RESUMEN
Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.
Asunto(s)
Burkholderia pseudomallei/inmunología , Hemo-Oxigenasa 1/metabolismo , Leucocitos Mononucleares/inmunología , Melioidosis/inmunología , Talasemia beta/inmunología , Adulto , Anciano , Células Cultivadas , Femenino , Voluntarios Sanos , Hemo-Oxigenasa 1/genética , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Melioidosis/microbiología , Persona de Mediana Edad , Cultivo Primario de Células , ARN Mensajero/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Tailandia , Adulto Joven , Talasemia beta/sangre , Talasemia beta/complicacionesRESUMEN
Type 2 diabetes mellitus (T2DM) is an important risk factor for development of tuberculosis (TB). Our previous study showed glibenclamide, an anti-diabetic drug used to control blood glucose concentration, reduced interleukin (IL)-8 secretion from primary human monocytes challenged with M. tuberculosis (Mtb). In mice infected with Mtb, IL-1ß is essential for host resistance through the enhancement of cyclooxygenase that limits excessive Type I interferon (IFN) production and fosters Mtb containment. We hypothesize that glibenclamide may also interfere with monocyte mediated immune responses against Mtb and alter the balance between IL-1ß and IFNα-mediated immunity. Purified monocytes from non-diabetic and diabetic individuals were infected with Mtb or M. bovis BCG. We demonstrate that monocytes from diabetes patients who were being treated with glibenclamide showed reduced IL-1ß and IL-8 secretion when exposed to Mtb. Additionally, these responses also occurred when monocytes from non-diabetic individuals were pre-treated with glibenclamide in vitro. Moreover, this pre-treatment enhanced IFNa1 expression but was not involved with prostaglandin E2 (PGE2) expression in response to Mtb infection. Taken together, our data show that glibenclamide might exacerbate susceptibility of diabetes patients to Mtb infection by reducing IL-1ß and IL-8 production by monocytes.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gliburida/toxicidad , Hipoglucemiantes/toxicidad , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Monocitos/efectos de los fármacos , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/microbiología , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Dinoprostona/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Interferón-alfa/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Medición de Riesgo , Tuberculosis/inmunologíaRESUMEN
Tuberculosis still claims more lives than any other pathogen, and a vaccine better than BCG is urgently needed. One of the challenges for novel TB vaccines is to protect against all Mycobacterium tuberculosis lineages, including the most virulent ones, such as the Beijing lineage. Here we developed a live attenuated M. tuberculosis mutant derived from GC1237, a Beijing strain responsible for tuberculosis outbreaks in the Canary Islands. The mutant strain is inactivated both in the Rv1503c gene, responsible for surface glycolipid synthesis, and in the two-component global regulator PhoPR. This double mutant is as safe as BCG in immunodeficient SCID mice. In immune-competent mice and guinea pigs, the mutant is as protective as BCG against M. tuberculosis strains of common lineage 4 (Euro-American). By contrast, in mice the vaccine is protective against a M. tuberculosis strain of lineage 2 (East-Asian, Beijing), while BCG is not. These results highlight differences in protection efficacy of live attenuated M. tuberculosis-derived vaccine candidates depending on their genetic background, and provide insights for the development of novel live vaccines against TB, especially in East-Asian countries where M. tuberculosis strains of the Beijing family are highly dominant.
Asunto(s)
Vacunas contra la Tuberculosis/inmunología , Tuberculosis , Animales , Vacuna BCG , Cobayas , Ratones , Ratones SCID , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas Atenuadas/inmunologíaRESUMEN
We have resequenced the genomes of four Burkholderia pseudomallei K96243 laboratory cultures and compared them to the reported genome sequence that was published in 2004. Compared with the reference genome, these laboratory cultures harbored up to 42 single-nucleotide variants and up to 11 indels, including a 31.7-kb deletion in one culture.
RESUMEN
Tuberculosis (TB) is a global public health problem, which is caused by Mycobacterium tuberculosis (Mtb). Type 2 diabetes mellitus (T2DM) is one of the leading predisposing factors for development of TB after HIV/AIDS. Glibenclamide is a widely used anti-diabetic drug in low and middle-income countries where the incidence of TB is very high. In a human macrophage cell line, glibenclamide, a K+ATP-channel blocker, promoted alternative activation of macrophages by enhancing expression of the M2 marker CD206 during M2 polarization. M2 macrophages are considered poorly microbicidal and associated with TB susceptibility. Here, we investigated the effect of glibenclamide on M1 and M2 phenotypes of primary human monocytes and further determined whether specific drug treatment for T2DM individuals influences the antibacterial function of monocytes in response to mycobacterial infection. We found that glibenclamide significantly reduced M1 (HLA-DR+ and CD86+) surface markers and TNF-α production on primary human monocytes against mycobacterial infection. In contrast, M2 (CD163+ and CD206+) surface markers and IL-10 production were enhanced by pretreatment with glibenclamide. Additionally, reduction of bactericidal activity also occurred when primary human monocytes from T2DM individuals who were being treated with glibenclamide were infected with Mtb in vitro, consistent with the cytokine responses. We conclude that glibenclamide reduces M1 and promotes M2 polarization leading to impaired bactericidal ability of primary human monocytes of T2DM individuals in response to Mtb and may lead to increased susceptibility of T2DM individuals to TB and other bacterial infectious diseases.
Asunto(s)
Gliburida/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Monocitos/inmunología , Tuberculosis/inmunología , Adulto , Anciano , Células Cultivadas , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/inmunología , Femenino , Humanos , Hipoglucemiantes/farmacología , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Lectinas de Unión a Manosa/metabolismo , Persona de Mediana Edad , Monocitos/metabolismo , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/fisiología , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Tuberculosis/complicaciones , Tuberculosis/microbiologíaRESUMEN
Burkholderia pseudomallei, a gram-negative intracellular bacillus, is the causative agent of a tropical infectious disease called melioidosis. Bacterial ATP-binding cassette (ABC) transporters import and export a variety of molecules across bacterial cell membranes. At present, their significance in B. pseudomallei pathogenesis is poorly understood. We report here characterization of the BPSL1039-1040 ABC transporter. B. pseudomallei cultured in M9 medium supplemented with nitrate, demonstrated that BPSL1039-1040 is involved in nitrate transport for B. pseudomallei growth under anaerobic, but not aerobic conditions, suggesting that BPSL1039-1040 is functional under reduced oxygen tension. In addition, a nitrate reduction assay supported the function of BPSL1039-1040 as nitrate importer. A bpsl1039-1040 deficient mutant showed reduced biofilm formation as compared with the wild-type strain (P = 0.027) when cultured in LB medium supplemented with nitrate under anaerobic growth conditions. This reduction was not noticeable under aerobic conditions. This suggests that a gradient in oxygen levels could regulate the function of BPSL1039-1040 in B. pseudomallei nitrate metabolism. Furthermore, the B. pseudomallei bpsl1039-1040 mutant had a pronounced effect on plaque formation (P < 0.001), and was defective in intracellular survival in both non-phagocytic (HeLa) and phagocytic (J774A.1 macrophage) cells, suggesting reduced virulence in the mutant strain. The bpsl1039-1040 mutant was found to be attenuated in a BALB/c mouse intranasal infection model. Complementation of the bpsl1039-1040 deficient mutant with the plasmid-borne bpsl1039 gene could restore the phenotypes observed. We propose that the ability to acquire nitrate for survival under anaerobic conditions may, at least in part, be important for intracellular survival and has a contributory role in the pathogenesis of B. pseudomallei.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/fisiología , Espacio Intracelular/microbiología , Macrófagos/microbiología , Melioidosis/inmunología , Transportadoras de Casetes de Unión a ATP/genética , Anaerobiosis , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Células HeLa , Humanos , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Mutación , Nitritos/metabolismo , Fenotipo , VirulenciaRESUMEN
The NRPS/PKS cluster encodes the enzymes necessary for glidobactin synthesis it is partially conserved in various members of the Burkholderia genus including B. pseudomallei. In this study we have shown that the insertional inactivation or deletion of glbC in this cluster in B. pseudomallei could reduce the ability of the bacterium to survive or grow in murine macrophages or in human neutrophils. Exogenously added proteasome inhibitors were able to chemically complement the mutation. The insertional inactivation or deletion of glbC increased virulence in an acute model of infection in Balb/c or C57BL/6 mice but virulence in a chronic model of infection was similar to that of the wild type. Our findings contrast with the previous finding that inactivation of the glb gene cluster in B. pseudomallei strain 1026b resulted in marked attenuation, and provides evidence of differential roles for some genes in virulence of different strains of B. pseudomallei.
Asunto(s)
Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Lisina/análogos & derivados , Inhibidores de Proteasoma/metabolismo , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/patogenicidad , Línea Celular , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Femenino , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Humanos , Lisina/efectos de los fármacos , Lisina/genética , Macrófagos/microbiología , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Familia de Multigenes/genética , Mutagénesis Insercional/métodos , Mutación , Neutrófilos/microbiología , Péptido Sintasas/genética , Sintasas Poliquetidas/genética , Eliminación de Secuencia , Sobrevida , VirulenciaRESUMEN
B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic.
Asunto(s)
Vacunas Bacterianas/inmunología , Burkholderia mallei/inmunología , Burkholderia pseudomallei/inmunología , Muermo/inmunología , Muermo/prevención & control , Melioidosis/inmunología , Melioidosis/prevención & control , Animales , Ensayos Clínicos como Asunto , HumanosRESUMEN
Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3- CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Interleucina-10/farmacología , Melioidosis/inmunología , Adulto , Anciano , Burkholderia pseudomallei/inmunología , Células Cultivadas , Enfermedades Endémicas , Femenino , Voluntarios Sanos , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Tailandia , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis.
Asunto(s)
Linfocitos B/inmunología , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antibacterianos/sangre , Linfocitos B/microbiología , Proteínas de la Membrana Bacteriana Externa/inmunología , Células Cultivadas , Enfermedades Endémicas , Proteínas Fimbrias/inmunología , Flagelina/inmunología , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Melioidosis/epidemiología , Ratones , Ratones SCID , Linfocitos T/microbiología , TailandiaRESUMEN
The major risk factor for melioidosis, an infectious disease caused by B. pseudomallei, is diabetes mellitus. More than half of diabetic melioidosis patients in Thailand were prescribed glibenclamide. Recent evidence demonstrates that glibenclamide reduces pro-inflammatory cytokine production by polymorphonuclear neutrophils (PMNs) of diabetic individuals in response to this bacterial infection. However, the mechanisms by which glibenclamide affects cytokine production are unknown. We found that PMNs from glibenclamide-treated diabetic individuals infected with live B. pseudomallei in vitro showed lower free glutathione (GSH) levels compared with those of healthy individuals. Glibenclamide decreased GSH levels and glutathione peroxidase (GPx) of PMNs after exposed to live B. pseudomallei. Moreover, glibenclamide reduced cytokine production and migration capacity of infected PMNs, whereas GSH could restore these functions. Taken together, our data show a link between the effect of glibenclamide on GSH and PMN functions in response to B. pseudomallei that may contribute to the susceptibility of diabetic individuals to B. pseudomallei infection.
Asunto(s)
Burkholderia pseudomallei/patogenicidad , Glutatión/metabolismo , Gliburida/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Acetilcisteína/farmacología , Adulto , Burkholderia pseudomallei/efectos de los fármacos , Estudios de Casos y Controles , Movimiento Celular/efectos de los fármacos , Citocinas/sangre , Diabetes Mellitus/tratamiento farmacológico , Femenino , Glutatión/sangre , Glutatión/farmacología , Humanos , Hipoglucemiantes/farmacología , Masculino , Melioidosis/tratamiento farmacológico , Melioidosis/patología , Neutrófilos/microbiología , Fagocitosis/efectos de los fármacosRESUMEN
Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-α, and IL-1ß are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α, and IL-1ß, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1ß and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.
Asunto(s)
Citocinas/biosíntesis , Inflamación/inmunología , Interferón Tipo I/biosíntesis , Interleucina-10/análisis , Macrófagos/metabolismo , Animales , Burkholderia pseudomallei/inmunología , Citocinas/inmunología , Interferón Tipo I/inmunología , Interleucina-10/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Mycobacterium tuberculosis (Mtb), possesses at least three type VII secretion systems, ESX-1, -3 and -5 that are actively involved in pathogenesis and host-pathogen interaction. We recently showed that an attenuated Mtb vaccine candidate (Mtb Δppe25-pe19), which lacks the characteristic ESX-5-associated pe/ppe genes, but harbors all other components of the ESX-5 system, induces CD4+ T-cell immune responses against non-esx-5-associated PE/PPE protein homologs. These T cells strongly cross-recognize the missing esx-5-associated PE/PPE proteins. Here, we characterized the fine composition of the functional cross-reactive Th1 effector subsets specific to the shared PE/PPE epitopes in mice immunized with the Mtb Δppe25-pe19 vaccine candidate. We provide evidence that the Mtb Δppe25-pe19 strain, despite its significant attenuation, is comparable to the WT Mtb strain with regard to: (i) its antigenic repertoire related to the different ESX systems, (ii) the induced Th1 effector subset composition, (iii) the differentiation status of the Th1 cells induced, and (iv) its particular features at stimulating the innate immune response. Indeed, we found significant contribution of PE/PPE-specific Th1 effector cells in the protective immunity against pulmonary Mtb infection. These results offer detailed insights into the immune mechanisms underlying the remarkable protective efficacy of the live attenuated Mtb Δppe25-pe19 vaccine candidate, as well as the specific potential of PE/PPE proteins as protective immunogens.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Reacciones Cruzadas , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Células TH1RESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, which is associated with a range of clinical manifestations, including sepsis and fatal pneumonia and is endemic in Southeast Asia and Northern Australia. Treatment can be challenging and control of infection involves prolonged antibiotic therapy, yet there are no approved vaccines available to prevent infection. Our aim was to develop and assess the potential of a prophylactic vaccine candidate targeted against melioidosis. The identified candidate is the 22kDa outer membrane protein, OmpW. We previously demonstrated that this protein was immunoprotective in mouse models of Burkholderia cepacia complex (Bcc) infections. We cloned Bp_ompW in Escherichia coli, expressed and purified the protein. Endotoxin free protein administered with SAS adjuvant protected Balb/C mice (75% survival) relative to controls (25% survival) (p<0.05). A potent serological response was observed with IgG2a to IgG1 ratio of 6.0. Furthermore C57BL/6 mice were protected for up to 80 days against a lethal dose of B. pseudomallei and surpassed the efficacy of the live attenuated 2D2 positive control. BpompW is homologous across thirteen sequenced B. pseudomallei strains, indicating that it should be broadly protective against B. pseudomallei. In conclusion, we have demonstrated that BpOmpW is able to induce protective immunity against melioidosis and is likely to be an effective vaccine antigen, possibly in combination with other subunit antigens.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Melioidosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Burkholderia pseudomallei , Femenino , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Vacunas de Subunidad/inmunologíaRESUMEN
Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises tryptophan to anthranilate. This metabolite can be used for downstream production of 2-alkyl-4-quinolone (AQ) signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate the role of kynB in the production of AQs and virulence-associated phenotypes of Burkholderia pseudomallei K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced AQ production, increased biofilm formation, decreased swarming and increased tolerance to ciprofloxacin. Addition of exogenous anthranilic acid restored the biofilm phenotype, but not the persister phenotype. This study suggests the kynurenine pathway is a critical source of anthranilate and signalling molecules that may regulate B. pseudomallei virulence.
Asunto(s)
Arilformamidasa/metabolismo , Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/fisiología , Locomoción , Quinolonas/metabolismo , Transducción de Señal , Burkholderia pseudomallei/genética , Eliminación de Gen , Triptófano/metabolismo , Virulencia , ortoaminobenzoatos/metabolismoRESUMEN
Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/metabolismo , Melioidosis/microbiología , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Factores de Virulencia/genéticaRESUMEN
Melioidosis, a severe human disease caused by the bacterium Burkholderia pseudomallei, has a wide spectrum of clinical manifestations ranging from acute septicemia to chronic localized illness or latent infection. Murine models have been widely used to study the pathogenesis of infection and to evaluate novel therapies or vaccines, but how faithfully they recapitulate the biology of human melioidosis at a molecular level is not known. In this study, mice were intranasally infected with either high or low doses of B. pseudomallei to generate either acute, chronic, or latent infection and host blood and tissue transcriptional profiles were generated. Acute infection was accompanied by a homogeneous signature associated with induction of multiple innate immune response pathways, such as IL-10, TREM1, and IFN signaling, largely found in both blood and tissue. The transcriptional profile in blood reflected the heterogeneity of chronic infection and quantitatively reflected the severity of disease. Genes associated with fibrosis and tissue remodeling, including matrix metalloproteases and collagen, were upregulated in chronically infected mice with severe disease. Transcriptional signatures of both acute and chronic melioidosis revealed upregulation of iNOS in tissue, consistent with the expression of IFN-γ, but also Arginase-1, a functional antagonist of the iNOS pathway, and was confirmed by immunohistochemistry. Comparison of these mouse blood datasets by pathway and modular analysis with the blood transcriptional signature of patients with melioidosis showed that many genes were similarly perturbed, including Arginase-1, IL-10, TREM1, and IFN signaling, revealing the common immune response occurring in both mice and humans.
Asunto(s)
Burkholderia pseudomallei/inmunología , Inmunidad Innata/inmunología , Melioidosis/inmunología , Animales , Arginasa/biosíntesis , Arginasa/sangre , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-10/sangre , Interleucina-10/genética , Interleucina-10/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Metaloproteinasa 9 de la Matriz/sangre , Melioidosis/microbiología , Melioidosis/patología , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Receptores Inmunológicos/sangre , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Transducción de Señal/inmunología , Transcriptoma/genética , Receptor Activador Expresado en Células Mieloides 1RESUMEN
BACKGROUND: Mycobacterium bovis BCG is presently the only available anti-tuberculosis vaccine used, worldwide. While BCG protects against miliary tuberculosis (TB) and tuberculoid meningitis in children, it often fails to protect against adult pulmonary TB. It is thus imperative that new improved anti-TB vaccines are developed. The integration of the ESX-1 secretion system, absent from BCG due to the deletion of region of difference 1 (RD1), into the genome of BCG has been shown to confer to BCG::ESX-1 enhanced protection against TB as compared to BCG. METHODS: In the present study, to counterbalance the increase in virulence resulting from the integration of the RD1 region into BCG, we have constructed and evaluated several BCG::ESX-1 variants that carry selected amino-acid changes in the ESX-1-secreted antigen ESAT-6. In order to find the candidate that combines low virulence with high protective efficacy, these novel recombinant BCG::ESX-1 strains were tested for their virulence properties and their protective efficacy against Mycobacterium tuberculosis in two different animal models (mouse and guinea-pig). RESULTS: Among several candidates tested, the BCG::ESAT-L28A/L29S strain, carrying modifications at residues Leu(28)-Leu(29) of the ESAT molecule, showed strong attenuation in mice and high protective efficiency both in mouse and guinea-pig vaccination-infection models. CONCLUSION: This strain thus represents a promising candidate that merits further investigations and development. Our research also provides the proof of concept that selected ESX-1-complemented BCG strains may show low virulence and increased protective potential over parental strains.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Vacuna BCG/inmunología , Proteínas Bacterianas/biosíntesis , Tuberculosis/prevención & control , Animales , Antígenos Bacterianos/genética , Vacuna BCG/administración & dosificación , Vacuna BCG/efectos adversos , Vacuna BCG/genética , Proteínas Bacterianas/genética , Recuento de Colonia Microbiana , Femenino , Cobayas , Pulmón/microbiología , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Bazo/microbiología , Análisis de Supervivencia , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , VirulenciaRESUMEN
Polymorphonuclear neutrophils (PMNs) are terminally differentiated cells that are involved in innate immune responses and form an early line of defense against pathogens. More recently, it has been shown that PMNs have immunosuppressive abilities on other immune cells. However, the effect of PMNs on T cell responses during bacterial infection remains to be determined. In this report, we examined the interaction of PMNs and T cells in response to infection with Burkholderia pseudomallei, the causative agent of human melioidosis. We observed that CD4(+) T cell proliferation and IFN-γ production in response to polyclonal activators is significantly inhibited by uninfected PMNs, and to a greater extent B. pseudomallei-infected PMNs. Programmed death ligand 1 (PD-L1), a known regulator of T cell activation, is increased in mRNA expression in the blood of patients and upon infection of PMNs in vitro. The increased expression of PD-L1 was correlated with the degree of T cell inhibition in individuals with type 2 diabetes, a major risk factor of melioidosis. In vitro, addition of anti-PD-L1 Abs blocked this inhibitory activity and restored proliferation of CD4(+) T cells and IFN-γ production, suggesting that PD-L1 on B. pseudomallei-infected PMNs is a regulatory molecule for the functions of T cells and may be involved in pathogenesis versus control of melioidosis.
Asunto(s)
Antígeno B7-H1/metabolismo , Burkholderia pseudomallei/inmunología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Adulto , Anciano , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Comunicación Celular , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Inmunomodulación , Masculino , Melioidosis/inmunología , Melioidosis/metabolismo , Persona de Mediana Edad , Neutrófilos/microbiología , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de SeñalRESUMEN
Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.