Biomedical subjects

Cynthia Nau Cornelissen

Publications and source records attributed to Cynthia Nau Cornelissen.

10 recordsLinked to original sources

PubMed2026

Genomic analysis of Neisseria gonorrhoeae strains from disseminated, urogenital, and rectal infections reveals differences in genes for iron acquisition and type IV secretion.

Neisseria gonorrhoeae, an obligate human pathogen, causes the sexually transmitted infection gonorrhea. Delayed treatment or immunodeficiencies can result in a higher risk of disseminated gonococcal infection (DGI), where the infection spreads to normally sterile anatomic sites, including the bloodstream. The gonococcus produces TonB-dependent transporters (TdTs) to sequester metals from host nutritional immunity proteins. These transporters are critical for survival and are important virulence factors. We characterized genes encoding iron-regulated TdTs in the genomes of strains from disseminated (n = 47), urogenital (n = 86), and rectal (n = 12) infections. We found that gonococcal strains isolated from DGI infections were more likely to express a functional hemoglobin-iron utilization operon (hpuAB wild type and phase on [32%, n = 15/47]), to harbor nonsense mutations in tdfF (57%, n = 27/47), and were predicted to express low levels of fetA (43%, n = 20/47) when compared to strains from urogenital or rectal infections. In contrast, gonococcal strains from localized urogenital infections were associated with a higher frequency of functional tdfF genes (66%, n = 57/86) and high fetA expression (30%, n = 26/86). Additionally, strains from rectal infections were more frequently found to have high fetA expression (33%, n = 4/12) and a functional tdfF (75%, n = 9/12). Furthermore, we identified associations between the presence and absence of tdfF and the gonococcal genetic island, which encodes a type IV secretion system. These findings inform our evolving understanding of the molecular mechanisms underlying disseminated gonococcal infections.IMPORTANCEGonorrhea is an emerging, global health threat, with over 100 million new cases each year. Delayed treatment, immunosuppressive medications, and immunodeficiencies can contribute to the spread of infection to the blood, presenting as a serious disseminated manifestation. With rising antimicrobial resistance and no licensed preventative vaccine, untreatable gonorrhea is a possibility in the near future. TonB-dependent transporters are highly conserved and vital for metal acquisition and survival, making them promising targets for therapeutic or preventative strategies. Associated surface-exposed lipoproteins display greater sequence variation but contribute to metal acquisition. We analyzed the genes encoding TonB-dependent transporters and associated lipoproteins from strains associated with distinct disease manifestations. We conclude that gonococci isolated from disseminated, urogenital, and rectal sites demonstrate different phase and allelic variations in genes encoding iron transport systems and the gonococcal genetic island.

Humans↗

PubMed2006

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.

Bacterial Outer Membrane Proteins↗

PubMed2005

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.

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.

Administration, Intranasal↗

PubMed2004

Immunogenicity of gonococcal transferrin binding proteins during natural infections.

In this study, we examined the immune response during gonococcal infection to the individual transferrin binding proteins by using a quantitative enzyme-linked immunosorbent assay (ELISA). Recombinant transferrin binding protein A (rTbpA) and rTbpB were purified under nondenaturing conditions for use as ELISA antigens. Sera and secretions from culture-positive individuals were analyzed for antibodies to rTbpA and rTbpB and compared to samples from individuals with no history of gonococcal infection. Although antibodies to both rTbpA and rTbpB were detected in serum, in most cases the antibody levels were not significantly different from those measured in the control population. Also, previous history of gonococcal infection did not increase antibody levels in serum, suggesting the lack of an anamnestic response. Analysis of secretion samples revealed antibody levels that were generally below the limits of detection in our assay. Overall, this study demonstrated a paucity of systemic and local antibody responses to rTbps as a result of natural infection and represents a baseline over which a protective antibody response will have to be generated in order to develop an efficacious gonococcal vaccine.

Antibodies, Bacterial↗

PubMed2004

Determination of surface-exposed, functional domains of gonococcal transferrin-binding protein A.

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and beta strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative beta strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA.

Amino Acid Sequence↗

PubMed2003

Transferrin-iron uptake by Gram-negative bacteria.

Members of the families Neisseriaceae, Pasteurellaceae and Moraxellaceae are capable of transferrin-iron acquisition in the absence of siderophore production. They do so via expression of a bi-partite receptor composed of two dissimilar proteins, TbpA and TbpB. Both proteins are surface exposed, iron-regulated and capable of binding transferrin. However, other physiochemical, antigenic, and immunogenic characteristics of the proteins are quite distinct. TbpB is a lipoprotein, which like the mammalian transferrin receptor is capable if discriminating between apo- and holo-transferrin. Expression of TbpB is not essential for transferrin-iron uptake, and in rare situations, the gene that encodes this protein is not linked to the gene encoding the second component. TbpA is a member of a family of TonB-dependent transporters, others of which accomplish ferric-siderophore and vitamin B12 uptake at the expense of a proton gradient across the cytoplasmic membrane. However, unlike the other TonB-dependent receptors where vitamins or ferric-siderophores are wholly internalized, the bacterial transferrin receptor must remove iron from transferrin at the cell surface. This review focuses on the structure-function relationships in the transferrin-binding proteins, their sequence and antigenic diversity, and the mechanisms by which they accomplish transferrin-iron uptake. The contribution of these proteins to pathogenesis and vaccine development based on TbpA and TbpB are also discussed.

Amino Acid Sequence↗

PubMed2003

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.

ATP-Binding Cassette Transporters↗

PubMed2002

Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.

The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.

Animals↗

PubMed2002

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.

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.

Amino Acid Sequence↗

PubMed2002

Demonstration and characterization of a specific interaction between gonococcal transferrin binding protein A and TonB.

Iron scavenging by Neisseria gonorrhoeae is accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin. Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB. TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake. Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques. In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa. The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB. The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers. Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction. In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated. We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including a Neisseria-specific, glycine-rich domain. This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin. Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain. The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB.

Amino Acid Sequence↗