Optimization of molecular techniques to obtain mutants in Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis

ALONSO MARIA NATALIA; MARIANA VIALE; ROBERTO DAMIÁN MOYANO; MARIA ALEJANDRA COLOMBATTI OLIVIERI; MARIA LAURA MON; MARIANA PIURI; MARY JACKSON; ROMANO MARIA ISABEL; MARIA DE LA PAZ SANTANGELO

Nantes

Congreso; 13th International Colloquium on Paratuberculosis; 2016

International association for paratuberculosis

Mycobacterial infections are major causes of morbidity and mortality in cattle and are also potential zoonotic agents withimplications for human health. Mycobacterium avium subsp.paratuberculosis(MAP) is the causative agent of Paratuberculosis(PTB) in ruminants, a chronic granulomatous disease of the intestines that is transmitted via the fecal-oral route,and causes important economic losses in Argentina and worldwide. Despite the implementation of comprehensive animalsurveillance programs, it is necessary to emphasize the PTB controls on health systems and also to develop new solutionsto control, prevent and eradicate the disease. Therefore, it is crucial to deepen the study of virulence genes involved in thepathogenesis of MAP. It is noteworthy, that obtaining mutants in slow growing mycobacteria is a very difficult process,due to the extremely low rate of recombination and efficiency of transformation. The aim of this study is to optimize themolecular strategies for obtaining mutants in virulence-related genes of M. avium complex (MAC) species. For this purpose,we compared three different strategies for the deletion of the mce4 operon: 1. SacB negative selection technique describedby Pelicic and collaborators in 1997 using the pPR27 vector; 2. Specialized transduction using the recombinant phasmidphAE87, described in Bardarov et al., 2002 and Park et al., 2008; 3. Recombineering technique described by van Kessel etal in 2007 that optimizes the efficiency of homologous recombination.Results: in this study we compared three differentmethods, successfully used in Mycobacteriumtuberculosis complex, for their use in MAC species. So far we have obtainedMAA transformed with the plasmid pPR27. However, transformation with the pPR27 plasmid could not be achieved in MAP,probably because this bacterium is difficult to transform. We obtained the recombinant phasmid expressing the upstreamand downstream regions of the operon mce4 and the obtention of the mutants in MAA and MAP is in progress. Finally, weare optimizing the conditions for the efficient usage of the recombineering strategy in Map. The pros and cons of the threemethods are further discussed