RESUMEN
Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), the third most common mycobacterial infection. Virulent M. ulcerans secretes mycolactone, a polyketide toxin. Most observations of M. ulcerans infection are described as an extracellular milieu in the form of a necrotic ulcer. While some evidence exists of an intracellular life cycle for M. ulcerans during infection, the exact role that mycolactone plays in this process is poorly understood. Many previous studies have relied upon the addition of purified mycolactone to cell-culture systems to study its role in M. ulcerans pathogenesis and host-response modulation. However, this sterile system drastically simplifies the M. ulcerans infection model and assumes that mycolactone is the only relevant virulence factor expressed by M. ulcerans. Here we show that the addition of purified mycolactone to macrophages during M. ulcerans infection overcomes the bacterial activation of the mechanistic target of rapamycin (mTOR) signaling pathway that plays a substantial role in regulating different cellular processes, including autophagy and apoptosis. To further study the role of mycolactone during M. ulcerans infection, we have developed an inducible mycolactone expression system. Utilizing the mycolactone-deficient Mul::Tn118 strain that contains a transposon insertion in the putative beta-ketoacyl transferase (mup045), we have successfully restored mycolactone production by expressing mup045 in a tetracycline-inducible vector system, which overcomes in-vitro growth defects associated with constitutive complementation. The inducible mycolactone-expressing bacteria resulted in the establishment of infection in a murine footpad model of BU similar to that observed during the infection with wild-type M. ulcerans. This mycolactone inducible system will allow for further analysis of the roles and functions of mycolactone during M. ulcerans infection.
Asunto(s)
Toxinas Bacterianas , Úlcera de Buruli , Mycobacterium ulcerans , Animales , Toxinas Bacterianas/metabolismo , Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Macrólidos/farmacología , Ratones , Mycobacterium ulcerans/metabolismoRESUMEN
Buruli ulcer (BU) vaccine design faces similar challenges to those observed during development of prophylactic tuberculosis treatments. Multiple BU vaccine candidates, based upon Mycobacterium bovis BCG, altered Mycobacterium ulcerans (MU) cells, recombinant MU DNA, or MU protein prime-boosts, have shown promise by conferring transient protection to mice against the pathology of MU challenge. Recently, we have shown that a recombinant BCG vaccine expressing MU-Ag85A (BCG MU-Ag85A) displayed the highest level of protection to date, by significantly extending the survival time of MU challenged mice compared to BCG vaccination alone. Here we describe the generation, immunogenicity testing, and evaluation of protection conferred by a recombinant BCG strain which overexpresses a fusion of two alternative MU antigens, Ag85B and the MU ortholog of tuberculosis TB10.4, EsxH. Vaccination with BCG MU-Ag85B-EsxH induces proliferation of Ag85 specific CD4+ T cells in greater numbers than BCG or BCG MU-Ag85A and produces IFNγ+ splenocytes responsive to whole MU and recombinant antigens. In addition, anti-Ag85A and Ag85B IgG humoral responses are significantly enhanced after administration of the fusion vaccine compared to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival times compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this highly safe and ubiquitous vaccine.
Asunto(s)
Antígenos Bacterianos/genética , Vacuna BCG/genética , Vacunas Bacterianas/inmunología , Úlcera de Buruli/prevención & control , Inmunogenicidad Vacunal , Mycobacterium ulcerans/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Vacuna BCG/efectos adversos , Vacuna BCG/inmunología , Vacunas Bacterianas/genética , Úlcera de Buruli/inmunología , Úlcera de Buruli/microbiología , Linfocitos T CD4-Positivos/inmunología , Expresión Génica , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Ratones , Mycobacterium ulcerans/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette-Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4+ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Úlcera de Buruli/inmunología , Úlcera de Buruli/prevención & control , Mycobacterium bovis/inmunología , Mycobacterium ulcerans/inmunología , Animales , Antígenos Bacterianos/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Interferón gamma/metabolismo , Ratones Endogámicos C57BL , Mycobacterium bovis/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/inmunología , Mycobacterium ulcerans/genética , Análisis de Supervivencia , Resultado del Tratamiento , Vacunación/métodos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.
Asunto(s)
Antígenos Virales/biosíntesis , Portadores de Fármacos , Productos del Gen gag/biosíntesis , Inestabilidad Genómica , Proteína gp120 de Envoltorio del VIH/biosíntesis , Mycobacterium bovis/genética , Vacunas contra el SIDA/genética , Vacunas contra el SIDA/inmunología , Animales , Antígenos Virales/genética , Productos del Gen gag/genética , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH/genética , Ratones Endogámicos C57BL , Vacunas contra el SIDAS/genética , Vacunas contra el SIDAS/inmunología , Linfocitos T/inmunologíaRESUMEN
We recently identified I602S as a frequent single-nucleotide polymorphism of human TLR1 that greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Because mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHC class II, CD64, and IFN-γ responses compared with individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1, an enzyme that depletes cellular arginine stores required for the production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria.
Asunto(s)
Macrófagos/inmunología , Monocitos/inmunología , Mycobacterium tuberculosis/inmunología , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 1/metabolismo , Arginasa/genética , Arginasa/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/microbiología , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Isoleucina/genética , Isoleucina/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Monocitos/efectos de los fármacos , Monocitos/microbiología , Mycobacterium tuberculosis/patogenicidad , Polimorfismo de Nucleótido Simple/inmunología , Transporte de Proteínas/efectos de los fármacos , Receptores de IgG/genética , Receptores de IgG/inmunología , Serina/genética , Serina/inmunología , Receptor Toll-Like 1/genética , Receptor Toll-Like 1/inmunologíaRESUMEN
Genetic association studies of leprosy cohorts across the world have identified numerous polymorphisms which alter susceptibility and outcome to infection with Mycobacterium leprae. As expected, many of the polymorphisms reside within genes that encode components of the innate and adaptive immune system. Despite the preponderance of these studies, our understanding of the mechanisms that underlie these genetic associations remains sparse. Toll-like receptors (TLRs) have emerged as an essential family of innate immune pattern recognition receptors which play a pivotal role in host defense against microbes, including pathogenic strains of mycobacteria. This paper will highlight studies which have uncovered the association of specific TLR gene polymorphisms with leprosy or tuberculosis: two important diseases resulting from mycobacterial infection. This analysis will focus on the potential influence these polymorphic variants have on TLR expression and function and how altered TLR recognition or signaling may contribute to successful antimycobacterial immunity.
RESUMEN
The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression.