Non-tuberculous Mycobacteria (NTM) are an important but often overlooked group of pathogens, especially important in the immunocompromised and patients with pre-existing pulmonary disease. Their condition of environmental bacteria enables them to persist in a wide range of habitats. Although multiple virulence factors of M. avium have been proposed, the virulence strategies of M. avium are still not fully clear including the mechanisms allowing this environmental bacterium to cause chronic infections in humans. Lysyl-phosphatidylglycerol, a component of the mycobacterial membrane, contributes to the resistance towards cationic antimicrobial peptides. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. The main objective of the doctoral project was to characterize the role of the lysX gene for growth and host cell interaction of M. avium subsp. hominissuis (MAH). A considerable impact of the gene lysX on the different functional pathways of M. avium in particular the central carbon metabolism was demonstrated. Proteomics studies revealed that the lysX mutation led to a metabolic shift which enhanced the suitability of the bacteria to be adaptive towards the living conditions inside host cells. In addition, the mutation also caused an upregulation of lipid synthesis genes which resulted in an intracellular lipid accumulation. The measure of mycobacterial virulence has been stated to depend on the ability of the bacteria to invade, persist and replicate within the hostile macrophage environment. In accordance the lysX mutant already displayed a hypervirulent phenotype, exhibiting an excessive intracellular growth in in-vitro (human blood monocytes) and invivo (Galleria mellonella). Additionally, the lysX mutation also resulted in an hyperinflammatory behaviour (increased secretion of cytokines and increased MGC formation), which also indicates a novel functional role of lysX in regards to virulence in M. avium species. Interestingly, the results with respect to the host-pathogen interaction of an MAH with a deficient lysX gene obtained in this study contrasted with the results obtained by other authors with a lysX mutant from MTB. This makes it more interesting to further explore on the differential survival strategies of mycobacterial species. The lysX gene may also be instrumental in identifying factors involved in molecular pathogenesis of different mycobacterial diseases, thus benefitting the health systems for developing strategies to combat these hardy pathogens.