RESUMEN
Self-emulsification is routinely used for oral delivery of lipophilic drugs in vivo, with the emulsion forming in vivo. We modified this technique to prepare novel oil-in-water emulsions of varying droplet size and composition on bench to enable adjuvanted vaccine delivery. We used these formulations to show that smaller droplets (20 nm) were much less effective as adjuvants for an influenza vaccine in mice than the emulsion droplet size of commercial influenza vaccine adjuvants (~160 nm). This was unexpected, given the many claims in the literature of the advantages of smaller particulates. We also undertook cell-recruitment mechanistic studies at site of injection and draining lymph nodes to directly address the question of why the smaller droplets were less effective. We discovered that emulsion droplet size and composition have a considerable impact on the ability to recruit immune cells to the injection site. We believe that further work is warranted to more extensively explore the question of whether, the smaller is not 'better', is a more common observation for particulate adjuvants.
Asunto(s)
Adyuvantes Farmacéuticos/administración & dosificación , Emulsiones/química , Vacunas contra la Influenza/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Composición de Medicamentos , Femenino , Inmunidad Celular , Vacunas contra la Influenza/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Tamaño de la PartículaRESUMEN
A rapidly acting, single dose vaccine against Staphylococcus aureus would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent S. aureus candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4+ T cells not only reduced survival but also increased S. aureus load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by in vivo neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against S. aureus through the combined actions of antibodies, CD4+ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-17/metabolismo , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Receptor Toll-Like 7/metabolismo , Adyuvantes Inmunológicos , Animales , Anticuerpos Neutralizantes/inmunología , Linfocitos T CD4-Positivos/citología , Citocinas/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Bazo/metabolismo , Bazo/patología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/genética , Tasa de Supervivencia , Células TH1/inmunología , Células Th17/inmunología , Receptor Toll-Like 7/inmunologíaRESUMEN
Staphylococcus aureus (S. aureus) is an important opportunistic pathogen that may cause invasive life-threatening infections, like sepsis and pneumonia. Due to the increasing antibiotic resistance, the development of an effective vaccine against S. aureus is needed. Although a correlate of protection against staphylococcal diseases is not yet established, several findings suggest that both antibodies and CD4 T cells might contribute to optimal immunity. In this study, we show that adjuvanting a multivalent vaccine (4C-Staph) with MF59, an oil-in-water emulsion licensed in human vaccines, further potentiated antigen-specific IgG titers and CD4 T-cell responses compared to alum and conferred protection in the peritonitis model of S. aureus infection. Moreover, we showed that MF59- and alum-adjuvanted 4C-Staph vaccines induced persistent antigen-specific humoral and T-cell responses, and protected mice from infection up to 4 months after immunization. Furthermore, 4C-Staph formulated with MF59 was used to investigate which immune compartment is involved in vaccine-induced protection. Using CD4 T cell-depleted mice or B cell-deficient mice, we demonstrated that both T and B-cell responses contributed to 4C-Staph vaccine-mediated protective immunity. However, the role of CD4 T cells seemed more evident in the presence of low-antibody responses. This study provides preclinical data further supporting the use of the adjuvanted 4C-Staph vaccines against S. aureus diseases, and provides critical insights on the correlates of protective immunity necessary to combat this pathogen.
RESUMEN
Induction of persistent protective immune responses is a key attribute of a successful vaccine formulation. MF59 adjuvant, an oil-in-water emulsion used in human vaccines, is known to induce persistent high-affinity functional Ab titers and memory B cells, but how it really shapes the Ag-specific B cell compartment is poorly documented. In this study, we characterized the Ab- and Ag-specific B cell compartment in wild-type mice immunized with HlaH35L, a Staphylococcus aureus Ag known to induce measurable functional Ab responses, formulated with MF59 or aluminum salts, focusing on germinal centers (GC) in secondary lymphoid organs. Taking advantage of single-cell flow cytometry analyses, HlaH35L-specific B cells were characterized for the expression of CD38 and GL-7, markers of memory and GC, respectively, and for CD80 and CD73 activation markers. We demonstrated that immunization with MF59-, but not aluminum salt-adjuvanted HlaH35L, induced expanded Ag-specific CD73(+)CD80(-) GC B cells in proximal- and distal-draining lymph nodes, and promoted the persistence of GC B cells, detected up to 4 mo after immunization. In addition to increasing GC B cells, MF59-adjuvanted HlaH35L also increased the frequency of T follicular helper cells. This work extends previous knowledge regarding adaptive immune responses to MF59-adjuvanted vaccines, and, to our knowledge, for the first time an adjuvant used in human licensed products is shown to promote strong and persistent Ag-specific GC responses that might benefit the rational design of new vaccination strategies.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Diferenciación Celular , Centro Germinal/citología , Centro Germinal/inmunología , Polisorbatos , Escualeno , Vacunación , Adyuvantes Inmunológicos , Animales , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos CD/metabolismo , Linfocitos B/metabolismo , Toxinas Bacterianas/inmunología , Quimiotaxis de Leucocito/inmunología , Femenino , Proteínas Hemolisinas/inmunología , Inmunofenotipificación , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Fenotipo , Escualeno/inmunología , Vacunas EstafilocócicasRESUMEN
Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/química , Receptor Toll-Like 7/química , Absceso/patología , Inmunidad Adaptativa , Animales , Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Antígenos/inmunología , Humanos , Ratones , Modelos Animales , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus , Células TH1/inmunologíaRESUMEN
Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Diseño de Fármacos , Vacunas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Disponibilidad BiológicaRESUMEN
BACKGROUND: Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. RESULTS: We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. CONCLUSION: Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Células Epiteliales/microbiología , Neisseria gonorrhoeae/fisiología , Adhesinas Bacterianas/genética , Proteínas de la Membrana Bacteriana Externa/genética , Células Cultivadas , Femenino , Eliminación de Gen , Humanos , Masculino , Neisseria gonorrhoeae/genéticaRESUMEN
The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacunas contra la Influenza/inmunología , Interferón-alfa/inmunología , Lipopéptidos/farmacología , Polisorbatos/farmacología , Escualeno/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Anticuerpos Monoclonales/farmacología , Quimiotaxis/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos BALB C , Músculos/inmunología , Músculos/metabolismo , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Toxoide Tetánico/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Interleucina-12/biosíntesis , Interleucina-23/biosíntesis , Receptores Toll-Like/agonistas , Familia-src Quinasas/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Imidazoles/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Pirimidinas/farmacología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/metabolismo , Receptores Toll-Like/metabolismo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
CpG-containing oligodeoxynucleotides are potent mucosal adjuvants and effective as stand-alone treatment of respiratory infections in mice. Although CpG is also used as a type 1 helper immunomodulator in the treatment of asthma and allergic disease, immune modulation following intranasal application has not been fully characterized yet. Using a B-type CpG, we monitored RNA expression profiles, cytokine production and cellular activation in lung tissue and bronchoalveolar lavages ex vivo and cytokine production of purified cell populations in vitro. CpG triggered the upregulation of many transcripts, including interferon response genes and proinflammatory cytokine genes, between 3 h and 4 days. Overlapping subsets of these cytokine proteins were induced in vitro in purified CD11c+ cells, B cells and alveolar macrophages from the lung, thus identifying these cells as direct targets of CpG. While lung B cells strongly respond to CpG in vitro, less activation is found ex vivo, suggesting efficient CpG sequestering or rapid B cell migration after activation. In contrast, a type II alveolar epithelial cell line did not respond to CpG in vitro. We noted selective recruitment of plasmacytoid dendritic cells (DCs) into the lung tissue, and of conventional DCs and natural killer (NK) cells into the lung tissue and bronchoalveolar space. Furthermore, CpG induced activation of intrapulmonary DCs, NK and T cells. We hypothesize that CpG-linked adjuvanticity and clearance of respiratory pathogens are mediated by two major mechanisms: transient induction of the interferon pathway limiting microbial survival and selective recruitment of DCs and NK cells, which allows for better adaptive responses.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Pulmón/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Administración Intranasal , Animales , Linfocitos B/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Antígeno CD11c/metabolismo , Femenino , Pulmón/citología , Activación de Linfocitos/inmunología , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/inmunologíaRESUMEN
Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.
Asunto(s)
Adyuvantes Inmunológicos , Hidróxido de Aluminio/inmunología , Quimiocinas CC/metabolismo , Células Dendríticas/inmunología , Granulocitos/inmunología , Monocitos/inmunología , Escualeno/inmunología , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Diferenciación Celular , Quimiocinas CC/inmunología , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Endocitosis , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Receptores de Lipopolisacáridos/inmunología , Receptores de Lipopolisacáridos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , PolisorbatosRESUMEN
LTK63, a nontoxic mutant of Escherichia coli heat labile enterotoxin (LT), is a potent and safe mucosal adjuvant that has also been shown to confer generic protection to several respiratory pathogens. To understand the mechanisms of action underlying the LTK63 protective effect, we analyzed the molecular and cellular events triggered by its administration in vivo. We show here that LTK63 intrapulmonary administration induced in the mouse lung a specific gene expression signature characterized by the up-regulation of cell cycle genes, several host defense genes, chemokines, chemokine receptors, and immune cell-associated genes. Such a transcriptional profile reflected the activation of alveolar macrophages and the recruitment to the lung of T and B cells and innate immune cells such as granulocytes, NK, and dendritic cells. All of these events were T cell dependent and specific for LTK63 because they were absent in SCID and nude mice. Additionally, we showed that LTK63 induces a potent adaptive immune response against itself directed to the lung. We propose that acquired response to LTK63 is the driving force for the local recruitment of both adaptive and innate immune cells. Our data suggest that LTK63 acts as an airway infection mimic that establishes a generic protective environment limiting respiratory infection by innate immune mechanisms and by improving adaptive responses to invading pathogens.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/inmunología , Enterotoxinas/administración & dosificación , Enterotoxinas/inmunología , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/inmunología , Inmunidad Mucosa , Pulmón/inmunología , Pulmón/metabolismo , Animales , Subgrupos de Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/inmunología , Quimiocinas/biosíntesis , Quimiocinas/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Inmunidad Celular , Inmunidad Innata , Intubación Intratraqueal , Pulmón/citología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Transcripción Genética/inmunologíaRESUMEN
Outer membrane protein As (OmpAs) are highly conserved proteins within the Enterobacteriaceae family. OmpA contributes to the maintenance of structural membrane integrity and invasion into mammalian cells. In Escherichia coli K1 OmpA also contributes to serum resistance and is involved in the virulence of the bacterium. Here we describe the identification of an OmpA-like protein in Neisseria gonorrhoeae (Ng-OmpA). We show that the gonococcal OmpA-like protein, similarly to E. coli OmpA, plays a significant role in the adhesion and invasion into human cervical carcinoma and endometrial cells and is required for entry into macrophages and intracellular survival. Furthermore, the isogenic knockout ompA mutant demonstrates reduced recovery in a mouse model of infection when compared with the wild-type strain, suggesting that Ng-OmpA plays an important role in the in vivo colonization. All together, these data suggest that the newly identified surface exposed protein Ng-OmpA represents a novel virulence factor of gonococcus.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Neisseria gonorrhoeae/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana/fisiología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/genética , Línea Celular , Línea Celular Tumoral , Secuencia de Consenso , Secuencia Conservada , Células Epiteliales/microbiología , Humanos , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/fisiología , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fagocitosis , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Macrophage infectivity potentiators (MIPs) are a family of surface-exposed virulence factors of intracellular microorganisms such as Legionella, Chlamydia and Trypanosoma. These proteins display peptidyl-prolyl cis/trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng-MIP, a surface-exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycin-inhibited PPIase activity confirming that it is a functional member of the MIP family. A knock-out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng-MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.