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

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

doi:10.1371/journal.pone.0215123

Abstract

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.

Highlights

Worldwide, atleast a million people die of Tuberculosis (TB) [1]
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)
Since it is (i) reported to be secreted into spent media (SM) [4]; (ii) not essential for Mycobacterium tuberculosis (Mtb)’s in vitro growth [22], and (iii) essential for Mtb’s growth in vivo [24], we speculated that this might be a potential effector in hosts

Summary

Introduction

Worldwide, atleast a million people die of Tuberculosis (TB) [1]. To establish infection and hijack its host, Mycobacterium tuberculosis (Mtb) injects a battery of arsenal [2,3,4,5]. Mtb’s stockpile is predicted to include lipids, proteins, sugars and small molecules. 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 [2,3,4,5,6]. SapM is a secreted lipid phosphatase that prevents phagosome-lysosome fusion [6]. ManLAM is a mannose-capped Lipoarabinomannan that inhibits phagosome-lysosome fusion and T-cell receptor-mediated signaling [10]

Methods

Results

Conclusion