Construction of auxotrophic marker in Mycobacteriumbovis BCG, knockout strain for the DPPD and proteomic study oftuberculin
Mycobacterium bovis BCG has the potential to be an effective live vector formultivalent vaccines. However, there are two problems regarding the utilizationof recombinant BCG as vaccine. The first one is that most mycobacterialcloning vectors rely on antibiotic resistance gene as selectable marker, which isused for genetic transformation. The second one is the limited use of BCG inanimals because it interferes in the tuberculosis diagnosis by tuberculin skintest, which elicits delayed type hypersensitivity to the purified protein derivative(PPD). In this work we developed and evaluated the use of auxotrophiccomplementation as a new selectable marker, characterized the proteins thatare present in the bovine and avium PPD and developed a knockout BCG strainby homologous recombination. To test the auxotrophic complementation asselectable marker, an auxotrophic BCG strain for the amino acid leucine wasconstructed by knocking out the leuD gene by homologous recombination.Expression of leuD on a plasmid acted as a selectable marker in theauxotrophic M. bovis BCG amp;#61508;leuD and M. smegmatis mc2144. The auxotrophiccomplementation selection was similar to selection by antibiotic resistance, butwith the advantage of promoting stability of the plasmid. The new system washighly stable even during in vivo BCG growth. The identification of proteins fromPPD was archived by LC-MS/MS (Liquid Chromatography/MassSpectrometry/Mass Spectrometry). A total of 147 proteins among five PPDsamples (2 bovine PPD and 3 avium PPD) were identified. The bovine PPD hada considerable higher number of proteins comparing to the avium PPD. Weidentifying a group of 28 proteins present only in bovine PPD and a group of fiveproteins deleted in M. bovis BCG vaccinal strain. These two groups are ofspecial interest as they can be used in tests with improved specificity, andpotentially able to differentiate vaccinated and infected individuals. A mutantBCG strain with the DPPD antigen deleted was constructed. The Mb0092coding sequence was knocked out by homologous recombination. The11sequences flanking the target gene were cloned into a suicide vector. Doublecrossovers were selected using sacB. The knockout genotype was determinedby PCR and by Southern blot. This mutant BCG strain can be useful in animalvaccination as it will not interfere in the tuberculosis diagnostic test, whenperformed using recombinant DPPD. The results show alternatives for theproblems related to the use of M. bovis BCG as a recombinant vaccine. Theauxotrophic complementation system was highly stable, efficient and it issuitable for expressing heterologous antigens in BCG. The identification ofproteins present in PPD preparations and the mutant BCG obtained provide thepossibility for the development of differential diagnostic test, thus allowing theuse of BCG as vaccine also in animals.
Advisor:Odir Antônio Dellagostin; Fabrício Rochedo Conceição
School:Universidade Federal de Pelotas
Source Type:Master's Thesis
Date of Publication:02/28/2008