subspecies (MAH) is an opportunistic pathogen that is ubiquitous in the environment and often isolated from faucets and showerheads. MAH mostly infects humans with an underlying disease, such as chronic pulmonary disorder, cystic fibrosis, or individuals that are immunocompromised. In recent years, MAH infections in patients without concurrent disease are increasing in prevalence as well. This pathogen is resistant to many antibiotics due to the impermeability of its envelope and due to the phenotypic resistance established within the host macrophages, making difficult to treat MAH infections. By screening a MAH transposon library for mutants that are susceptible to killing by reactive nitrogen intermediaries, we identified the _4644 (_4644:Tn) gene knockout clone that was also significantly attenuated in growth within the host macrophages. Complementation of the mutant restored the wild-type phenotype. The _4644 gene encodes a dual-function protein with a putative pore-forming function and ADP-ribosyltransferase activity. Protein binding assay suggests that MAV_4644 interacts with the host lysosomal peptidase cathepsin Z (CTSZ), a key regulator of the cell signaling and inflammation. Pathogenic mycobacteria have been shown to suppress the action of many cathepsins to establish their intracellular niche. Our results demonstrate that knocking-down the cathepsin Z in human macrophages rescues the attenuated phenotype of _4644:Tn clone. Although, the purified cathepsin Z by itself does not have any killing effect on MAH, it contributes to bacterial killing in the presence of the nitric oxide (NO). Our data suggest that the cathepsin Z is involved in early macrophage killing of MAH, and the virulence factor MAV_4644 protects the pathogen from this process.