Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.

Stealthy microbes: How Neisseria gonorrhoeae hijacks bulwarked iron during infection.

Transition metals are essential for metalloprotein function among all domains of life. Humans utilize nutritional immunity to limit bacterial infections, employing metalloproteins such as hemoglobin, transferrin, and lactoferrin across a variety of physiological niches to sequester iron from invading bacteria. Consequently, some bacteria have evolved mechanisms to pirate the sequestered metals and thrive in these metal-restricted environments. Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, causes devastating disease worldwide and is an example of a bacterium capable of circumventing human nutritional immunity. Via production of specific outer-membrane metallotransporters, N. gonorrhoeae is capable of extracting iron directly from human innate immunity metalloproteins. This review focuses on the function and expression of each metalloprotein at gonococcal infection sites, as well as what is known about how the gonococcus accesses bound iron.

Acclimation to Nutritional Immunity and Metal Intoxication Requires Zinc, Manganese, and Copper Homeostasis in the Pathogenic Neisseriae.

Neisseria gonorrhoeae and Neisseria meningitidis are human-specific pathogens in the Neisseriaceae family that can cause devastating diseases. Although both species inhabit mucosal surfaces, they cause dramatically different diseases. Despite this, they have evolved similar mechanisms to survive and thrive in a metal-restricted host. The human host restricts, or overloads, the bacterial metal nutrient supply within host cell niches to limit pathogenesis and disease progression. Thus, the pathogenic Neisseria require appropriate metal homeostasis mechanisms to acclimate to such a hostile and ever-changing host environment. This review discusses the mechanisms by which the host allocates and alters zinc, manganese, and copper levels and the ability of the pathogenic Neisseria to sense and respond to such alterations. This review will also discuss integrated metal homeostasis in N. gonorrhoeae and the significance of investigating metal interplay.

Mutagenesis of the Loop 3 α-Helix of Neisseria gonorrhoeae TdfJ Inhibits S100A7 Binding and Utilization.

Neisseria gonorrhoeae causes the sexually transmitted infection (STI) gonorrhea, which afflicts over 80 million people each year. No vaccine is available to prevent gonorrhea. The pathogen alters the expression and antigenic presentation of key surface molecules, making the identification of suitable vaccine targets difficult. The human host utilizes metal-binding proteins to limit free essential transition metal ions available to invading pathogens, limiting their infective potential, a process called nutritional immunity. To overcome this, N. gonorrhoeae employs outer membrane TonB-dependent transporters (TdTs) that bind host nutritional immunity proteins and strip them of their metal cargo. The TdTs are well conserved, and some play key roles in establishing infections, making them promising vaccine targets. One TdT, TdfJ, recognizes human S100A7, a zinc-binding protein that inhibits the proliferation of other pathogens via zinc sequestration. N. gonorrhoeae uses TdfJ to strip and internalize zinc from S100A7. TdfJ contains a conserved α-helix finger in extracellular loop 3; a similar α-helix in loop 3 of another gonococcal TdT, TbpA, plays a critical role in the interaction between TbpA and human transferrin. Therefore, we hypothesized that the TdfJ loop 3 helix (L3H) participates in interactions with S100A7. We determined the affinity between wild-type TdfJ and S100A7 and then generated a series of mutations in the TdfJ L3H. Our study revealed that mutagenesis of key residues within the L3H reduced S100A7 binding and zinc piracy by the gonococcus, with profound effects seen with substitutions at residues K261 and R262. Taken together, these data suggest a key role for the TdfJ L3H in subverting host metal restriction. IMPORTANCE Gonorrhea is a global threat to public health due to the increasing incidence of antimicrobial drug resistance, rising treatment costs, and lack of a protective vaccine. The prospect of untreatable gonococcal infections has spurred efforts to identify targets for novel therapeutic and prevention strategies, and members of the family of outer membrane TonB-dependent metal transporters have emerged as promising candidates. These conserved surface molecules play a critical role in establishing infection by facilitating nutrient uptake in the human host that dedicates considerable efforts to restricting nutrient availability. In this study, we characterized the binding interaction between the zinc importer TdfJ and its human zinc source, S100A7. We went on to identify a key region of TdfJ that mediates this interaction. With a more thorough understanding of the intricate relationships between these bacterial nutrient receptors and their host nutrient sources, we may help pave the way toward identifying effective prophylaxis and treatment for an important human disease.

Recent Progress Towards a Gonococcal Vaccine.

Gonorrhea is a global health concern. Its etiological agent, Neisseria gonorrhoeae, rapidly acquires antimicrobial resistance and does not confer protective immunity as a consequence of infection. Attempts to generate an effective vaccine for gonorrhea have thus far been unsuccessful, as many structures on the bacterial envelope have the propensity to rapidly change, thus complicating recognition by the human immune system. In response to recent efforts from global health authorities to spur the efforts towards development of a vaccine, several new and promising steps have been made towards this goal, aided by advancements in computational epitope identification and prediction methods. Here, we provide a short review of recent progress towards a viable gonococcal vaccine, with a focus on antigen identification and characterization, and discuss a few of the tools that may be important in furthering these efforts.

TdfH selectively binds metal-loaded tetrameric calprotectin for zinc import.

To combat nutritional immunity, N. gonorrhoeae has evolved systems to hijack zinc and other metals directly from host metal-binding proteins such as calprotectin (CP). Here, we report the 6.1 Å cryoEM structure of the gonococcal surface receptor TdfH in complex with a zinc-bound CP tetramer. We further show that TdfH can also interact with CP in the presence of copper and manganese, but not with cobalt.

Adherence Enables Neisseria gonorrhoeae to Overcome Zinc Limitation Imposed by Nutritional Immunity Proteins.

Neisseria gonorrhoeae (Gc) must overcome the limitation of metals such as zinc to colonize mucosal surfaces in its obligate human host. While the zinc-binding nutritional immunity proteins calprotectin (S100A8/A9) and psoriasin (S100A7) are abundant in human cervicovaginal lavage fluid, Gc possesses TonB-dependent transporters TdfH and TdfJ that bind and extract zinc from the human version of these proteins, respectively. Here we investigated the contribution of zinc acquisition to Gc infection of epithelial cells of the female genital tract. We found that TdfH and TdfJ were dispensable for survival of strain FA1090 Gc that was associated with Ect1 human immortalized epithelial cells, when zinc was limited by calprotectin and psoriasin. In contrast, suspension-grown bacteria declined in viability under the same conditions. Exposure to murine calprotectin, which Gc cannot use as a zinc source, similarly reduced survival of suspension-grown Gc, but not Ect1-associated Gc. We ruled out epithelial cells as a contributor to the enhanced growth of cell-associated Gc under zinc limitation. Instead, we found that attachment to glass was sufficient to enhance bacterial growth when zinc was sequestered. We compared the transcriptional profiles of WT Gc adherent to glass coverslips or in suspension, when zinc was sequestered with murine calprotectin or provided in excess, from which we identified open reading frames that were increased by zinc sequestration in adherent Gc. One of these, ZnuA, was necessary but not sufficient for survival of Gc under zinc-limiting conditions. These results show that adherence protects Gc from zinc-dependent growth restriction by host nutritional immunity proteins.

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

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.

Metal-Limited Growth of Neisseria gonorrhoeae for Characterization of Metal-Responsive Genes and Metal Acquisition from Host Ligands.

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

The novel interaction between Neisseria gonorrhoeae TdfJ and human S100A7 allows gonococci to subvert host zinc restriction.

Neisseria gonorrhoeae causes the sexually-transmitted infection gonorrhea, a global disease that is difficult to treat and for which there is no vaccine. This pathogen employs an arsenal of conserved outer membrane proteins called TonB-dependent transporters (TdTs) that allow the gonococcus to overcome nutritional immunity, the host strategy of sequestering essential nutrients away from invading bacteria to handicap infectious ability. N. gonorrhoeae produces eight known TdTs, of which four are utilized for acquisition of iron or iron chelates from host-derived proteins or xenosiderophores produced by other bacteria. Of the remaining TdTs, two of them, TdfH and TdfJ, facilitate zinc uptake. TdfH was recently shown to bind Calprotectin, a member of the S100 protein family, and subsequently extract its zinc, which is then internalized by N. gonorrhoeae. Like Calprotectin, other S100s are also capable of binding transition metals such as zinc and copper, and thus have demonstrated growth suppression of numerous other pathogens via metal sequestration. Considering the functional and structural similarities of the TdTs and of the S100s, as well as the upregulation in response to Zn limitation shown by TdfH and TdfJ, we sought to evaluate whether other S100s have the ability to support gonococcal growth by means of zinc acquisition and to frame this growth in the context of the TdTs. We found that both S100A7 and S10012 are utilized by N. gonorrhoeae as a zinc source in a mechanism that depends on the zinc transport system ZnuABC. Moreover, TdfJ binds directly to S100A7, from which it internalizes zinc. This interaction is restricted to the human version of S100A7, and zinc presence in S100A7 is required to fully support gonococcal growth. These studies highlight how gonococci co-opt human nutritional immunity, by presenting a novel interaction between TdfJ and human S100A7 for overcoming host zinc restriction.

Generation of Metal-Depleted Conditions for In Vitro Growth of Neisseria gonorrhoeae.

Neisseria gonorrhoeae employs high-affinity metal acquisition systems to obtain necessary nutrients, such as iron (Fe) and zinc (Zn) from the environment. Because growth and replication depend upon successful metal acquisition, these high-affinity uptake systems are important virulence factors. Expression of metal acquisition systems is tightly controlled and preferentially expressed under low-metal conditions. Therefore, in order to optimally produce these transport proteins and study them in vitro, growth media must be deployed that mimic low-metal conditions. This chapter describes the chelators, media, and culturing conditions that can generate low-metal in vitro growth conditions.

Structural Basis for Evasion of Nutritional Immunity by the Pathogenic Neisseriae.

The pathogenic Neisseria species are human-adapted pathogens that cause quite distinct diseases. Neisseria gonorrhoeae causes the common sexually transmitted infection gonorrhea, while Neisseria meningitidis causes a potentially lethal form of bacterial meningitis. During infection, both pathogens deploy a number of virulence factors in order to thrive in the host. The focus of this review is on the outer membrane transport systems that enable the Neisseriae to utilize host-specific nutrients, including metal-binding proteins such as transferrin and calprotectin. Because acquisition of these critical metals is essential for growth and survival, understanding the structures of receptor-ligand complexes may be an important step in developing preventative or therapeutic strategies focused on thwarting these pathogens. Much can also be learned by comparing structures with antigenic diversity among the transporter sequences, as conserved functional domains in these essential transporters could represent the pathogens' "Achilles heel." Toward this goal, we present known or modeled structures for the transport systems produced by the pathogenic Neisseria species, overlapped with sequence diversity derived by comparing hundreds of neisserial protein sequences. Given the concerning increase in N. gonorrhoeae incidence and antibiotic resistance, these outer membrane transport systems appear to be excellent targets for new therapies and preventative vaccines.

Subversion of nutritional immunity by the pathogenic Neisseriae.

The pathogenic Neisseria species, including Neisseria meningitidis and Neisseria gonorrhoeae, are obligate human pathogens that cause significant morbidity and mortality. The success of these pathogens, with regard to causing disease in humans, is inextricably linked to their ability to acquire necessary nutrients in the hostile environment of the host. Humans deploy a significant arsenal of weaponry to defend against bacterial pathogens, not least of which are the metal-sequestering proteins that entrap and withhold transition metals, including iron, zinc and manganese, from invaders. This review will discuss the general strategies that bacteria employ to overcome these metal-sequestering attempts by the host, and then will focus on the relatively uncommon 'metal piracy' approaches utilized by the pathogenic Neisseria for this purpose. Because acquiring metals from the environment is critical to microbial survival, interfering with this process could impede growth and therefore disease initiation or progression. This review will also discuss how interfering with metal uptake by the pathogenic Neisseriae could be deployed in the development of novel or improved preventative or therapeutic measures against these important pathogens.

The genes that encode the gonococcal transferrin binding proteins, TbpB and TbpA, are differentially regulated by MisR under iron-replete and iron-depleted conditions.

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two-component regulatory system that also includes the sensor kinase MisS. The tbp genes were up-regulated in the misR mutant under iron-replete conditions but were conversely down-regulated in the misR mutant under iron-depleted conditions. The misR mutant was capable of transferrin-iron uptake at only 50% of wild-type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR-regulated in previous studies using this related pathogen. This is the first report of a trans-acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue.

Identification of regulatory elements that control expression of the tbpBA operon in Neisseria gonorrhoeae.

Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.

Conserved regions of gonococcal TbpB are critical for surface exposure and transferrin iron utilization.

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

Structural insight into the lactoferrin receptors from pathogenic Neisseria.

Neisseria are pathogenic bacteria that cause gonorrhea, septicemia, and meningitis. Like other pathogenic bacteria, Neisseria must acquire iron for survival from their local environment within the human host. Instead of secreting siderophores to scavenge iron, Neisseria steal iron from human iron binding proteins such as hemoglobin, transferrin and lactoferrin for survival. Recently we reported the crystal structures of the Neisseria meningitidis transferrin receptors TbpA and TbpB, as well as the structures of apo and holo human transferrin. We also analyzed these proteins using small angle X-ray scattering and electron microscopy to provide the molecular details explaining how Neisseria are able to interact with and extract iron from transferrin. Here, we utilize the structural reports, as well as the recently reported structure of the N-lobe of LbpB from Moraxella bovis, to assemble improved 3D homology models for the neisserial lactoferrin import receptors LbpA and LbpB, both of which are important vaccine targets against N. meningitidis. We then analyzed these models to gain structural insights into the lactoferrin-iron import system and form a mechanistic model fashioned in parallel to the homologous transferrin-iron import system.

The transferrin-iron import system from pathogenic Neisseria species.

Two pathogenic species within the genus Neisseria cause the diseases gonorrhoea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and, therefore, these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis.

The iron-repressed, AraC-like regulator MpeR activates expression of fetA in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an obligate human pathogen that causes the common sexually transmitted infection gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy, and infertility. In the last few years, antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity, nor is there an effective vaccine to prevent the disease. Thus, further understanding of the expression, function, and regulation of surface antigens could lead to better treatment and prevention modalities in the future. In the current study, we determined that an iron-repressed regulator, MpeR, interacted specifically with the DNA sequence upstream of fetA and activated FetA expression. Interestingly, MpeR was previously shown to regulate the expression of gonococcal antimicrobial efflux systems. We confirmed that the outer membrane transporter FetA allows gonococcal strain FA1090 to utilize the xenosiderophore ferric enterobactin as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated that fetA is part of an iron-repressed, MpeR-activated operon which putatively encodes other iron transport proteins. This is the first study to describe a regulatory linkage between antimicrobial efflux and iron transport in N. gonorrhoeae. The regulatory nidus that links these systems, MpeR, is expressed exclusively by pathogenic neisseriae and is therefore expected to be an important virulence factor.