RESUMO
CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1 to 3 subunits that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of 16 amino acids that was derived from the N terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e., CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including the generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6 subunit protein in a subunit vaccine strategy against ETEC.
Assuntos
Antígenos de Bactérias/imunologia , Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Escherichia coli Enterotoxigênica/imunologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Subunidades Proteicas/imunologiaRESUMO
UNLABELLED: Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE: Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron-sulfur cluster regulator, IscR. Furthermore, we demonstrate that the apo form of IscR, lacking an Fe-S cluster, is able to directly fix the corresponding promoter region. These results provide further evidence implicating IscR in bacterial virulence and suggest that IscR may represent a more general regulator mediating the iron response in enteropathogens.
Assuntos
Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/farmacologia , Fatores de Transcrição/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Enterotoxinas/genética , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.
RESUMO
INTRODUCTION: Pertussis vaccines have drastically reduced the disease burden in humans since their implementation. Despite their success, pertussis remains an important global public health challenge. Bordetella pertussis resurgence could be a result of greater surveillance combined with improved diagnosis methods, changes in Bordetella pertussis biology, vaccine schedules, and/or coverage. Additionally, mechanisms of protection conferred by acellular pertussis (aP) and whole-cell pertussis (wP) vaccines differ qualitatively. There are no clear immune correlates of protection for pertussis vaccines. Pertussis antigens can induce toxin neutralizing antibodies, block adherence or engage complement mediated phagocytic/bactericidal killing. AREAS COVERED: We reviewed the existing evidence on antibody-mediated serum bactericidal and opsonophagocytic activity and discussed the relevance of these functional antibodies in the development of next-generation pertussis vaccines. EXPERT OPINION: Current paradigm proposes that wP vaccines may confer greater herd protection than aP vaccines due to their enhanced clearance of bacteria from the nasopharynx in animal models. Functional antibodies may contribute to the reduction of nasal colonization, which differentiates aP and wP vaccines. Understanding the intrinsic differences in protective immune responses elicited by each class of vaccines will help to identify biomarkers that can be used as immunological end points in clinical trials.
Assuntos
Bordetella pertussis , Coqueluche , Animais , Humanos , Coqueluche/prevenção & controle , Vacina contra Coqueluche , Proteínas do Sistema Complemento , Anticorpos AntibacterianosRESUMO
Bordetella pertussis, a gram-negative beta-proteobacterium, is the agent of whooping cough in humans. Whooping cough remains a public health problem worldwide, despite well-implemented infant/child vaccination programs. It continues to be endemic and is observed cyclically in vaccinated populations. Classical molecular subtyping methods indicate that genome diversity among B. pertussis isolates is limited. Although the whole bacterial genome has been studied by pulsed-field gel electrophoresis, the genes implicated in the diversity have not been identified. We developed a B. pertussis whole-genome DNA microarray representing over 91% of the predicted coding sequences of the sequenced strain Tohama I. Genomic DNA from clinical isolates with various pulsed-field gel electrophoresis profile patterns was competitively hybridized with the DNA microarray and coding sequences were classified as present, absent or duplicated. Our data strongly suggest that the B. pertussis population is dynamic. In France, with highly vaccinated population, the genetic diversity is low and decreasing with time, and clonal expansion correlates with cycles of the disease. This decrease in diversity is essentially due to loss of genes and pseudogenes. The genes deleted are most of the time flanked by insertion sequences.
Assuntos
Bordetella pertussis/classificação , Bordetella pertussis/genética , Genoma Bacteriano , Hibridização de Ácido Nucleico/métodos , Coqueluche/microbiologia , Bordetella pertussis/isolamento & purificação , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Evolução Molecular , França , Deleção de Genes , Variação Genética , Epidemiologia Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de TempoRESUMO
Colonization factors (CFs) mediate early adhesion of Enterotoxigenic Escherichia coli (ETEC) in the small intestine. Environmental signals including bile, glucose, and contact with epithelial cells have previously been shown to modulate CF expression in a strain dependent manner. To identify novel components modulating CF surface expression, 20 components relevant to the intestinal environment were selected for evaluation. These included mucin, bicarbonate, norepinephrine, lincomycin, carbon sources, and cations. Effects of individual components on surface expression of the archetype CF, CFA/I, were screened using a fractional factorial Hadamard matrix incorporating 24 growth conditions. As most CFs agglutinate erythrocytes, surface expression was evaluated by mannose resistant hemagglutination. Seven components, including porcine gastric mucin, lincomycin, glutamine, and glucose were found to induce CFA/I surface expression in vitro in a minimal media while five others were inhibitory, including leucine and 1,10-phenanthroline. To further explore the effect of components positively influencing CFA/I surface expression, a response surface methodology (RSM) was designed incorporating 36 growth conditions. The optimum concentration for each component was identified, thereby generating a novel culture media, SP1, for CFA/I expression. CFs closely related to CFA/I, including CS4 and CS14 were similarly induced in SP1 media. Other epidemiologically relevant CFs were also induced when compared to the level obtained in minimal media. These results indicate that although CF surface expression is complex and highly variable among strains, the CF response can be predicted for closely related strains. A novel culture media inducing CFs in the CF5a group was successfully identified. In addition, mucin was found to positively influence CF expression in strains expressing either CFA/I or CS1 and CS3, and may function as a common environmental cue.
Assuntos
Meios de Cultura/farmacologia , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Proteínas de Escherichia coli/biossíntese , Proteínas de Fímbrias/biossíntese , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana , Toxinas Bacterianas/análise , Técnicas Bacteriológicas , Bovinos , Parede Celular/química , Meios de Cultura/química , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/análise , Ensaio de Imunoadsorção Enzimática , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Glutamina/farmacologia , Testes de Hemaglutinação , Humanos , Intestinos/química , Intestinos/microbiologia , Lincomicina/farmacologia , Mucinas/farmacologia , Sus scrofa , Suínos , Virulência/efeitos dos fármacosRESUMO
Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.
Assuntos
Biofilmes , Escherichia coli/genética , Escherichia coli/fisiologia , Genes Bacterianos/genética , Klebsiella pneumoniae/fisiologia , Simbiose , Animais , Feminino , Camundongos , Microbiota/genética , Microbiota/fisiologia , Especificidade da EspécieRESUMO
The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT) domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The repertoire of BvgS sequences will pave the way for functional analyses of this prototypic system.
Assuntos
Proteínas de Bactérias/genética , Bordetella/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Animais , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Filogenia , Estrutura Terciária de Proteína , Transdução de Sinais , Especificidade da Espécie , VirulênciaRESUMO
Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious human pathogen capable of inducing mucosal and systemic immune responses upon a single intranasal administration. In an attenuated, pertussis toxin (PTX)-deficient recombinant form, it may therefore constitute an efficient bacterial vector that is particularly well adapted for the delivery of heterologous antigens to the respiratory mucosa. Filamentous hemagglutinin (FHA) has been used as a carrier to present foreign antigens at the bacterial surface, thereby inducing local, systemic, and protective immune responses to these antigens in mice. Both full-length and truncated (Fha44) forms of FHA have been used for antigen presentation. To investigate the effect of the carrier (FHA or Fha44) on antibody responses to passenger antigens, we genetically fused the HtrA protein of nontypeable Haemophilus influenzae to either FHA form. The fha-htrA and Fha44 gene-htrA hybrids were expressed as single copies inserted into the chromosome of PTX-deficient B. pertussis. Both chimeras were secreted into the culture supernatants of the recombinant strains and were recognized by anti-FHA and anti-HtrA antibodies. Intranasal infection with the strain producing the FHA-HtrA hybrid led to significantly higher anti-HtrA and anti-FHA antibody titers than those obtained in mice infected with the Fha44-HtrA-producing strain. Interestingly, the B. pertussis strain producing the Fha44-HtrA chimera colonized the mouse lungs more efficiently than the parental, Fha44-producing strain and gave rise to higher anti-FHA antibody titers than those induced by the parental strain.
Assuntos
Adesinas Bacterianas/metabolismo , Bordetella pertussis/genética , Haemophilus influenzae/patogenicidade , Hemaglutininas/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência/biossíntese , Animais , Anticorpos Antibacterianos/sangue , Linhagem Celular , Haemophilus influenzae/imunologia , Humanos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/fisiologia , Proteínas Recombinantes/biossínteseRESUMO
To investigate the effect of the autoinducer AI-2 on protein expression in Neisseria meningitidis, a luxS mutant of strain MC58 was grown in the presence and absence of in vitro-produced AI-2, and differential protein expression was assessed by two-dimensional differential gel electrophoresis. N. meningitidis did not show a global response to AI-2 signaling activity.
Assuntos
Proteínas de Bactérias/análise , Homosserina/análogos & derivados , Homosserina/fisiologia , Neisseria meningitidis/metabolismo , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre , Eletroforese em Gel Bidimensional , Lactonas , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively. Inactivated forms of these virulence factors are the protein components of vaccines against these diseases. Here we report microcalorimetric studies to characterize these proteins. The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic. For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM). This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al. [Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J. Biol. Chem. 275, 8889-8894]. In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2. The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C. The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form). Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively). Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility. The latter could account for the observed inhibition of ATP binding after binding to fetuin. Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing. The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential denaturation of four structural domains.
Assuntos
Adesinas Bacterianas/química , Calorimetria/métodos , Gangliosídeos/química , Hemaglutininas/química , Metaloendopeptidases/química , Microquímica/métodos , Toxina Pertussis/química , Toxina Tetânica/química , Titulometria/métodos , Fatores de Virulência de Bordetella/química , Adesinas Bacterianas/análise , Sequência de Aminoácidos , Sítios de Ligação , Gangliosídeos/análise , Hemaglutininas/análise , Cinética , Metaloendopeptidases/análise , Conformação Molecular , Dados de Sequência Molecular , Peso Molecular , Toxina Pertussis/análise , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Toxina Tetânica/análise , Fatores de Virulência de Bordetella/análiseRESUMO
Neisseria meningitidis acquires iron through the action of the transferrin (Tf) receptor, which is composed of the Tf-binding proteins A and B (TbpA and TbpB). Meningococci can be classified into isotype I and II strains depending on whether they harbor a type I or II form of TbpB. Both types of TbpB have been shown to differ in their genomic, biochemical, and antigenic properties. Here we present a comparative study of isogenic mutants deficient in either or both Tbps from the isotype I strain B16B6 and isotype II strain M982. We show that TbpA is essential in both strains for iron uptake and growth with iron-loaded human Tf as a sole iron source. No growth has also been observed for the TbpB- mutant of strain B16B6, as shown previously, whereas the growth of the analogous mutant in M982 was similar to that in the wild type. This indicates that TbpB in the latter strain plays a facilitating but not essential role in iron uptake, which has been observed previously in similar studies of other bacteria. These data are discussed in relation to the fact that isotype II strains represent more than 80% of serogroup B meningococcal strains. The contribution of both subunits in the bacterial virulence of strain M982 has been assessed in a murine model of bacteremia. Both the TbpB- TbpA- mutant and the TbpA- mutant are shown to be nonvirulent in mice, whereas the virulence of the TbpB- mutant is similar to that of the wild type.
Assuntos
Neisseria meningitidis Sorogrupo B/patogenicidade , Receptores da Transferrina/metabolismo , Proteína A de Ligação a Transferrina/metabolismo , Proteína B de Ligação a Transferrina/metabolismo , Transferrina/metabolismo , Animais , Bacteriemia/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/imunologia , Ferro/metabolismo , Infecções Meningocócicas/microbiologia , Camundongos , Mutação , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/crescimento & desenvolvimento , Neisseria meningitidis Sorogrupo B/metabolismo , Coelhos , Transferrina/imunologia , Proteína A de Ligação a Transferrina/genética , Proteína A de Ligação a Transferrina/imunologia , Proteína B de Ligação a Transferrina/genética , Proteína B de Ligação a Transferrina/imunologia , VirulênciaRESUMO
The transferrin receptor of Neisseria meningitidis is composed of the transmembrane protein TbpA and the outer membrane protein TbpB. Both receptor proteins have the capacity to independently bind their ligand human transferrin (htf). To elucidate the specific role of these proteins in receptor function, isothermal titration calorimetry was used to study the interaction between purified TbpA, TbpB or the entire receptor (TbpA + TbpB) with holo- and apo-htf. The entire receptor was shown to contain a single high affinity htf-binding site on TbpA and approximately two lower affinity binding sites on TbpB. The binding sites appear to be independent. Purified TbpA was shown to have strong ligand preference for apo-htf, whereas TbpA in the receptor complex with TbpB preferentially binds the holo form of htf. The orientation of the ligand specificity of TbpA toward holo-htf is proposed to be the physiological function of TbpB. Furthermore, the thermodynamic mode of htf binding by TbpB of isotypes I and II was shown to be different. A protocol for the generation of active, histidine-tagged TbpB as well as its individual N- and C-terminal domains is presented. Both domains are shown to strongly interact with each other, and isothermal titration calorimetry and circular dichroism experiments provide clear evidence for this interaction causing conformational changes. The N-terminal domain of TbpB was shown to be the site of htf binding, whereas the C-terminal domain is not involved in binding. Furthermore, the interactions between TbpA and the different domains of TbpB have been demonstrated.
Assuntos
Neisseria meningitidis/química , Receptores da Transferrina/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Varredura Diferencial de Calorimetria , Ligação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Receptores da Transferrina/química , Receptores da Transferrina/metabolismo , Termodinâmica , Proteína A de Ligação a Transferrina/metabolismo , Proteína B de Ligação a Transferrina/química , Proteína B de Ligação a Transferrina/isolamento & purificação , Proteína B de Ligação a Transferrina/metabolismoRESUMO
A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.