Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

Abstract

Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis.

PPE37 Is Essential for Mycobacterium tuberculosis Heme-Iron Acquisition (HIA), and a Defective PPE37 in Mycobacterium bovis BCG Prevents HIA

“…It was recently reported that PPE37 is exported, with the protein found primarily in the cell membrane fraction, and that it possesses a eukaryotic-type signal sequence from residues 1 to 40, although the details for how the signal sequence was determined were not reported (31). We analyzed the M. tuberculosis PPE37 sequence using SignalP, TatP, LipoP, and TargetP to predict potential secretion signals.…”

“…Thus, PPE37 does not have a conventional signal peptide that can be detected by the SignalP, TatP, or LipoP algorithm, but the higher score than nonsecretory proteins and the cleavage site predictions suggest that it may possess a nonclassical signal peptide. As PPE37 has two transmembrane domains and as the protein was detected in the cell membrane fraction (31), it is likely to be an integral membrane protein. Interestingly, the cleavage site predictions occur either in the amino acids deleted from the BCG PPE37 protein or immediately adjacent to the deletion, suggesting that the BCG PPE37 protein might be processed differently.…”

“…Genetic variation of ppe37 in M. tuberculosis. Recently, Ahmad et al reported significant variability in PPE37 from an analysis of 28 M. tuberculosis strains with completed genomes and presented a protein alignment of PPE37 proteins from three representative strains (H37Rv, F11, and 49-02) that demonstrated that the F11 sequence is identical to that of H37Rv PPE37 on the N terminus but has an altered C terminus and that the 49-02 sequence is identical to that of H37Rv PPE37 on the C terminus but has an altered N terminus (31). However, further explanation of the variability was not discussed.…”

“…Thus, M. tuberculosis clearly has additional means of releasing iron from heme that do not require MhuD. However, the localization of PPE37 in the cell membrane fraction (31) and the likely presence of a signal peptide in PPE37 argue against a role for PPE37 in cytosolic degradation of heme although this possibility cannot be ruled out at present. Finally, deletion of ppe37 (or expression of a nonfunctional PPE37 by BCG) might have a general effect on cell wall permeability which impacts HIA in a nonspecific manner; i.e., it might make the cell wall less permeable to diffusion of heme.…”

“…In one study, expression of M. tuberculosis PPE37 by M. smegmatis resulted in a lower level of proinflammatory cytokines secreted by infected macrophages, but the mechanism by which expression of recombinant PPE37 interfered with the proinflammatory cytokine response was not determined (44). Recently, a second study examining the function of PPE37 demonstrated that PPE37 appears to be exported as it is predominantly located in the cell membrane fraction of M. tuberculosis grown under low-iron conditions (31). The authors also ascribed different host-modulating functions to the N-terminal and C-terminal segments of PPE37 when the segments are transfected into the human monocytic THP-1 cell line.…”