Date Published: April 10, 2019
Publisher: Public Library of Science
Author(s): Nishant Sharma, Suruchi Aggarwal, Saravanan Kumar, Rahul Sharma, Konika Choudhury, Niti Singh, Praapti Jayaswal, Renu Goel, Saima Wajid, Amit Kumar Yadav, Krishnamohan Atmakuri, Yung-Fu Chang.
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 PepNMtb 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 PepNMtb is required for modulating virulence in vivo, PepNMsm is essential for Msm growth in vitro. Despite such essentiality difference, both PepNMtb and PepNMsm 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 pepNMsm reveals PepNMsm also retaining the ability to reach host cytosol. Lastly, but notably, we determined the PepNMtb and PepNMsm interactomes and found them to barely coincide. While PepNMtb chiefly interacts with Mtb’s secreted proteins, PepNMsm 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.
Annually, worldwide, atleast a million people die of Tuberculosis (TB) . To establish infection and hijack its host, Mycobacterium tuberculosis (Mtb) injects a battery of arsenal [2–5]. Mtb’s stockpile is predicted to include lipids, proteins, sugars and small molecules. Over the years, though several aspects of the Mtb’s biology have been discovered, to this day, only few of Mtb’s effectors that manipulate host cellular processes have been identified and their roles determined [2–6]. For example, SapM is a secreted lipid phosphatase that prevents phagosome-lysosome fusion . ESAT-6 is an early secretory antigenic target protein that induces apoptosis , inhibits generation of reactive oxygen species  and suppresses antigen presentation by MHC1 . ManLAM is a mannose-capped Lipoarabinomannan that also inhibits phagosome-lysosome fusion and T-cell receptor-mediated signaling .
In this study, we selected PepNMtb and PepNMsm as representative PepNs of pathogenic and non-pathogenic mycobacteria respectively. We independently BLASTed (BLAST P)  them as query sequences and found PepN widely conserved across all mycobacterial species. We then aligned (CLUSTALW)  PepNMtb and PepNMsm protein sequences for comparative analysis. Though, they align substantially well (~78% identity at amino acids level; 10.6084/m9.figshare.7873274), their N- and C-terminal end sequences show some divergence (10.6084/m9.figshare.7873274). To probe this divergence further, because of unavailability of their quaternary structures, employing Expresso , we aligned their secondary structures. Expresso aligns multiple protein sequences using structural information. Interestingly, while the N-terminal halves (Peptidase M1 N-terminal domain + Peptidase family M1 domain; https://pfam.xfam.org/protein/L7N655) of both pathogenic and non-pathogenic PepNs structurally aligned ‘good’ (S1 Fig), their C-terminal ERAP1_C-like domains aligned “average to bad” (S1 Fig; Good’, ‘average’ and ‘bad’ are algorithm outputs displayed by Expresso to indicate high, medium and poor levels of structure-based sequence homology). In contrast, both M1 peptidase and ERAP1_C-like domains of pathogenic PepNs (Mtb complex) structurally aligned ‘good’ throughout their lengths (10.6084/m9.figshare.7873442).
Our search for potential Mtb effectors among its aminopeptidases pool led us to PepN. Since it is (i) reported to be secreted into SM ; (ii) not essential for Mtb’s in vitro growth , and (iii) essential for Mtb’s growth in vivo , we speculated that this might be a potential effector in hosts. Our in vitro macrophage infection studies with Mtb showed that PepN not only secretes into SM  but also reaches macrophages (Fig 1). Presence of (i) a C-terminal ERAP1_C-like domain  that resembles ERAP1, an essential host aminopeptidase of ER  and (ii) identification of ER-homing like signals along its length (S2 Fig) furthered us to hypothesize that PepN not only enters macrophages but also reaches ER. However, surprisingly, our THP-1 infection experiments showed that PepN barely co-localizes with GRP94, an ER-specific marker. (Fig 1). Further evaluation also indicated that PepN fails to localize with THP-1’s nuclei (Fig 1) and lysosomes (S3 Fig). Thus, our in vitro infection experiments with THP-1 indicate that PepN is largely localized to host cell cytosol. Himar 1 insertion into pepNMtb showed that mutated PepN led to attenuation of Mtb-mediated virulence in mice . In contrast, a Tn5370 insertion into pepN facilitated Mtb to become hypervirulent in BALB-C mice . It is unclear at this point as to what probably is the function of PepNMtb post its delivery into host macrophages.