RESUMEN
Mycobacterium tuberculosis secretes several hundred proteins; many of which elicit immune responses. As a result, many of these proteins have been explored for their potential as diagnostic and vaccine candidates. Of these, the Antigen 85 complex proteins, represented by Antigen85 A, B, and C, are the most studied from the mycobacterial secretome. However, vaccine constructs exploiting Antigen 85 as the sole antigen repertoire have not experienced the pre-clinical and clinical trials success originally anticipated. Anecdotal and biochemical evidence suggests that differences in protein abundance may explain this phenomenon. Here, biochemical, molecular, and mass spectrometry approaches were used to quantify Antigen 85 among six M. tuberculosis strains from four phylogenetically distinct clades. Our data demonstrates that the greatest variation in Antigen 85 is ascribed to protein quantities, whereas few transcriptional differences were found. In addition, the ratio of Antigen 85 A, to B, to C is conserved within clades and phylogenetic neighbors. In contrast, no such relationship between individual protein quantities was observed, and in the case of Antigen85 B, this variation even extends within biological replicates of individual isolates. The relevance of Antigen 85 protein quantities and vaccine efficacy remains to be defined. BIOLOGICAL SIGNIFICANCE: Absolute quantitation via multiple reaction monitoring mass spectrometry was used to determine the exact molar concentrations of Antigen 85A, B, and C; three key immunodominant proteins present in M. tuberculosis. Further, the concentration of these three proteins was compared among various clades of M. tuberculosis, and demonstrated differences in abundance of two of the three proteins. These proteins have been identified as key antigens in multiple vaccine and diagnostic platforms, thus the potential relevance of their abundance in various M. tuberculosis clades to the successful outcome of these interventions is discussed. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
Asunto(s)
Antígenos Bacterianos/genética , Variación Genética , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis/genética , Antígenos Bacterianos/inmunología , Biomarcadores , Humanos , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
BACKGROUND: The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (ori(R6Kγ)) is dependent on the pir gene-encoded π protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support replication of plasmids harboring ori(R6Kγ). In Escherichia coli this is commonly achieved by chromosomal integration of pir either via lysogenization with a λpir phage or homologous recombination at a pre-determined locus. FINDINGS: Current methods for construction of host strains for ori(R6Kγ)-containing plasmids involve procedures that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition, we isolated pir+ and pir-116 expressing derivatives of a clinical isolate of Salmonella enterica serovar Typhimurium. In both E. coli and S. enterica serovar Typhimurium, the presence of the pir+ wild-type or pir-116 alleles allowed the replication of ori(R6Kγ)-containing plasmids. CONCLUSIONS: A mini-Tn7 system was employed for rapid and reliable engineering of E. coli and S. enterica serovar Typhimurium host strains for plasmids containing ori(R6Kγ). Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with ori(R6Kγ).