Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall

Alessandro Cascioferro,Marcello Ventura,Wilbert Bitter,Riccardo Manganelli,Giovanni Delogu,Maria H Daleke,Giorgio Palù,Valentina Donà

doi:10.1371/journal.pone.0027713

Alessandro Cascioferro, Marcello Ventura + Show 6 more

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https://doi.org/10.1371/journal.pone.0027713

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Abstract

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PERv1818c), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PERv1818c domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.

Highlights

PE, together with PPE, are peculiar mycobacterial proteins over-represented in pathogenic mycobacterial species
Any of the about 100 PE proteins encoded by the Mycobacterium tuberculosis genome have been associated with a physiological function, with the exceptions of LipY (Rv3097c), whose C-terminal domain shows lipase activity [13], PE_PGRS11, which was recently shown to encode a functional phosphoglycerate mutase [14] and PE_PGRS33, which might be involved in induction of macrophage necrosis and apoptosis through interaction with Toll-like receptor 2 [15,16]
We show that PE_PGRS33 secretion in M. marinum is ESX-5 dependent, and by characterizing the cellular localization of several PE_PGRS33 mutants and PE-based chimeric proteins in M. smegmatis, M. marinum, M. bovis BCG and M. tuberculosis we identify portions of the PE domain that are required for protein translocation

Summary

Introduction

PE, together with PPE, are peculiar mycobacterial proteins over-represented in pathogenic mycobacterial species. In PE_PGRS proteins, the PE domain is followed by a C-terminal domain with a highly variable Gly-Ala rich sequence [6,7], which has been suggested to be involved in antigenic variation [8,9]. In the other PE proteins the PE domain can be followed by an unrelated Cterminal domain, or the PE domain represents the entire protein [7] In the latter case the PE-encoding gene is usually in tandem with a PPE-encoding gene, and at least in one case the PE and PPE domains encoded by the coupled genes have been shown to interact [10,11,12]. Any of the about 100 PE proteins encoded by the Mycobacterium tuberculosis genome have been associated with a physiological function, with the exceptions of LipY (Rv3097c), whose C-terminal domain shows lipase activity [13], PE_PGRS11, which was recently shown to encode a functional phosphoglycerate mutase [14] and PE_PGRS33, which might be involved in induction of macrophage necrosis and apoptosis through interaction with Toll-like receptor 2 [15,16]

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