Cynthia Nau Cornelissen

Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.

The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin‐binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB‐dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin‐binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter‐species genetic exchange.

The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers

Iron, an essential nutrient for most microorganisms, is sequestered by the host to decrease the concentration of iron available to bacterial pathogens. Neisseria gonorrhoeae, the causative agent of gonorrhoea, can acquire iron by direct interaction with human iron‐binding proteins, including the serum glycoprotein, transferrin. Iron internalization from host transferrin requires the expression of a bacterial receptor, which specifically recognizes the human form of transferrin. Two gonococcal transferrin‐binding proteins have been implicated in transferrin receptor function, TbpA and TbpB. We constructed a gonococcal transferrin receptor mutant without the introduction of additional antibiotic resistance markers and tested its ability to cause experimental urethritis in human male volunteers. The transferrin receptor mutant was incapable of initiating urethritis, although the same inoculum size of the wild‐type parent strain, FA1090, causes urethritis in >90% of inoculated volunteers. To our knowledge, this is the first experimental demonstration that a bacterial iron acquisition system is an essential virulence factor for human infection.

The sloABCR Operon of Streptococcus mutans Encodes an Mn and Fe Transport System Required for Endocarditis Virulence and Its Mn-Dependent Repressor

Streptococcus mutans belongs to the viridans group of oral streptococci, which is the leading cause of endocarditis in humans. The LraI family of lipoproteins in viridans group streptococci and other bacteria have been shown to function as virulence factors, adhesins, or ABC-type metal transporters. We previously reported the identification of the S. mutans LraI operon, sloABCR, which encodes components of a putative metal uptake system composed of SloA, an ATP-binding protein, SloB, an integral membrane protein, and SloC, a solute-binding lipoprotein, as well as a metal-dependent regulator, SloR. We report here the functional analysis of this operon. By Western blotting, addition of Mn to the growth medium repressed SloC expression in a wild-type strain but not in a sloR mutant. Other metals tested had little effect. Cells were also tested for aerobic growth in media stripped of metals then reconstituted with Mg and either Mn or Fe. Fe at 10 micro M supported growth of the wild-type strain but not of a sloA or sloC mutant. Mn at 0.1 micro M supported growth of the wild-type strain and sloR mutant but not of sloA or sloC mutants. The combined results suggest that the SloABC proteins transport both metals, although the SloR protein represses this system only in response to Mn. These conclusions are supported by (55)Fe uptake studies with Mn as a competitor. Finally, a sloA mutant demonstrated loss of virulence in a rat model of endocarditis, suggesting that metal transport is required for endocarditis pathogenesis.

Intranasal Administration of Recombinant Neisseria gonorrhoeae Transferrin Binding Proteins A and B Conjugated to the Cholera Toxin B Subunit Induces Systemic and Vaginal Antibodies in Mice

ABSTRACT The transferrin binding proteins (TbpA and TbpB) comprise the gonococcal transferrin receptor and are considered potential antigens for inclusion in a vaccine against Neisseria gonorrhoeae. Intranasal (IN) immunization has shown promise in development of immunity against sexually transmitted disease pathogens, in part due to the induction of antigen-specific genital tract immunoglobulin A (IgA) and IgG. Conjugation of antigens to the highly immunogenic cholera toxin B subunit (Ctb) enhances antibody responses in the serum and mucosal secretions following IN vaccination. In the current study, we characterized the anti-Tbp immune responses following immunization of mice IN with recombinant transferrin binding proteins (rTbpA and rTbpB) conjugated to rCtb. We found that both rTbpA-Ctb and rTbpB-Ctb conjugates administered IN induced antibody responses in the serum and genital tract. IN immunization resulted in both IgA and IgG in the genital tract; however, subcutaneous immunization mainly generated IgG. Surprisingly, rTbpA alone was immunogenic and induced serum and mucosal antibody responses similar to those elicited against the rTbpA-Ctb conjugate. Overall, rTbpB was much more immunogenic than rTbpA, generating serum IgG levels that were greater than those elicited against rTbpA. Bactericidal assays conducted with sera collected from mice immunized IN with TbpA and/or TbpB indicated that both antigens generated antibodies with bactericidal activity. Anti-TbpA antibodies were cross-bactericidal against heterologous gonococcal strains, whereas TbpB-specific antibodies were less cross-reactive. By contrast, antibodies elicited via subcutaneous immunization were not cross-bactericidal against heterologous strains, indicating that IN vaccination could be the preferred route for elicitation of biologically functional antibodies.

Energy-dependent changes in the gonococcal transferrin receptor

The pathogenic Neisseria spp. are capable of iron utilization from host iron‐binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy‐dependent, receptor‐mediated event in which two identified transferrin‐binding proteins participate. One of these proteins, TbpA, is homologous to the TonB‐dependent family of outer membrane receptors that are required for high‐affinity uptake of vitamin B12 and ferric siderophores. The ‘TonB box’ is a conserved domain near the amino‐terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy‐dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin‐binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de‐energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de‐energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton‐dependent changes in the binding parameters and exposures of TbpA and TbpB.

Growth of Neisseria gonorrhoeae in the Female Mouse Genital Tract Does Not Require the Gonococcal Transferrin or Hemoglobin Receptors and May Be Enhanced by Commensal Lactobacilli

ABSTRACT Neisseria gonorrhoeae is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for N. gonorrhoeae in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor “on” (Hg+) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg+ phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of N. gonorrhoeae in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.

Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells

Neisseria gonorrhoeae has evolved a repertoire of iron acquisition systems that facilitate essential iron uptake in the human host. Acquisition of iron requires both the energy‐harnessing cytoplasmic membrane protein, TonB, as well as specific outer membrane TonB‐dependent transporters (TdTs.) Survival within host epithelial cells is important to the pathogenesis of gonococcal disease and may contribute to the persistence of infection. However, the mechanisms by which gonococci acquire iron within this intracellular niche are not currently understood. In this study, we investigated the survival of gonococcal strain FA1090 within ME180 human cervical epithelial cells with respect to high affinity iron acquisition. Intracellular survival was dependent upon iron supplied by the host cell. TonB was expressed in the host cell environment and this protein was critical to gonococcal intracellular survival. Furthermore, expression of the characterized outer membrane transporters TbpA, FetA and LbpA and putative transporters TdfG, TdfH and TdfJ were not necessary for intracellular survival. Conversely, intracellular survival was dependent on expression of the putative transporter, TdfF. Expression of TdfF was detected in the presence of epithelial cell culture media containing fetal bovine serum. Expression was further modulated by iron availability. To our knowledge, this study is the first to demonstrate the specific requirement for a single iron transporter in the survival of a bacterial pathogen within host epithelial cells.

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.

TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae

Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention.

Antigenic and Sequence Diversity in Gonococcal Transferrin-Binding Protein A

ABSTRACT Neisseria gonorrhoeae is a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additional tbpA genes from other N. gonorrhoeaestrains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera.