Interaction mechanism of Mycobacterium tuberculosis GroEL2 protein with macrophage Lectin-like, oxidized low-density lipoprotein receptor-1: An integrated computational and experimental study
Introduction
The cell wall of Gram positive bacteria have a large number of covalently and non-covalently attached proteins. Proteins with N- terminal signal (leader) peptide get translocated across the cytoplasmic membrane, then secreted outside or get anchored to the cell wall [1]. In recent years, evidence for extracellular localization of few cytoplasmic proteins lacking N-terminal secretion signals has been also reported from several bacteria. Cytoplasmic proteins devoid of N-terminal signal (leader) peptide are reported to exist on the surface of Staphylococcus aureus, Streptococcus pneumoniae, Lactobacillus plantarum, Salmonella enterica, Helicobacter pylori, Clostridium difficile and Mycobacterium tuberculosis (Mtb) [[2], [3], [4], [5], [6], [7], [8]]. In case of Mtb the known anchorless cell surface associated proteins include malate synthase (MS), which functions as an adhesin binding with the laminin of host tissue, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), a receptor for human lactoferrin and the chaperone heat shock protein GroEL2 [7,[9], [10], [11]]. GroEL protein was also found on the surface of Chlamydia pneumoniae, Clostridium difficile and, Escherichia coli where it aids in adhesion and uptake of these bacteria into host cells [5,12,13]. The mycobacteria are unusual in encoding two forms of the GroEL (Hsp60) chaperones, GroEL1 (cpn60.1) and GroEL2 (cpn60.2) [14,15]. Presence of GroEL2 on the surface of Mtb has been shown by Hickey et al [10]. Mtb GroEL2 shares high level sequence similarity with GroEL proteins expressed by other pathogens (Supplementary Fig. 1A). Structurally, Mtb GroEL2 protein consists of apical, intermediate and equatorial domains (Supplementary Fig. 1B) [16,17]. Apart from the structural similarity, 62% sequence identity was observed between the GroEL of E. coli and GroEL2 of Mtb. It has been previously shown that surface associated GroEL protein of E. coli interacts with host scavenger receptor Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1) expressed on macrophages and thereby increases uptake of the bacteria by these cells [13]. Similarly, Lin et al. reported that, purified GroEL1 of Chlamydia pneumoniae enhances the uptake of Oxidized Low-Density Lipoprotein (Ox-LDL) through LOX-1 receptor in human coronary endothelial cells (HCAECs) [18]. The main objective of the present study was to understand the biological significance of the direct interaction of Mtb GroEL2 with host macrophage LOX-1 receptor protein.
The crystal structure of the human LOX-1 receptor was solved using X-ray crystallographic technique. Human LOX-1 is a 50 kDa disulfide-linked homo dimeric type II transmembrane protein consisting of a short cytoplasmic region, a transmembrane region and an extracellular region. The extracellular portion of the receptor protein consists of a stalk or neck domain, followed by a C-type lectin-like domain (CTLD) at the C-terminal (Supplementary Fig. 2A). The CTLD is highly conserved among mammals and bears the key ligand binding regions of LOX-1 consisting of hydrophobic tunnel, basic spine and the saddle hydrophobic patch (Supplementary Fig. 2B-E, 3A) [19,20]. LOX-1 is expressed on the surface of fibroblasts, platelets, smooth muscle cells, endothelial cells, dendritic cells and macrophages [21,22]. Though LOX-1 is the receptor for Ox-LDL, it can bind with other ligands such as platelets, apoptotic cells, aged cells and some bacterial surface molecules [[23], [24], [25]].
In order to study the interaction between GroEL2 of Mtb and host macrophage receptor LOX-1 we have adopted a combinatorial strategy involving computational analyses and biological assays.
Section snippets
In vitro Experimental Methods:
Bacterial strains, cell lines and chemicals.
E. coli BL21 was cultured in Luria Bertani broth (HiMedia) in a shaking incubator (160 rpm) at 37 °C. RAW 264.7 (murine macrophage cell line) was obtained from National Centre for Cell Sciences, India and was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 4.5 g/l glucose, 2–4 mM l-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin at 37 °C in a 5% CO2 atmosphere. The plasmid pMRLB1
Results
In vitro Experimental Studies:
GroEL2 promotes uptake by macrophages via LOX-1 receptor.
GroEL2 protein of Mtb (60 kDa) was isolated and purified as recombinant 6×-His-GroEL2 from E. coli carrying plasmid pMRLB1 (5225 bp; Fig. 1A). Fluorescent latex beads were coated using purified GroEL2 protein. Coating of GroEL2 over the latex beads was confirmed using SDS-PAGE analysis and immunofluorescence assays. Following protein extraction from the suspension of beads by boiling, SDS PAGE analysis showed
Discussion
Present study shows the significance of interaction between GroEL2 protein of Mtb and LOX-1 receptor using a combination of in vitro and computational techniques. We show that macrophage uptake of GroEL2 coated latex beads (84.5%) was higher than that of the uncoated beads (54.0%) by 30.5%. The uptake of latex beads was also analyzed after blocking the receptor LOX-1 using a known inhibitor - Polyinosinic acid (PIA), which was shown to competitively inhibit LOX-1 receptor and decrease the
Author contribution
VV performed all in vitro experiments; VV and ACP performed in silico studies; AK, CGM and RB wrote the manuscript.
Declaration of Competing Interest
None declared.
Acknowledgement
This work is supported by an extramural grant from Department of Biotechnology (DBT), Government of India (102/IFD/SAN/2644/2012-2013) to RB as Principal Investigator. Authors acknowledge gratefully Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi for the infrastructure support. We thank Dr. Ullas Mony for his valuable inputs and help with the flow cytometry analysis.
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