Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae, responsible for the sexually transmitted infection gonorrhea, is an obligate human pathogen exquisitely adapted for survival on mucosal surfaces of humans. This host-pathogen relationship has resulted in evolution by N. gonorrhoeae of pathways that enable the use of host metalloproteins as required nutrients through the deployment of outer membrane-bound TonB-dependent transporters (TdTs). Recently, a TdT called TdfH was implicated in binding to calprotectin (CP) and in removal of the bound zinc (Zn), enabling gonococcal growth. TdfH is highly conserved among the pathogenic Neisseria species, making it a potentially promising candidate for inclusion into a gonococcal vaccine. Currently, the nature and specificity of the TdfH-CP interaction have not been determined. In this study, we found that TdfH specifically interacted with human calprotectin (hCP) and that growth of the gonococcus was supported in a TdfH-dependent manner only when hCP was available as a sole zinc source and not when mouse CP was provided. The binding interactions between TdfH and hCP were assessed using isothermal titration calorimetry where we observed a multistate model having both high-affinity and low-affinity sites of interaction. hCP has two Zn binding sites, and gonococcal growth assays using hCP mutants deficient in one or both of the Zn binding sites revealed that TdfH exhibited a site preference during Zn piracy and utilization. This report provides the first insights into the molecular mechanism of Zn piracy by neisserial TdfH and further highlights the obligate human nature of N. gonorrhoeae and the high-affinity interactions occurring between TdTs and their human ligands during pathogenesis. IMPORTANCE The dramatic rise in antimicrobial resistance among Neisseria gonorrhoeae isolates over the last few decades, paired with dwindling treatment options and the lack of a protective vaccine, has prompted increased interest in identifying new bacterial targets for the treatment and, ideally, prevention of gonococcal disease. TonB-dependent transporters are a conserved set of proteins that serve crucial functions for bacterial survival within the host. In this study, binding between the gonococcal transporter, TdfH, and calprotectin was determined to be of high affinity and host restricted. The current study identified a preferential TdfH interaction at the calprotectin dimer interface. An antigonococcal therapeutic could potentially block this site on calprotectin, interrupting Zn uptake by N. gonorrhoeae and thereby prohibiting continued bacterial growth. We describe protein-protein interactions between TdfH and calprotectin, and our findings provide the building blocks for future therapeutic or prophylactic targets.

Regulation of zinc-dependent enzymes by metal carrier proteins

“…Due to its role in nutritional immunity through its ability to sequester Zn 2+ , CALP has become a target for zinc piracy by certain bacterial species much as LTF has. The TdfH protein of Neisseria gonorrhoeae binds to and extracts a zinc ion from human calprotectin (Kammerman et al 2020 ; Stork et al 2013 ) or the closely related psoriasin/S100A7 (Maurakis et al 2019 ). Likewise, Salmonella typhimurium employs a similar strategy, using the zinc transporter ZnuABC to sequester Zn 2+ away from CALP (Liu et al 2012 ).…”

Epidemiology, Treatments, and Vaccine Development for Antimicrobial-Resistant Neisseria gonorrhoeae: Current Strategies and Future Directions

“…Other investigators have focused on intrinsic mechanisms important for N. gonorrhoeae survival. One such mechanism is the outer-membrane bound TonB -dependent transporters (TdTs) that N. gonorrhoeae use for uptake of metalloproteins from hosts [ 129 ]. Kammerman et al found that one specific TdT, TdfH , was found to be highly conserved in Neisseria species, making this an ideal target for antimicrobial therapy [ 129 ].…”

“…One such mechanism is the outer-membrane bound TonB -dependent transporters (TdTs) that N. gonorrhoeae use for uptake of metalloproteins from hosts [ 129 ]. Kammerman et al found that one specific TdT, TdfH , was found to be highly conserved in Neisseria species, making this an ideal target for antimicrobial therapy [ 129 ]. A separate highly conserved component of N. gonorrhoeae is a multicomponent protein complex (BAM) critical to the synthesis of β-barrel outer membrane proteins [ 130 ].…”

Recent Progress Towards a Gonococcal Vaccine

“…As generation of a nonbinding Ngo TbpA appears possible, using it as an immunogen to test protection relative to the wildtype may be a promising way forward. Indeed, as researchers gain more insight into other TdT/host protein pairs ( Jean et al., 2016 ; Maurakis et al., 2019 ) and further characterize TdT structures and binding propensities ( Sikora et al., 2018 ; Yadav et al., 2019 ; Kammerman et al., 2020 ), similar studies with the other TdTs are feasible.…”