Mycobacterium tuberculosis Hip 1 Modulates Macrophage Responses through Proteolysis of GroEL 2

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. Citation: Naffin-Olivos JL, Georgieva M, Goldfarb N, Madan-Lala R, Dong L, et al. (2014) Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2. PLoS Pathog 10(5): e1004132. doi:10.1371/journal.ppat. 1004132 Editor: Helena Ingrid Boshoff, National Institutes of Health, United States of America Received November 27, 2013; Accepted April 3, 2014; Published May 15, 2014 Copyright: 2014 Naffin-Olivos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from National Institutes of Health grants R00TW008043 and 5R01AI083366 (to JR), GM 32415 (to GAP), and R37AI28571 (to BMD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jrengar@emory.edu . These authors contributed equally to this work. Introduction The outcome of infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is determined by a complex interplay between the host immune response and pathogen factors that promote virulence. In response to multiple stresses encountered during infection, Mtb employs a wide array of strategies to evade the first-line defenses mounted by host innate immune responses and successfully replicates intracellularly within host macrophages [1]. Mtb hinders macrophage functions by inhibiting phagosome maturation and acidification, interferes with IFN-cmediated activation, counters toxic reactive oxygen (ROI) and nitrogen intermediates (RNI) and resists antimicrobial agents that damage the mycobacterial cell envelope [2,3]. The lipid-rich Mtb cell envelope effectively protects the pathogen from antimicrobial agents and immune toxins and provides a potent hydrophobic barrier against several antibiotics [4]. Remodeling of its cell envelope in response to the rapidly changing immune milieu allows Mtb to adapt to intracellular macrophage environments and to persist within granulomatous lesions in the lung. Mtb also secretes a number of gene products extracellularly, many of which can serve as effector molecules to modulate host cells and promote disease progression [5]. Delineating the molecular functions of cell envelope-associated and extracellular Mtb factors that are required for evading host immunity is therefore critical for understanding Mtb pathogenesis. We have recently identified the cell envelope-associated serine hydrolase, Hip1 (Hydrolase important for pathogenesis 1; Rv2224c), as a key immunomodulatory protein that prevents robust activation of macrophages following Mtb infection and controls the onset and magnitude of pro-inflammatory responses induced by Mtb [6–8]. This strategy of dampening early proinflammatory responses is likely to be advantageous to the pathogen by allowing it to escape immune detection. In addition, Mtb Hip1 and its Mycobacterium smegmatis (M. smegmatis) ortholog are important for maintaining Mtb cell envelope integrity and confer resistance to cell envelope-directed stresses [7,9,10]. In this study, we provide key insights into the molecular and biochemical mechanisms underlying Hip1 enzymatic activity and its immunomodulatory functions. Hip1 is predicted to encode a serine protease based on its similarity to the tripeptidyl-peptidases TPP B (SlpD) and TPP C (SlpE) from Streptomyces lividans, which are mycelium-associated proteases involved in cell growth [11]. Hip1 PLOS Pathogens | www.plospathogens. org 1 May 2014 | Volume 10 | Issue 5 | e1004132 possesses the catalytic triad S228, D463, H490, present in a/b hydrolase family members, including esterases, lipases and proteases, but the presence of 11 cysteine residues and 5 predicted disulfide bonds within the protein, complicated previous efforts to purify this protein and, thus, the true enzymatic activity of Hip1 has remained unclear [12,13]. We now conclusively demonstrate that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a substrate of Hip1 protease activity. While several proteases have been implicated in promoting Mtb virulence, identification of their physiological substrates has been largely lacking and the interplay between proteases and their substrates during Mtb infection is poorly understood [14–23]. Here, we show that Hip1 proteolytically cleaves GroEL2 in the N-terminus of the protein and we have mapped the cleavage site within GroEL2. Interestingly, cleavage of GroEL2, which encodes a chaperone-like immunomodulatory protein, converts the protein from a multimeric form to a monomeric form. Remarkably, while GroEL2 remains uncleaved in the hip1 mutant, ectopic expression of cleaved GroEL2 monomers within the hip1 mutant strain restores wild type levels of cytokine responses in infected macrophages. Our studies implicate Hip1-dependent proteolysis of its substrate as a novel regulatory mechanism in Mtb that helps the pathogen respond rapidly to changing host immune environments during infection.