Comparative genomic and proteomic analyses of PE/PPE multigene family of Mycobacterium tuberculosis H₃₇Rv and H₃₇Ra reveal novel and interesting differences with implications in virulence

Abstract

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease taking one human life every 15 s globally. The two well-characterized strains H(37)Rv and H(37)Ra, derived from the same parental strain M. tuberculosis H(37), show dramatically different pathogenic phenotypes. PE/PPE gene family, comprising of 176 open reading frames and present exclusively in genus Mycobacterium, accounts for ∼10% of the M. tuberculosis genome. Our comprehensive in silico analyses of PE/PPE family of H(37)Ra and virulent H(37)Rv strains revealed genetic differences between these strains in terms of several single nucleotide variations and InDels and these manifested in changes in physico-chemical properties, phosphorylation sites, and protein: protein interacting domains of the corresponding proteomes. Similar comparisons using the 13 sigma factor genes, 36 members of the mammalian cell entry family, 13 mycobacterial membrane protein large family members and 11 two-component signal transduction systems along with 5 orphaned response regulators and 2 orphaned sensor kinases failed to reveal very significant difference between H(37)Rv and H(37)Ra, reinforcing the importance of PE/PPE genes. Many of these changes between H(37)Rv and H(37)Ra can be correlated to differences in pathogenesis and virulence of the two strains.

Figure 1.

Nucleotide variations between H₃₇Rv and H₃₇Ra manifest in changes in GRAVY values. GRAVY values of PE/PPE proteins in H₃₇Rv and their homologs in H₃₇Ra were plotted where ΔGRAVY was >50%. (A) A major transition from hydrophobic to hydrophilic protein is observed in Rv2408/MRA_2433 and Rv0878c/MRA_0885, whereas Rv2371/MRA_2394 only showed a significant difference in GRAVY value. A hydrophilic to hydrophobic transition was observed in Rv0388c/MRA_0395. (B) Non-PE/PPE proteins. H₃₇Rv proteins MMPL13b (Rv1146), SigM (Rv3911), PhoP (Rv0757) and their homologs in H₃₇Ra (MRA_1156, MRA_3950, MRA_0767), respectively. ΔGRAVY value >50% was observed only in case of Rv3911/MRA_3950.

Figure 2.

Variations in nucleotide sequences between H₃₇Rv and H₃₇Ra affect the stability of PE/PPE proteins but not the non-PE/PPE proteins. Instability index value >40 is indicative of unstable protein and value <40 means the protein is stable. (A) PE/PPE proteins in H₃₇Rv and their homologs in H₃₇Ra show a change from stable to unstable (Rv1091/MRA_1102 and Rv2408/MRA_2433) or the reverse (Rv1195/MRA_1205 and Rv1807/MRA_1819). (B) Non-PE/PPE proteins MMPL13b (Rv1146), SigM (Rv3911), PhoP (Rv0757), and their homologs in H₃₇Ra (MRA_1156, MRA_3950, MRA_0767), respectively, did not show any transition from stable to unstable or reverse.

Figure 3.

Gain or loss of globular domain is a function of variation between H₃₇Rv and H₃₇Ra. Disorder propensity of the protein stretch was calculated by GlobPlot analyses to identify the globular domain (shown in pink). Amino acid sequence alignment above the figure illustrates the differences between H₃₇Rv and H₃₇Ra. (A) N-terminal extension in MRA_2394 (shown in right panel) renders the protein globular. (B) Deletion in MRA_3384 leads to a loss of globular domain.

Figure 4.

Nucleotide variations between H₃₇Rv and H₃₇Ra result in gain or loss of serine/threonine phosphorylation sites. (A) The relative percentage of members of PE/PPE, mce, MmpL, Sigma, TCSS families exhibiting difference in phosphorylation sites between H₃₇Rv and H₃₇Ra is shown. (B, C) Gain of serine/threonine phosphorylation sites. The predictive phosphorylation site(s) in proteins is represented by blue circle (in the case of H₃₇Rv) or red dots (in case of H₃₇Ra). Overlap of blue and red circle indicates no gain or loss of phosphorylation sites in H₃₇Rv and H₃₇Ra proteins. Horizontal line represents the cutoff (threshold) above which it is considered as a putative phosphorylation site. The actual sequence of amino acids and the potential phosphorylation site (highlighted in red) is shown above the respective figures. N-terminal extension in MRA_1801 (B) or MRA_2433 (C) leads to gain of additional serine/threonine phosphorylation sites.