RESUMEN
Lactobacillus paracasei are diverse Gram-positive bacteria that are very closely related to Lactobacillus casei, belonging to the Lactobacillus casei group. Due to extreme genome similarities between L. casei and L. paracasei, many strains have been cross placed in the other group. We had earlier sequenced and analyzed the genome of Lactobacillus paracasei Lbs2, but mistakenly identified it as L. casei. We re-analyzed Lbs2 reads into a 2.5 MB genome that is 91.28% complete with 0.8% contamination, which is now suitably placed under L. paracasei based on Average Nucleotide Identity and Average Amino Acid Identity. We took 74 sequenced genomes of L. paracasei from GenBank with assembly sizes ranging from 2.3 to 3.3 MB and genome completeness between 88% and 100% for comparison. The pan-genome of 75 L. paracasei strains hold 15,945 gene families (21,5232 genes), while the core genome contained about 8.4% of the total genes (243 gene families with 18,225 genes) of pan-genome. Phylogenomic analysis based on core gene families revealed that the Lbs2 strain has a closer relationship with L. paracasei subsp. tolerans DSM20258. Finally, the in-silico analysis of the L. paracasei Lbs2 genome revealed an important pathway that could underpin the production of thiamin, which may contribute to the host energy metabolism.
RESUMEN
We report here a 3.2-Mb draft assembled genome of Lactobacillus casei Lbs2. The bacterium shows probiotic and immunomodulatory activities. The genome assembly and annotation will help to identify molecules and pathways responsible for interaction between the host immune system and the microbe.
RESUMEN
Vibrio cholerae induces acute inflammatory response at intestinal epithelial surface; the underlying cellular immune mechanisms for such effects are largely unexplored. Mucosal immune response is controlled by crosstalk between the intestinal epithelial cells (ECs) and dendritic cells (DCs). An EC-derived cytokine thymic stromal lymphopoietin (TSLP) has been found a critical regulator of several human inflammatory conditions. TSLP is highly elevated in ECs stimulated with V. cholerae and its recombinant flagellin (rFlaA). V. cholerae treated human ECs produce DC-attracting chemokine MIP-3α (CCL20). Flagellin, a potent V. cholerae factor was responsible for maximum stimulation of epithelial CCL20 production and subsequent DC activation. Activated DCs express high levels of costimulatory molecules and secrete inflammatory cytokines TNF-α, IL-6 and IL-1ß. Bacteria stimulated ECs conditioned DCs to produce Th2 cell-attracting chemokines CCL17 and CCL22. TSLP and other mediators present in the V. cholerae stimulated EC-culture filtrate potently activated DCs, which subsequently primed CD4(+)T cells to differentiate into T helper type 2 (Th2) cells that produce high amounts of IL-4, IL-13 and TNF-α and low IFN-γ. TSLP-induced proinflammatory response in DCs involved the transcriptional mechanisms, MAPKs (ERK1/2, p38 and JNK) and STAT3 activation. This study suggests TSLP and other mediators released from ECs in response to V. cholerae colonization actively influence DCs in initiating inflammatory response.
Asunto(s)
Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Inflamación/patología , Células Th2/inmunología , Vibrio cholerae/inmunología , Línea Celular , Quimiocinas/biosíntesis , Cólera/complicaciones , Cólera/inmunología , Cólera/microbiología , Técnicas de Cocultivo , Recuento de Colonia Microbiana , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/microbiología , Regulación hacia Arriba , Vibrio cholerae/crecimiento & desarrollo , Linfopoyetina del Estroma TímicoRESUMEN
Interleukin (IL)-8 is an important mediator in neutrophil-mediated acute inflammation. We previously demonstrated that incubation of intestinal epithelial cells with Vibrio cholerae O395 resulted in increased IL-8 mRNA expression and IL-8 secretion, which was associated with the adherence and motility of bacteria. However, the mechanisms responsible for transcriptional regulation of the IL-8 gene in epithelial cells during V. cholerae infections were not explored. Transient transfection analysis of 5' deletions and mutations of the IL-8 promoter driving expression of the luciferase reporter gene indicates that multiple binding sites contribute to IL-8 promoter induction in response to V. cholerae and that cooperation among these different sites is required for full activation of the promoter. Stimulation with V. cholerae O395 insertional mutants, defective in adherence and motility, significantly reduced IL-8 promoter activity compared with the wild-type strain. We further demonstrate maximal involvement of extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase pathways to regulate IL-8 gene transcription. This study will help to design agents which could reduce the V. cholerae-induced inflammatory response and in the generation of safe vaccines.
Asunto(s)
Adhesión Bacteriana , Movimiento Celular , Regulación de la Expresión Génica , Interleucina-8/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas/genética , Elementos Reguladores de la Transcripción , Vibrio cholerae/patogenicidad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células HeLa , Humanos , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Luciferasas/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/inmunologíaRESUMEN
Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration-rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-gamma. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines.
Asunto(s)
Antígenos de Protozoos/administración & dosificación , Leishmania donovani/inmunología , Leishmaniasis Visceral/prevención & control , Vacunas Antiprotozoos/administración & dosificación , Vacunación , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Células Cultivadas , Química Farmacéutica , Modelos Animales de Enfermedad , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/parasitología , Inmunidad Celular , Inmunidad Humoral , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Liposomas , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Vacunas Antiprotozoos/química , Vacunas Antiprotozoos/inmunología , Células TH1/inmunología , Células TH1/parasitología , Factores de TiempoRESUMEN
Vibrio cholerae activates proinflammatory response in cultured intestinal epithelial cells. In this study, we demonstrate that V. cholerae O395 infection of intestinal epithelial cells results in the activation of Akt. Inhibition of Akt significantly decreases IL-1alpha, IL-6, and TNF-alpha production in V. cholerae infected Int407 cells. Analysis of the mechanisms of Akt influences on cytokine response demonstrates that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with virulence, adherence, or motility. Insertion mutants in the virulence genes coding for CtxA, ToxT, and OmpU of V. cholerae modulate the activation of PI3K/Akt signaling pathway, whereas an aflagellate non-motile mutant (O395FLAN) and a adherent and less motile mutant (O395Y3N/O395Y4N) of V. cholerae both show very significant down-regulation of Akt activity in Int407 cells. Together, these observations indicate that Akt promotes proinflammatory cytokine production by V. cholerae infected human intestinal epithelial cells through its influences on NF-kappaB.
Asunto(s)
Cólera/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Vibrio cholerae/fisiología , Línea Celular , Cólera/inmunología , Cólera/microbiología , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Intestinos/citología , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 mug of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-gamma and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8+ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/prevención & control , Liposomas/administración & dosificación , Metaloendopeptidasas/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Antiprotozoarios/sangre , Linfocitos T CD8-positivos/inmunología , Relación Dosis-Respuesta Inmunológica , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Liposomas/farmacología , Hígado/parasitología , Ratones , Ratones Endogámicos BALB C , Fosfatidilcolinas/administración & dosificación , Fosfatidilcolinas/farmacología , Bazo/parasitología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Factores de TiempoRESUMEN
Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.