Investigating the importance of selected surface-exposed loops in HpuB for hemoglobin binding and utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the etiological agent of the sexually transmitted infection gonorrhea. The pathogen is a global health challenge since no protective immunity results from infection, and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising vaccine antigens as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs is part of the hemoglobin transport system comprised of HpuA and HpuB. This system allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHb, although the growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme-binding motifs and a hypothetical α-helical region, both of which may be important for the use of hHb. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb and extracting and internalizing iron.

Investigating the importance of surface exposed loops in the gonococcal HpuB transporter for hemoglobin binding and utilization.

Neisseria gonorrhoeae is the etiological agent of the sexually-transmitted infection gonorrhea and a global health challenge since no protective immunity results from infection and far fewer treatment options are available with increasing antimicrobial resistance. With no efficacious vaccines, researchers are exploring new targets for vaccine development and innovative therapeutics. The outer membrane TonB-dependent transporters (TdTs) produced by N. gonorrhoeae are considered promising antigen targets as they are highly conserved and play crucial roles in overcoming nutritional immunity. One of these TdTs, the hemoglobin transport system comprised of HpuA and HpuB, allows N. gonorrhoeae to acquire iron from hemoglobin (hHb). In the current study, mutations in the hpuB gene were generated to better understand the structure-function relationships in HpuB. This study is one of the first to demonstrate that N. gonorrhoeae can bind to and utilize hemoglobin produced by animals other than humans. This study also determined that when HpuA is absent, mutations targeting extracellular loop 7 of HpuB led to defective hHb binding and utilization. However, when the lipoprotein HpuA is present, these loop 7 mutants recovered their ability to bind hHB, although their growth phenotype remained significantly impaired. Interestingly, loop 7 contains putative heme binding motifs and a hypothetical α-helical region. Taken together, these results highlight the importance of loop 7 in the functionality of HpuB in binding hHb, and extracting and internalizing iron.

Transcriptional activation of ompA in Neisseria gonorrhoeae mediated by the XRE family member protein NceR.

Increasing antibiotic resistance of Neisseria gonorrhoeae, the causative agent of gonorrhea, is a growing global concern that has renewed vaccine development efforts. The gonococcal OmpA protein was previously identified as a vaccine candidate due to its surface exposure, conservation, stable expression, and involvement in host-cell interactions. We previously demonstrated that the transcription of ompA can be activated by the MisR/MisS two-component system. Interestingly, earlier work suggested that the availability of free iron also influences ompA expression, which we confirmed in this study. In the present study, we found that iron regulation of ompA was independent of MisR and searched for additional regulators. A DNA pull-down assay with the ompA promoter from gonococcal lysates obtained from bacteria grown in the presence or absence of iron identified an XRE (Xenobiotic Response Element) family member protein encoded by NGO1982. We found that an NGO1982 null mutant of N. gonorrhoeae strain FA19 displayed a reduced level of ompA expression compared to the wild-type (WT) parent strain. Given this regulation, and the capacity of this XRE-like protein to regulate a gene involved in peptidoglycan biosynthesis (ltgA), along with its presence in other Neisseria sp., we termed the NGO1982-encoded protein as NceR (Neisseria cell envelope regulator). Critically, results from DNA-binding studies indicated that NceR regulates ompA through a direct mechanism. Thus, ompA expression is subject to both iron-dependent (NceR) and -independent (MisR/MisS) pathways. Hence, levels of the vaccine antigen candidate OmpA in circulating gonococcal strains could be influenced by transcriptional regulatory systems and the availability of iron. IMPORTANCE Herein, we report that the gene encoding a conserved gonococcal surface-exposed vaccine candidate (OmpA) is activated by a heretofore undescribed XRE family transcription factor, which we term NceR. We report that NceR regulation of ompA expression in N. gonorrhoeae is mediated by an iron-dependent mechanism, while the previously described MisR regulatory system is iron-independent. Our study highlights the importance of defining the complexity of coordinated genetic and physiologic systems that regulate genes encoding vaccine candidates to better understand their availability during infection.

Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH.

Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)-outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time that N. gonorrhoeae exploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity.

GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease.

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections.

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a ß-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the ß-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

Type IV secretion machinery promotes ton-independent intracellular survival of Neisseria gonorrhoeae within cervical epithelial cells.

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.

Hijacking transferrin bound iron: protein-receptor interactions involved in iron transport in N. gonorrhoeae.

Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.