RESUMO
Vibrio cholerae O139 variants with different surface phenotypes have been compared for their resistance to complement and also for their susceptibility to antibody-dependent, complement-mediated bacteriolysis (ACB). While both capsular polysaccharide (CPS) and lipopolysaccharide (LPS) contribute to complement resistance in the absence of antibody, the relative survival of variants expressing only one of these surface polysaccharides reveals CPS to be of much greater significance in this respect. Variants with LPS+/CPS- or LPS-/CPS+ surface phenotypes were both susceptible to ACB, showing that antibody binding to either of the O139 polysaccharides can initiate ACB. Demonstration of the lytic potential of antibodies bound to the capsule is novel and necessitates a revision of the suggested mechanism by which CPS confers partial protection of wild-type V. cholerae O139 against ACB. This finding also raised the possibility that there may be CPS-restricted epitopes which can function as substrates for ACB, which would invalidate the common practice of using unencapsulated O139 variants for estimating bactericidal responses. Since our data did not support this scenario, we evaluated several unencapsulated strains - including variants producing normal or elongated polysaccharide chains, and a hybrid O1/O139 strain - for their utility as indicators of bactericidal antibodies to V. cholerae O139. Our findings support the use of the recently-isolated CIRS134, which ensures reproducible titrations and allows assignment of high lytic titres in assays requiring only 2% complement. However low-level, cross-serogroup lytic activity was detected when CIRS134 was used to assay responses of mice injected with O1 serogroup V. cholerae, suggesting that this indicator should be used with caution when evaluating the immunogenicity of bivalent O1/O139 vaccines.
Assuntos
Anticorpos Antibacterianos/análise , Cápsulas Bacterianas/imunologia , Bacteriólise/imunologia , Proteínas do Sistema Complemento/imunologia , Vibrio cholerae O139/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Cólera/imunologia , Cólera/microbiologia , Cólera/prevenção & controle , Vacinas contra Cólera/imunologia , Cobaias , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Coelhos , Teste Bactericida do Soro , Vibrio cholerae O139/genética , Vibrio cholerae O139/metabolismoRESUMO
Salmonella typhimurium possesses two wzz genes conferring long (wzz(ST)) and very long (wzz(fepE)) lipopolysaccharide O antigen modal chain lengths. While the long O antigen modal length was essential for complement resistance, the very long modal length was found to have a minor role. However, when grown in the presence of serum, S. typhimurium demonstrated a wzz(fepE)-dependent increase in the density of very long O antigen chains, resulting in a significant increase in serum resistance. Similar phenotypic changes were observed after growth under iron-limiting conditions, though iron limitation is unlikely to be the sole signalling mechanism behind the changes induced in serum. A wzz(fepE)::lacZ promoter fusion was used to determine that regulation of wzz(fepE) transcription is unlikely to be the mechanism behind the variation in O antigen length. Since systemic infection occurs in a small but significant percentage of human non-typhoid salmonellosis, the phenomenon identified in this study may be significant during the bacteraemic phase of infection.
Assuntos
Antígenos de Bactérias/análise , Proteínas do Sistema Complemento/imunologia , Antígenos O/imunologia , Salmonella typhimurium/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Meios de Cultura/química , Ferro/metabolismo , Testes de Sensibilidade Microbiana , Antígenos O/química , Salmonella typhimurium/patogenicidade , VirulênciaRESUMO
The Shigella flexneri autotransporter protein IcsA is essential for intra- and intercellular spread, and icsA mutants are attenuated in several models. However, the pathogenic significance of the outer membrane protease IcsP, which orchestrates the polar distribution of IcsA on the bacterial surface, remains unclear. To further examine this point, we constructed icsP mutants in the two most commonly studied S. flexneri strains and evaluated their in vitro and in vivo performance relative to wild type. Both icsP mutants showed aberrant surface distribution of IcsA, but the in vitro consequences depended upon the cell line being used to assess bacterial motility and plaque formation. Evaluating the behaviour of the mutants in two mouse models suggested functional expression of icsP might limit bacterial persistence and the associated inflammation in host tissues, consistent with the findings in one of the three cell lines used.
Assuntos
Proteínas de Bactérias/metabolismo , Shigella flexneri/genética , Shigella flexneri/patogenicidade , Actinas/genética , Aminoglicosídeos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Células CACO-2 , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/microbiologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fatores de Transcrição/metabolismoRESUMO
Shigella flexneri remains a significant human pathogen due to high morbidity among children < 5 years in developing countries. One of the key features of Shigella infection is the ability of the bacterium to initiate actin tail polymerisation to disseminate into neighbouring cells. Dynamin II is associated with the old pole of the bacteria that is associated with F-actin tail formation. Dynamin II inhibition with dynasore as well as siRNA knockdown significantly reduced Shigella cell to cell spreading in vitro. The ocular mouse Sereny model was used to determine if dynasore could delay the progression of Shigella infection in vivo. While dynasore did not reduce ocular inflammation, it did provide significant protection against weight loss. Therefore dynasore's effects in vivo are unlikely to be related to the inhibition of cell spreading observed in vitro. We found that dynasore decreased S. flexneri-induced HeLa cell death in vitro which may explain the protective effect observed in vivo. These results suggest the administration of dynasore or a similar compound during Shigella infection could be a potential intervention strategy to alleviate disease symptoms.
Assuntos
Dinamina II/antagonistas & inibidores , Disenteria Bacilar/tratamento farmacológico , Hidrazonas/farmacologia , Análise de Variância , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Vermelho Congo , Escherichia coli K12 , Olho/microbiologia , Células HeLa , Humanos , Hidrazonas/uso terapêutico , Proteínas Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Killed oral cholera vaccines (OCVs) have been licensed for use in developing countries, but protection conferred by licensed OCVs beyond two years of follow-up has not been demonstrated in randomized, clinical trials. METHODS/PRINCIPAL FINDINGS: We conducted a cluster-randomized, placebo-controlled trial of a two-dose regimen of a low-cost killed whole cell OCV in residents 1 year of age and older living in 3,933 clusters in Kolkata, India. The primary endpoint was culture-proven Vibrio cholerae O1 diarrhea episodes severe enough to require treatment in a health care facility. Of the 66,900 fully dosed individuals (31,932 vaccinees and 34,968 placebo recipients), 38 vaccinees and 128 placebo-recipients developed cholera during three years of follow-up (protective efficacy 66%; one-sided 95%CI lower boundâ=â53%, p<0.001). Vaccine protection during the third year of follow-up was 65% (one-sided 95%CI lower boundâ=â44%, p<0.001). Significant protection was evident in the second year of follow-up in children vaccinated at ages 1-4 years and in the third year in older age groups. CONCLUSIONS/SIGNIFICANCE: The killed whole-cell OCV conferred significant protection that was evident in the second year of follow-up in young children and was sustained for at least three years in older age groups. Continued follow-up will be important to establish the vaccine's duration of protection. TRIAL REGISTRATION: ClinicalTrials.gov NCT00289224.
Assuntos
Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Administração Oral , Adolescente , Criança , Pré-Escolar , Cólera/microbiologia , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/economia , Diarreia/microbiologia , Diarreia/prevenção & controle , Seguimentos , Humanos , Imunização Secundária/métodos , Índia , Lactente , Placebos/administração & dosagem , Fatores de Tempo , Vacinação/métodos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/economia , Vacinas de Produtos Inativados/imunologia , Vibrio cholerae O1/isolamento & purificaçãoRESUMO
Immune responses after one and two doses of the reformulated killed oral cholera vaccine were measured in a double-blind, randomized, placebo-controlled trial of 77 adults aged 18-40 years and 77 children aged 1-17 years residing in Kolkata, India. 65% of adults and 87% of children and 46% of adults and 82% of children exhibited a > or =4-fold rise in serum Vibrio cholerae O1 vibriocidal antibody titers from baseline following dose 1 and 2, respectively. Responses to V. cholerae O139 were less pronounced but followed a similar pattern. We demonstrate that in a cholera-endemic area, the vaccine elicited vibriocidal responses after a single-dose of the vaccine.
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Administração Oral , Adolescente , Adulto , Formação de Anticorpos , Criança , Pré-Escolar , Cólera/imunologia , Método Duplo-Cego , Feminino , Humanos , Imunização Secundária , Índia , Lactente , Masculino , Vacinas de Produtos Inativados/imunologia , Adulto JovemRESUMO
Protocols are described for the induction of strong, consistent serum and mucosal antibody responses to Vibrio cholerae O1 or O139 lipopolysaccharide (LPS) following intranasal or oral immunization of adult mice with viable or formalin-killed bacteria. A simplified two-dose schedule for intranasal immunization has been identified, whereby viable bacteria elicit strong serum responses and, most importantly, also induce significant, sustained intestinal IgA responses. Using higher doses of bacteria it was also possible to generate consistently high intestinal and serum anti-LPS responses by the oral route. The efficacy of these immunization schedules was not dependent on co-administration of adjuvant. Gut responses were estimated using two sampling techniques involving the collection of fresh faecal pellets or the preparation of intestinal tissue extracts. The significant correlation between these estimates validates the more convenient approach of measuring intestinal responses using faecal pellet extracts, which allows repeated sampling from the same animals. V. cholerae O1 and O139 were similarly immunogenic by either mucosal route. More intensive immunization schedules for administration of formalin-killed bacteria have also been defined. Using these regimes it was possible to generate serum and gut antibody responses comparable to those elicited by viable V. cholerae. The established immunization protocols will allow evaluation of the systemic and mucosal immunogenicity of new vaccine formulations.
Assuntos
Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/farmacologia , Imunidade nas Mucosas/imunologia , Imunização/métodos , Vibrio cholerae/imunologia , Animais , Anticorpos Antibacterianos/análise , Desinfetantes , Ensaio de Imunoadsorção Enzimática , Fezes/química , Formaldeído , Esquemas de Imunização , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologiaRESUMO
OBJECTIVES: An effective vaccine against cholera has been used for public health purposes in Vietnam since the 1990s. This vaccine was reformulated to meet WHO requirements. We assessed the safety and immunogenicity of the reformulated bivalent (Vibrio cholerae 01 and 0139) killed whole cell oral vaccine in a cholera endemic area in Kolkata, India. DESIGN: Double-blind, randomized, placebo controlled trial. SETTING: The trial was conducted in the clinical trial ward of the Infectious Diseases Hospital in Kolkata, India. PARTICIPANTS: The participants were 101 healthy adults (males and non-pregnant females) aged 18-40 years and 100 healthy children (males and non-pregnant females) aged 1-17 years. INTERVENTIONS: Participants were randomized to receive either the bivalent killed whole cell oral cholera vaccine or placebo (killed oral Escherichia coli K12). OUTCOME MEASURES: For safety: proportion of subjects with adverse events during the duration of study participation. For immunogenicity: Proportion of subjects who had a > or = 4-fold rise in serum vibriocidal antibody titers 14 days after the second dose of vaccine or placebo. RESULTS: Adverse reactions were observed with similar frequency among vaccine and placebo recipients in both age groups. Among adults 4% of vaccine and 8% of placebo recipients and among children 4% of vaccine and 2% of placebo recipients had at least one adverse event within 28 days of the first dose of the vaccine. Following immunization, 53% of adult and 80% of children vaccinees showed a > or = 4 fold rise in serum V. cholerae O1 vibriocidal antibody titers. A less pronounced response to V. cholerae O139 vibriocidal antibody titers post-immunization was noted among vaccinees. CONCLUSIONS: We found the vaccine to be safe and immunogenic in a cholera-endemic area in India. TRIAL REGISTRATION: ClinicalTrials.gov NCT00119197.
Assuntos
Vacinas contra Cólera/administração & dosagem , Cólera/prevenção & controle , Doenças Endêmicas/prevenção & controle , Administração Oral , Adolescente , Adulto , Criança , Pré-Escolar , Cólera/epidemiologia , Cólera/imunologia , Vacinas contra Cólera/imunologia , Método Duplo-Cego , Feminino , Humanos , Índia/epidemiologia , Lactente , Masculino , Avaliação de Resultados em Cuidados de Saúde , Placebos , Tamanho da AmostraRESUMO
Vietnam currently produces an orally administered, bivalent (O1 and O139) killed whole-cell vaccine and is the only country in the world with endemic cholera to use an oral cholera vaccine in public health practice. In order to allow international use, the vaccine had to be reformulated to meet World Health Organization (WHO) requirements. We performed a randomized, placebo controlled, safety and immunogenicity studies of this reformulated vaccine among Vietnamese adults. One hundred and forty-four subjects received the two-dose regimen and 143 had two blood samples obtained for analysis. We found that this reformulated oral killed whole-cell cholera vaccine was safe, well tolerated and highly immunogenic.
Assuntos
Vacinas contra Cólera/administração & dosagem , Vacinas contra Cólera/imunologia , Cólera/imunologia , Vibrio cholerae O139/imunologia , Vibrio cholerae O1/imunologia , Administração Oral , Adolescente , Adulto , Cólera/microbiologia , Cólera/prevenção & controle , Vacinas contra Cólera/efeitos adversos , Método Duplo-Cego , Feminino , Humanos , Masculino , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , VietnãRESUMO
A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzzST and wzzfepE) or three heterologous (wzzO139 of Vibrio cholerae and wzzSF and wzzpHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.
Assuntos
Proteínas do Sistema Complemento/imunologia , Macrófagos/imunologia , Antígenos O/química , Antígenos O/imunologia , Salmonella typhimurium/química , Salmonella typhimurium/imunologia , Animais , Linhagem Celular , Macrófagos/metabolismo , Antígenos O/metabolismo , Salmonella typhimurium/metabolismoRESUMO
Our previous studies have shown that mice which have been orally primed with an attenuated Salmonella vector [S. enterica serovar Stanley] are hypo-responsive to foreign antigens later delivered orally by the same vector strain, responding with significantly impaired serum and intestinal antibody responses compared with those seen in unprimed controls. Initial vector priming of the gut-associated lymphoid tissue (GALT) is likely to result in impaired persistence of recombinant Salmonella later administered orally. Delivery of recombinant bacteria by the intra-peritoneal or intra-nasal route, to avoid exposure to a primed GALT, did not allow vector-primed recipients to mount normal antibody responses to the foreign pilus protein K88. The negative impact of vector priming could be largely overcome, however, if mice were exposed to the foreign protein just prior to priming with the vector strain. Using this strategy, vector-primed mice displayed normal gut IgA and intermediate serum IgG responses to K88 following oral administration of recombinant Salmonella. Our findings are compatible with the concept of epitopic suppression, in which failure to respond to the foreign vaccine antigen reflects the clonal dominance of B cells specific for epitopes associated with the vector strain.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas de Fímbrias/imunologia , Vetores Genéticos/imunologia , Vacinas contra Salmonella/administração & dosagem , Salmonella enterica/imunologia , Animais , Antígenos de Bactérias/genética , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Vetores Genéticos/administração & dosagem , Imunoglobulina A Secretora/metabolismo , Intestinos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Recombinação Genética , Vacinas contra Salmonella/genética , Vacinas contra Salmonella/imunologia , Salmonella enterica/classificação , Salmonella enterica/genética , Vacinação , Vacinas SintéticasRESUMO
There are conflicting reports concerning the impact of prior vector priming on the immunogenicity of recombinant-Salmonella-based vaccines. A comparison of experimental protocols identified two variables which might account for this inconsistency: the potential of the vector strain to colonize the murine gut-associated lymphoid tissue (GALT) and the nature of the foreign antigen subsequently delivered by the recombinant Salmonella construct. The former was investigated by constructing an aroA mutant of the Salmonella enterica serovar Stanley vector previously used in our laboratory. Although the introduction of an aroA mutation had surprisingly little effect on GALT colonization, it did reduce the strength of antilipopolysaccharide (anti-LPS) antibody responses and the impact of vector priming. Studies were also performed to ascertain the extent to which any observed hyporesponsiveness consequent upon vector priming might be determined by the characteristics of the foreign antigen. S. enterica serovar Stanley was used to deliver either of two Escherichia coli antigens, K88 pilus protein or the LT-B toxin subunit, to vector-primed mice. Both serum immunoglobulin G (IgG) and intestinal IgA responses to K88 were completely abolished, and those to LT-B were significantly reduced, as a consequence of vector priming. When similar experiments were performed with an aroA S. enterica serovar Dublin vector, responses to K88 were significantly reduced but those to LT-B were unaffected by vector priming. Paradoxically, a priming infection with this vector induced stronger anti-LPS antibody responses but was less likely to elicit a state of hyporesponsiveness to subsequently presented foreign antigen. The impact of vector priming thus depends on both the Salmonella strain used and the nature of the foreign antigen, but our present data strengthen concerns that preexisting antivector immunity represents a serious threat to the Salmonella-based vaccine strategy.
Assuntos
Vacinas Bacterianas/imunologia , Vetores Genéticos/imunologia , Vacinas contra Salmonella/imunologia , Salmonella/imunologia , Vacinas Sintéticas/imunologia , 3-Fosfoshikimato 1-Carboxiviniltransferase , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/imunologia , Animais , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Proteínas de Fímbrias/imunologia , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/sangue , Intestinos/imunologia , Linfotoxina-alfa/imunologia , Linfotoxina-beta , Proteínas de Membrana/imunologia , Camundongos , Mutação , Salmonella/genética , Vacinas contra Salmonella/genética , Vacinas Tíficas-Paratíficas/imunologia , VacinaçãoRESUMO
The present studies in Nramp1(-/-) BALB/c and Nramp1(+/+) CBA mice question the significance of this genotype as a determinant of the level of gut colonization following oral administration of naturally attenuated or highly virulent Salmonella strains. In line with previous results in BALB/c hosts, vector priming of CBA mice with Salmonella enterica serovar Stanley was found to significantly compromise the immunogenicity of a recombinant construct expressing a foreign pilus protein.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Vetores Genéticos/imunologia , Vacinas contra Salmonella/imunologia , Salmonella enterica/patogenicidade , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Transporte de Cátions/genética , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Salmonelose Animal/imunologia , Salmonelose Animal/prevenção & controle , Salmonella enterica/classificação , Salmonella enterica/imunologia , Vacinas Atenuadas/imunologiaRESUMO
Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.
Assuntos
Proteínas de Bactérias/fisiologia , Antígenos O/metabolismo , Salmonella typhimurium/patogenicidade , Administração Oral , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/química , Gastroenterite/microbiologia , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Peritonite/microbiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Homologia de Sequência , Organismos Livres de Patógenos Específicos , Baço/microbiologia , VirulênciaRESUMO
Monoclonal antibodies (MAbs) were prepared against toxin-coregulated pili (TCP) isolated from Vibrio cholerae O1 El Tor. Despite their limited bactericidal potential, two MAbs were able to mediate biotype-specific protection against experimental cholera in infant mice. These MAbs were used in immunoblotting studies to assess seroconversion to El Tor TCP following cholera. Clear anti-pilus responses were observed in five of nine patients.
Assuntos
Anticorpos Antibacterianos/sangue , Toxinas Bacterianas/imunologia , Cólera/imunologia , Fímbrias Bacterianas/imunologia , Vibrio cholerae O1/imunologia , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Especificidade de Anticorpos , Antígenos de Bactérias , Toxinas Bacterianas/antagonistas & inibidores , Cólera/microbiologia , Cólera/prevenção & controle , Vacinas contra Cólera/administração & dosagem , Humanos , CamundongosRESUMO
A previously undescribed haemolysin, distinct from the major Vibrio cholerae O1 El Tor haemolysin, HlyA, was cloned from the O1 classical biotype strain Z17561. This novel haemolysin showed 71.5% overall similarity to the delta-thermostable direct haemolysin of Vibrio parahaemolyticus, and so it has been termed V. cholerae delta-thermostable haemolysin (Vc-deltaTH, encoded by the dth gene). An ORF found immediately downstream, which appears to be transcriptionally and translationally linked to dth, displayed strong homology to the family of acyl-CoA synthetases. When expressed from an inducible promoter in Escherichia coli, Vc-deltaTH was shown to be a 22.8 kDa protein active on sheep red blood cells. Co-expression of acs with dth had no effect on the haemolytic activity or cytoplasmic localization of Vc-deltaTH. A V. cholerae Z17561 dth::Km(R) mutant showed unaltered behaviour in the infant mouse cholera model.
Assuntos
Cólera/fisiopatologia , Clonagem Molecular , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Vibrio cholerae/patogenicidade , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Cólera/microbiologia , Modelos Animais de Doenças , Testes de Hemaglutinação , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNA , Frações Subcelulares/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , VirulênciaRESUMO
A microplate assay for the detection of bactericidal antibodies to Vibrio cholerae O139 is described. The assay is sensitive, highly reproducible, specific, and convenient to perform. It has been used to demonstrate the induction of serum bactericidal antibodies in Vietnamese recipients of an oral, inactivated, bivalent O1/O139 vaccine, as well as in Bangladeshi patients with O139 disease. In both study groups there was a significant inverse correlation between the preexposure level of antibodies in serum and the magnitude of the subsequent bactericidal response. Although infection generated stronger responses than vaccination, the proportion of responders was similar among individuals with low background titers.