Dataset Information

Expression of a Gene Encoding 34.9?kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli.

ABSTRACT: Mycobacterium avium subsp. paratuberculosis (Map) contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9?kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into E. coli M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9?kDa PPE and 2.2?kDa His-tag), which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1?kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.

SUBMITTER: Deb R

PROVIDER: S-EPMC3218304 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli.

Deb Rajib R Goswami P P PP

Molecular biology international 20100518

Mycobacterium avium subsp. paratuberculosis (Map) contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9 kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into Escherichia coli vector pQE30 UA. The recombinant plasmid desig ...[more]

PMID: 22110958

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Project description:Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.

Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.

Project description:BackgroundMycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study.ResultsTo characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical.ConclusionsWe present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Dataset Information

Expression of a Gene Encoding 34.9?kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli.

Publications

Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli.

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