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  • Next we elucidated that the Mtb enolase binds to host


    Next, we elucidated that the Mtb enolase binds to host plasminogen with a high affinity. The KD value of 360nM for this interaction suggested an avid binding between the two proteins in vivo. This observation is also in conciliation with the observed KD values for enolase-plasminogen interactions in streptococcal species, however, the affinity was slightly lower for Mtb enolase [38], [39], [49], [50]. It is well established that plasminogen LY 255283 in pathogenic plasminogen receptors are lysine-rich motifs. Consequently, the affinity of interaction between rEno and human plasminogen was abrogated in the presence of 0.1M lysine and reduced to >2-folds upon point mutating the C-terminal lysine to alanine. Lysine mediated binding of plasminogen to its receptors makes it susceptible to conversion into fibrinolytic plasmin [9]. In streptococcal surface enolase, which is one of the most extensively studied proteins in this class, an internal plasminogen binding site comprising of nine residues mediates binding to plasminogen, in synergy with the terminal lysine residues [38], [51]. Similarly, another study showed that the binding of Group A Streptococcus surface enolase to human plasminogen is mediated by two internal lysines (252 and 255) in addition to their C-terminal lysines [52]. Interestingly, Mtb enolase lacks these homologous residues important for streptococcal enolases (Supplementary Fig. S7). Further, the C-terminus of Mtb harbors a single lysine residue, as opposed to more than one in streptococcus. As a result, mutation of this C-terminal lysine severely reduces plasminogen binding in Mtb enolase. Also, absence of the internal plasminogen binding site in Mtb enolase might explain its lower affinity towards plasminogen, in comparison to streptococcal enolase. In the context of TB, plasmin mediated degradation of ECM components could have major consequences for granuloma stability. ECM is a major determinant in the genesis of granuloma formation in terms of migration of immune cells to and from the site of infection and formation of a stable granuloma [3]. MMPs are speculated to play a vital role in the pathology of TB by mediating degradation of collagen in a necrotic granuloma resulting in the spread of Mtb to distant sites [2]. Hence, surface recruited plasmin activity in Mtb could be instrumental in mediating bacterial escape from a caseating granuloma. This observation also suggested that enolase can be a potential immunomodulatory agent, as effective antibody mediated recognition of this moonlighting protein can decrease the infectious potential of Mtb. As expected, mice immunized with rEno compared to PBS group showed decreased Mtb load in lungs. The Mtb load post rEno immunization was similar to that of BCG. This result makes enolase a promising candidate to be further tested as a component of vaccine formulations against TB. Major extracellular proteins of Mtb like Ag85B, when emulsified with an adjuvant, have been shown before to induce a protective effect against Mtb challenge in mice [29], [30]. The response of rEno immunization was comparable to Ag85B, however, BCG imparted the best protection, as has been seen in a number of previous studies also [29], [30]. A successful subunit vaccine against TB must induce immune recognition of a variety of Mtb antigens, which are expressed during different stages of infection. This may be achieved by formulation of a cocktail of diverse mycobacterial immunodominant antigens, and enolase could be one of them. However, persistence of protection conferred by enolase immunization needs to be studied in detail in order to investigate the vaccine potential of rEno. Moreover, the protection conferred in an aerosol mode of infection remains to be explored. Further characterization of other plasmin/plasminogen binding proteins of Mtb can delineate the significance of pathophysiology of TB in relation with its tissue invasive potential. Chemical therapeutics against the lysine dependent binding of plasminogen to these surface localized moonlighting proteins can also be exploited. TB is more understood as an intracellular infection of the macrophages, which forms the first line of host innate defense mechanism. However, evasion from antibody mediated recognition and immunity is an important anti-host characteristic for both extracellular and intracellular pathogens. Moreover, anti-mycobacterial antibodies mediated opsonization of Mtb enhances the innate and cell-mediated immunity against it [53]. Studies on the effect of plasmin bound to Mtb in modulation of uptake by macrophages and phagocytosis can be done on the basis of findings of this report. In summary, this work now establishes the implication of tissue invasive potential of Mtb as one of the major regulators of TB and the crucial role of moonlighting glycolytic enzymes like enolase in it.