Comparative analysis of homologous aminopeptidase PepN from pathogenic and non-pathogenic mycobacteria reveals divergent traits

by Nishant Sharma, Suruchi Aggarwal, Saravanan Kumar, Rahul Sharma, Konika Choudhury, Niti Singh, Praapti Jayaswal, Renu Goel, Saima Wajid, Amit Kumar Yadav, Krishnamohan Atmakuri

Mycobacterium tuberculosis (Mtb) secretes proteases and peptidases to subjugate its host. Out of its sixty plus proteases, atleast three are reported to reach host macrophages. In this study, we show that Mtb also delivers a lysyl alanine aminopeptidase, PepN (Rv2467) into host macrophage cytosol. Our comparative in silico analysis shows PepN_Mtb highly conserved across all pathogenic mycobacteria. Non-pathogenic mycobacteria including M. smegmatis (Msm) also encode pepN. PepN protein levels in both Mtb (pathogenic) and Msm (non-pathogenic) remain uniform across all in vitro growth phases. Despite such tight maintenance of PepNs’ steady state levels, upon supplementation, Mtb alone allows accumulation of any excessive PepN. In contrast, Msm does not. It not only proteolyzes, but also secretes out the excessive PepN, be it native or foreign. Interestingly, while PepN_Mtb is required for modulating virulence in vivo, PepN_Msm is essential for Msm growth in vitro. Despite such essentiality difference, both PepN_Mtb and PepN_Msm harbor almost identical N-terminal M1-type peptidase domains that significantly align in their amino acid sequences and overlap in their secondary structures. Their C-terminal ERAP1_C-like domains however align much more moderately. Our in vitro macrophage-based infection experiments with MtbΔpepN-expressing pepN_Msm reveals PepN_Msm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepN_Mtb and PepN_Msm interactomes and found them to barely coincide. While PepN_Mtb chiefly interacts with Mtb’s secreted proteins, PepN_Msm primarily coimmunoprecipitates with Msm’s housekeeping proteins. Thus, despite high sequence homology and several common properties, our comparative analytical study reveals host-centric traits of pathogenic and bacterial-centric traits of non-pathogenic PepNs.