Mycobacterium tuberculosis protein PPE2 binds to DNA region containing promoter activity

Highlights

• A promoter is present upstream to ppe2 gene from putative ppe2-cobQ1-cobU operon.

• PPE2 protein of M.tb has DNA binding property.

• MST analysis revealed that PPE2 protein binds to promoter-bearing region.

• The study is important for studying operon organization of pe/ppe family in M.tb.

Abstract

ppe2 gene is predicted to be present in operon with non pe/ppe genes, cobq1 and cobu as ppe2-cobq1-cobu. Thus, we were interested to investigate the role of ppe2 in operonic organization. We performed microscale thermophoresis (MST) experiment which revealed that PPE2 protein could bind to upstream DNA segments of ppe2-cobq1-cobu operon. Upstream region of ppe2 had shown promoter activity in β-gal assay. In this study, for the first time, a physical interaction between PPE2 protein and DNA fragment was reported, suggesting that PPE2 protein plays a role in the regulation of the putative ppe2-cobq1-cobu operon, via unknown mechanism.

Introduction

Genomic studies on Mycobacteirum tuberculosis (M.tb) have discovered a new gene family known as PE/PPE family. There are approximately 100 pe genes and 69 ppe genes which have been identified in pathogenic strains of M.tb H₃₇Rv, in some saprophytic mycobacteria than non-pathogenic strains like M. smegmatis [1]. PE/PPE protein family is characterized by the occurrence of proline (P)-glutamate (E) (at the positions 8 and 9) or proline-proline-glutamate amino acids (at the 7–9 positions) at the conserved N-terminal region. The variable C-terminal of PE/PPE family member proteins gives rise to multiple subfamily lineages such as PE-PGRS, PPE-MPTR PPE-SVP, PPE-PPW [1]. PE/PPE family proteins are instrumental in imparting antigenic variability, confer virulence and contribute in the pathogenicity of M.tb [2,3]. The functions of PE/PPE protein family members functions are redundant and linked. Within PE/PPE protein family, there are some uncharacterized pe/ppe genes which are present in operon as a pe/ppe gene pair [4,5]. In pe/ppe operonic gene pairs in M.tb, pe gene is generally followed by ppe gene. pe/ppe gene products interact and co-regulate each other [4,5]. It has been observed that individual recombinant PE or PPE proteins are not very stable or do not come in soluble fraction, hence, structure-based study for PE/PPE proteins are not feasible. Till date, the only known structure for PE/PPE protein pair is the heterodimer of PE25/PPE41 protein complex [6]. Operonic PE25/PPE41 protein pair is co-transcribed. They form soluble heterodimer whereas overexpression of either protein individually in Escherichia coli form inclusion body [7]. In another protein pair, PE1/PE2, PE1 is cell-surface associated and PE2 is cytosolic protein [8] and both code for esterase enzyme in M.tb but unlike PE25/PPE41, they do not form heterodimer suggesting their independent functions. PPE50/PPE51 are involved in glycerol uptake as their expression is upregulated when cultures in minimal medium supplemented with glycerol [9]. ppe4−pe5 operon is involved in iron uptake [10]. pe32−ppe65 operon present in region of difference 8 (RD) 8 of M.tb [11]. pe8−ppe15 is involved in secretion of virulent proteins in M.tb and M. marinum [12].

Experimental evidences had identified that operons in M.tb tend to possess essential genes along with non-essential genes. But some pe or ppe genes are predicted to be present in an operon with non-pe/ppe family gene such as ppe2−cobq1−cobu operon, Mg²⁺ riboswitch (Mbox) [13]. In ppe2-cobq1-cobu operon, cobq1, cobu genes are member of Vitamin B₁₂ synthesis pathway whereas ppe2 is a member of the pe/ppe family in M.tb. Interestingly, ppe2 is located upstream of the cobq1−cobu [13] and there is no information available about the function of ppe2 within this operon. Therefore, the present study aims to investigate the role of ppe2 in ppe2−cobq1−cobu operon.