RESUMEN
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pnc A gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice.Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. The bbk46 gene was dispensable for B. burgdorferi infection in mice. Our findings highlight the power of the IVET-based approach for identification of B. burgdorferi in vivo-expressed genes, which might not be discovered using other genome-wide gene expression methods. Further investigation of the novel in vivo-expressed candidate genes will contribute to advancing the understanding of molecular mechanisms of B.burgdorferi survival and pathogenicity in the mammalian host.
Asunto(s)
Borrelia burgdorferi/inmunología , Borrelia burgdorferi/patogenicidad , Enfermedad de Lyme/inmunología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Virulencia/genética , Secuencia de Aminoácidos , Animales , Borrelia burgdorferi/genética , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano/genética , Genotipo , Enfermedad de Lyme/genética , Enfermedad de Lyme/patología , Ratones , Ratones Endogámicos C3H , Ratones SCID , Sistemas de Lectura Abierta/genética , Fenotipo , Alineación de Secuencia , Transcriptoma/genética , Factores de Virulencia/biosíntesis , Factores de Virulencia/inmunologíaRESUMEN
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidad , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Enfermedad de Lyme/metabolismo , Factores de Virulencia/biosíntesis , Animales , Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Modelos Animales de Enfermedad , Femenino , Enfermedad de Lyme/genética , Enfermedad de Lyme/patología , Ratones , Factores de Virulencia/genéticaRESUMEN
Therapeutic vaccines for the treatment of cancer are an attractive alternative to some of the conventional therapies that are currently used. More importantly, vaccines could be very useful to prevent recurrences when applied after primary therapy. Unfortunately, most therapeutic vaccines for cancer have performed poorly due to the low level of immune responses that they induce. Previous work done in our laboratory in cancer mouse models demonstrated that vaccines consisting of synthetic peptides representing minimal CD8 T-cell epitopes administered i.v. mixed with poly-IC and anti-CD40 antibodies (TriVax) were capable of inducing massive T cell responses similar to those found during acute infections. We now report that some peptides are capable of inducing similarly large T cell responses after vaccination with poly-IC alone (BiVax). The results show that amphiphilic peptides are more likely to function as strong immunogens in BiVax and that systemic immunizations (i.v. or i.m.) were more effective than local (s.c.) vaccine administration. The immune responses induced by BiVax were found to be effective against established tumors in two mouse cancer models. The roles of various immune-related pathways such as type-I IFN, CD40 costimulation, CD4 T cells, TLRs and the MDA5 RNA helicase were examined. The present findings could facilitate the development of simple and effective subunit vaccines for diseases where CD8 T cells provide a therapeutic benefit.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Péptidos/inmunología , Poli I-C/inmunología , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito T/inmunología , Femenino , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas E7 de Papillomavirus/inmunología , Fragmentos de Péptidos/inmunología , Tripsina/inmunologíaRESUMEN
The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD(+) homeostasis and contributes to numerous important biological processes in diverse organisms. In Borrelia burgdorferi, the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the in vivo importance of PncA, the current annotation for the pncA ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of B. burgdorferi PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that pncA translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this in vivo-essential protein.