Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

Abstract

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

“…In addition to esterases, we detected six proteases that included the two Clp protease subunits, ClpP1 and ClpP2, the immune modulating protease Hip1 (Naffin-Olivos et al, 2014), and the acid resistance protease Rv3671c (Vandal et al, 2008). In addition, we detected the probable peptidase Rv0457, providing experimental support for its tentative annotation.…”

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

“…Chimeras with mycobacterial equatorial domain EAI2 and EA2 displayed distinct tetradecameric form. The respective hinge variant versions EAI1 and EA1 existed in equilibrium between a tetradecamer and a dimer, a feature reminiscent of the parent M. tuberculosis GroEL2 (29,41). Interestingly, EAI1and EA2 were able to effectively rescue E. coli LG6, but EAI2 and EA1 failed to complement, suggesting that these chaperonin variants might be similar to M. tuberculosis GroEL2, wherein oligomeric assembly upon their abundance was required to rescue E. coli LG6 (Fig.…”

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…Moreover, GroEL2 has been shown to localize at the cell wall (Hickey et al 2009) as a multimer and upon cleavage by a serine protease, Hip1, separates into the secretion-competent monomeric form. This GroEL2-Hip1 interaction and further cleavage have been demonstrated to pacify macrophage response (Naffin-Olivos et al 2014). The unusual nature of mycobacterial GroELs has triggered curiosity to identify any moonlighting function (Mande et al 2013) to these chaperones and led to the discovery that dimeric GroEL1 interacts with DNA in a sequence-independent manner (Basu et al 2009).…”