RESUMO
Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease that facilitates the transmission of human immunodeficiency virus. In the human model of infection, the histopathology of infected sites in part resembles a delayed-type hypersensitivity (DTH) response. In this study, T cells were isolated from skin biopsy specimens obtained from 24 subjects who were infected for 7 to 14 days. One clone and 12 lines that responded to H. ducreyi antigens were obtained from 12 of the subjects. Fluorescence-activated cell sorter analysis showed that the antigen-responsive lines and clone were predominantly CD3(+) and CD4(+). The lines and clone responded to H. ducreyi antigen in a dose-dependent manner and produced gamma interferon (IFN-gamma) alone or IFN-gamma and interleukin-10 (IL-10) but no IL-4 or IL-5 in response to H. ducreyi. Proliferation of T cells was dependent on the presence of autologous antigen-presenting cells. The lines showed little response to antigens prepared from other members of the Pasteurellaceae and responded to different fractions of H. ducreyi separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that T cells that recognize H. ducreyi antigens are recruited to sites experimentally infected with the organism. The lack of cross-reactivity to the Pasteurellaceae and the response of the lines to different antigen fractions suggest that subjects are sensitized to H. ducreyi during the course of infection.
Assuntos
Cancroide/imunologia , Linfócitos T/imunologia , Antígenos de Bactérias/imunologia , Linhagem Celular , Cancroide/patologia , Fracionamento Químico , Haemophilus ducreyi/imunologia , Humanos , Pasteurellaceae/imunologiaRESUMO
The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.
Assuntos
Cancroide/fisiopatologia , Glucosiltransferases/metabolismo , Haemophilus ducreyi/patogenicidade , Lipopolissacarídeos/metabolismo , Mutação , Adulto , Cancroide/microbiologia , Feminino , Glucosiltransferases/genética , Haemophilus ducreyi/genética , Haemophilus ducreyi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , VirulênciaRESUMO
In a previous study, Haemophilus ducreyi was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis. H. ducreyi was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts. H. ducreyi colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular H. ducreyi. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that H. ducreyi remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.
Assuntos
Aderência Bacteriana , Colágeno/fisiologia , Fibrina/fisiologia , Haemophilus ducreyi/fisiologia , Fagócitos/microbiologia , Adulto , Feminino , Humanos , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/microbiologia , Pele/microbiologia , Linfócitos T/microbiologiaRESUMO
Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyi were subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, and lspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.
Assuntos
Cancroide/microbiologia , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidade , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Adulto , Feminino , Genes Bacterianos , Humanos , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Virulência/genéticaRESUMO
Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P = 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Cancroide/etiologia , Haemophilus ducreyi/patogenicidade , Mutação , Adulto , Biópsia , Cloranfenicol/farmacologia , Feminino , Haemophilus ducreyi/genética , Haemophilus ducreyi/isolamento & purificação , Humanos , Masculino , Testes de Sensibilidade MicrobianaRESUMO
Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.
Assuntos
Toxinas Bacterianas/biossíntese , Cancroide/patologia , Haemophilus ducreyi/patogenicidade , Proteínas Hemolisinas/biossíntese , Adulto , Toxinas Bacterianas/genética , Cancroide/etiologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Proteínas Hemolisinas/genética , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n = 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n = 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.
Assuntos
Proteínas da Membrana Bacteriana Externa , Infecções por Haemophilus/etiologia , Haemophilus ducreyi/patogenicidade , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Proteoglicanas , Adulto , Proteínas de Escherichia coli , Feminino , Haemophilus ducreyi/efeitos dos fármacos , Haemophilus ducreyi/genética , Humanos , Antígenos Comuns de Leucócito/análise , Lipoproteínas/genética , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fases de Leitura Aberta , Peptidoglicano/genética , Fenótipo , VirulênciaRESUMO
Haemophilus ducreyi expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that momp and ompA2 are transcribed independently. Sequences of the momp open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Omega-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into H. ducreyi 35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Omega-Km2 cassette in momp. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (P = 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by H. ducreyi in the human model of infection.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Haemophilus ducreyi/patogenicidade , Adulto , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Cancroide/imunologia , Cancroide/microbiologia , Cancroide/patologia , Feminino , Deleção de Genes , Haemophilus ducreyi/genética , Haemophilus ducreyi/imunologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fenótipo , Transcrição GênicaRESUMO
Haemophilus ducreyi expresses a conserved hemoglobin-binding outer-membrane protein (HgbA). To test the role of HgbA in pathogenesis, we infected 9 adults with isolate 35000 and its isogenic hgbA-inactivated mutant (FX504) on their upper arms in a double-blinded, escalating dose-response study. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule-formation rate was 55% (95% confidence interval [CI], 30. 8%-78.5%) at parent sites and 0 (95% CI, 0-10.5%) at mutant sites (P<.0001). The recovery rate of H. ducreyi from surface cultures was 16% (n=142) from parent sites and 0 (n=213) from mutant sites (P<. 0001). H. ducreyi was recovered at biopsy from 6 of 7 parent sites and from 0 of 3 mutant sites. The results indicate that hemoglobin may be a critical source of heme or iron for the establishment of H. ducreyi infection in humans.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias , Proteínas de Transporte/fisiologia , Cancroide/etiologia , Haemophilus ducreyi/patogenicidade , Adulto , Feminino , Hemoglobinas/fisiologia , Humanos , Hipersensibilidade Tardia/etiologia , Masculino , Pessoa de Meia-Idade , Mutação , FenótipoRESUMO
Haemophilus ducreyi expresses fine tangled pili, which are composed predominantly of a major subunit (FtpA). Confocal microscopy showed that an FtpA-specific monoclonal antibody bound to bacteria in biopsy samples obtained from infected human volunteers. To test the role of pili in pathogenesis, an isogenic mutant (35000HP-SMS1) was constructed by insertionally inactivating ftpA. 35000HP-SMS1 did not express FtpA and was nonpiliated but was otherwise identical to its parent, 35000HP. Seven healthy adults were challenged on the upper arm with the isogenic isolates in a double-blinded, escalating dose-response study. Sites inoculated with the mutant produced papules and pustules at rates similar to the rates observed at sites inoculated with the parent. The recovery rate of H. ducreyi from cultures and the histopathology of biopsy samples obtained from pustules inoculated with 35000HP or 35000HP-SMS1 were similar. Although pili are expressed in vivo, FtpA is not required for pustule formation in the human challenge model.
Assuntos
Cancroide/etiologia , Fímbrias Bacterianas/fisiologia , Haemophilus ducreyi/patogenicidade , Adulto , Método Duplo-Cego , Feminino , Haemophilus ducreyi/isolamento & purificação , Humanos , Mutação , Fenótipo , VirulênciaRESUMO
To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.
Assuntos
Haemophilus ducreyi/isolamento & purificação , Adulto , Anticorpos Monoclonais/imunologia , Cancroide/microbiologia , Derme/microbiologia , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência/métodos , Pele/microbiologiaRESUMO
BACKGROUND AND OBJECTIVES: To study Haemophilus ducreyi pathogenesis, the authors developed an experimental model of infection in human volunteers. The authors analyze their cumulative experience with strain 35000 in the model and calculate the papule and pustule formation rates for estimated delivered doses (EDDs) ranging from 1 cfu to 100 cfu. STUDY DESIGN: Sixty-five volunteers were included in the analysis. A total of 139 sites were available for calculation of the papule formation rate, and 117 sites were available for calculation of the pustule formation rates. RESULTS: The effect of EDDs and probabilities of papule formation and the pustule formation were dose-dependent. Increasing the EDD resulted in a higher probability of papule and pustule formation. CONCLUSION: H ducreyi is highly infectious for humans. Inoculation of an EDD of 1 cfu causes a papule formation rate of 50%. Pustule formation rates are approximately 50% for 27 cfu and 90% for 100 cfu.
Assuntos
Cancroide/fisiopatologia , Haemophilus ducreyi/patogenicidade , Doenças Bacterianas Sexualmente Transmissíveis/fisiopatologia , Adulto , Cancroide/patologia , Feminino , Humanos , Modelos Logísticos , Masculino , Probabilidade , VirulênciaRESUMO
The immune response to Haemophilus ducreyi is mediated in part by T cells infiltrating the site of infection. In this study, we show that H. ducreyi antigen preparations inhibited the proliferation of peripheral blood mononuclear cells and primary human T-cell lines. H. ducreyi also inhibited Jurkat T-cell proliferation and induced apoptosis of Jurkat T cells, confirmed through the detection of DNA degradation and membrane unpacking. The cytotoxic product(s) was present in cell-free culture supernatant and whole-cell preparations of H. ducreyi and was heat labile. H. ducreyi produces two known heat-labile toxins, a hemolysin and a cytolethal distending toxin (CDT). Whole cells and supernatants prepared from a hemolysin-deficient mutant had the same inhibitory and apoptotic effects on Jurkat T cells as did its isogenic parent. Preparations made from an H. ducreyi cdtC mutant were less toxic and induced less apoptosis than the parent. The toxic activity of the cdtC mutant was restored by complementation in trans. CdtC-neutralizing antibodies also inhibited H. ducreyi-induced toxicity and apoptosis. The data suggest that CDT may interfere with T-cell responses to H. ducreyi by induction of apoptosis.
Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Haemophilus ducreyi/patogenicidade , Linfócitos T/citologia , Linhagem Celular , Citometria de Fluxo , Haemophilus ducreyi/crescimento & desenvolvimento , Haemophilus ducreyi/imunologia , Humanos , Células Jurkat , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Linfócitos T/imunologiaRESUMO
The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.
Assuntos
Cancroide/patologia , Haemophilus ducreyi/patogenicidade , Lipopolissacarídeos/metabolismo , Adulto , Cancroide/microbiologia , Cancroide/fisiopatologia , Feminino , Globosídeos/química , Haemophilus ducreyi/genética , Haemophilus ducreyi/isolamento & purificação , Haemophilus ducreyi/metabolismo , Humanos , Lipopolissacarídeos/química , Masculino , Mutação , Sialiltransferases/genética , Sialiltransferases/metabolismo , Pele/patologia , VirulênciaRESUMO
We developed an enzyme-linked immunosorbent assay-based assay to assess Haemophilus ducreyi binding to extracellular matrix (ECM) proteins. H. ducreyi 35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in ftpA or losB bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for H. ducreyi to bind to ECM proteins.
Assuntos
Aderência Bacteriana/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Haemophilus ducreyi/fisiologia , Adulto , Aderência Bacteriana/genética , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Fibronectinas/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/fisiologia , Haemophilus ducreyi/genética , Haemophilus ducreyi/patogenicidade , Humanos , Técnicas In Vitro , Laminina/metabolismo , Lipopolissacarídeos/metabolismo , Mutação , Ligação Proteica , Pele/metabolismo , Pele/microbiologiaRESUMO
Two groups of human volunteers were inoculated with 2 doses of live Haemophilus ducreyi 35000HP. The reinfection group consisted of 7 subjects who previously had participated in experimental infection with 35000HP to the pustular stage of disease. The control group consisted of 7 naive subjects. Papules developed at 92.8% (95% confidence interval [CI], 66.1%-99.8%) of sites inoculated with live bacteria, in the reinfection group, and at 85.7% (95% CI, 57.2%-98. 2%) of sites in the control group. Sixty-nine percent (95% CI, 36. 8%-90.9%) of papules evolved into pustules in the reinfection group, compared with 41% (95% CI, 15.2%-72.3%) in the control group. The recovery rates of H. ducreyi from surface cultures and the histopathology of biopsies obtained from both groups were similar. Thus, experimental infection to the pustular stage of disease does not provide protective immunity against subsequent challenge.
Assuntos
Cancroide/imunologia , Cancroide/patologia , Haemophilus ducreyi/imunologia , Adulto , Anticorpos Antibacterianos/sangue , Cancroide/microbiologia , Feminino , Haemophilus ducreyi/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Leucócitos Mononucleares , Ativação Linfocitária , Masculino , Pele/microbiologia , Pele/patologiaRESUMO
A bactericidal assay was developed in order to test the effect of hyperimmune rabbit sera on the viability of serum-resistant Haemophilus ducreyi 35000HP. Testing of several lots of rabbit complement and time course experiments showed that the serum-sensitive H. ducreyi CIPA77 was killed efficiently by 25% complement at 35 degrees C in 3 h. We hypothesized that incubation of 35000HP under these conditions with the appropriate bactericidal antibody would kill this strain. A panel of high titre rabbit antisera was developed and tested against 35000HP. The panel included antisera raised to whole cells, total membranes, Sarkosyl-insoluble outer membrane proteins, the H. ducreyi lipoprotein, and the peptidoglycan-associated lipoprotein. None of the antisera convincingly showed bactericidal activity. The bactericidal assay was also used to determine the effect of normal human serum (NHS) on isogenic mutants of 35000HP. 35000HP-RSM2, an Omegakan insertion mutant that expresses a truncated lipooligosaccharide, was as resistant to NHS as its parent. A mutant deficient in expression of the major outer membrane protein (35000. 60) was sensitive to NHS. We conclude that 35000HP is relatively resistant to normal and hyperimmune sera, and that the major outer membrane protein contributes to this resistance.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Atividade Bactericida do Sangue/imunologia , Haemophilus ducreyi/crescimento & desenvolvimento , Lipopolissacarídeos/imunologia , Animais , Anticorpos Monoclonais , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Cancroide/imunologia , Contagem de Colônia Microbiana , Proteínas do Sistema Complemento/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Haemophilus ducreyi/genética , Haemophilus ducreyi/imunologia , Humanos , Imunização/veterinária , Mutação , CoelhosRESUMO
Human volunteers were challenged with Haemophilus ducreyi. Twenty subjects were inoculated with 2 doses (approximately 30 cfu) of live and 1 dose of heat-killed bacteria at 3 sites on the arm. Eight subjects were assigned to biopsy 1 or 4 days after inoculation, and 12 were biopsied after they developed a painful pustular lesion or were followed until disease resolved. Papules developed at 95% of 40 sites infected with live bacteria (95% confidence interval [CI], 83. 1%-99.4%). In 24 sites followed to end point, 27% of the papules resolved, 69% (95% CI, 47.1%-86.6%) evolved into pustules, and 4% remained at the papular stage. Recovery rates of H. ducreyi from surface cultures ranged from 13% to 41%. H. ducreyi was recovered from biopsies of 12 of 15 pustules and 1 of 7 papules, suggesting that H. ducreyi replicates between the papular and pustular stages of disease.
Assuntos
Cancroide/patologia , Cancroide/fisiopatologia , Haemophilus ducreyi , Adulto , Progressão da Doença , Feminino , Haemophilus ducreyi/isolamento & purificação , Humanos , Masculino , Pele/patologia , Fatores de TempoRESUMO
Previous work in 3 subjects infected for 2 weeks indicated that experimental infection with Haemophilus ducreyi recruits CD4 cells to the skin at the pustular stage of disease. In order to describe the kinetics of the host response, 23 subjects were infected at 2 sites with a standardized dose of H. ducreyi. Subjects were biopsied 1 or 4 days after inoculation or when they developed a painful pustular lesion (days 7-14). Papules and pustules contained a predominant T cell infiltrate that consisted of CD45RO and CD4 cells of the alpha beta lineage. Both papules and pustules contained mixed or T helper 1 type cytokine mRNA and interleukin-8 and tumor necrosis factor-alpha mRNA. Although the subjects had no history of chancroid, their immune responses resembled delayed-type hypersensitivity reactions that occurred within 24 h of inoculation and persisted throughout the course of experimental infection.
Assuntos
Linfócitos B/imunologia , Cancroide/imunologia , Haemophilus ducreyi/imunologia , Hipersensibilidade Tardia , Pele/imunologia , Linfócitos T/imunologia , Adulto , Formação de Anticorpos , Antígenos CD/análise , Sequência de Bases , Biópsia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Cancroide/patologia , Citocinas/genética , Primers do DNA , Sondas de DNA , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Celular , Cinética , Masculino , RNA Mensageiro/genética , Pele/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Fatores de Tempo , Transcrição GênicaRESUMO
Protegrins, potent antimicrobial peptides found in porcine leukocytes, have activity against the sexually transmitted pathogens Neisseria gonorrhoeae, Chlamydia trachomatis, and human immunodeficiency virus type 1. We tested synthetic protegrin 1 (PG-1) for activity against nine isolates of Haemophilus ducreyi, the etiologic agent of chancroid. The test organisms included CIP 542 (the type strain), 35000HP (a human-passaged variant of 35000), 35000HP-RSM2 (an isogenic D-glycero-D-manno-heptosyltransferase mutant of 35000HP), and six clinical isolates. The isolates were epidemiologically unrelated, represented three HindIII ribotypes, and had varying antimicrobial resistance patterns. In bactericidal assays, five isolates were rapidly killed by synthetic PG-1. In radial diffusion assays, all nine isolates were exquisitely sensitive to PG-1. These data highlight the potential of protegrins for development as topical agents to prevent many sexually transmitted diseases, including chancroid.