susceptibility. However, ramA overexpression increased CFDC MIC by 4-fold. Results from subsequent RT-(q)PCR analysis suggest the overexpression of ramA to indirectly induce the repression of cirA, fiu, fepA and fecA through an unspecified intermediary regulator, exposing an avenue for further research. Thus, the present work has highlighted fundamental implications of EnvZOmpR, BaeSR and RamA in Kp Fe3+ homeostasis, virulence and pathogenicity, which are imperative to investigate further as the global usage of CFDC increases.