Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection. J Intern Med 2017; https://doi.org/10.1111/joim.12714.
Original Article doi: 10.1111/joim.12714
Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection I. Palucci1,* E. Petruccioli3
, I. Matic2,* , D. Goletti3
, L. Falasca3 , M. Minerva1 , G. Maulucci4 , M. De Spirito4 , F. Rossin2 , M. Piacentini2,3 & G. Delogu1
,
1 From the Institute of Microbiology, Universit a Cattolica del Sacro Cuore – Fondazione Policlinico Gemelli ; 2Department of Biology, University of Rome “Tor Vergata” ; 3 National Institute for Infectious Diseases, IRCCS “Lazzaro Spallanzani” ; and 4Institute of Physics, Universit a Cattolica del Sacro Cuore – Fondazione Policlinico Gemelli, Rome, Italy
Abstract. Palucci I, Matic I, Falasca L, Minerva M, Maulucci G, De Spirito M, Petruccioli E, Goletti D, Rossin F, Piacentini M, Delogu G (Universit a Cattolica del Sacro Cuore – Fondazione Policlinico Gemelli; University of Rome “Tor Vergata”; IRCCS “Lazzaro Spallanzani”; Universit a Cattolica del Sacro Cuore – Fondazione Policlinico Gemelli, Rome, Italy). Transglutaminase type 2 plays a key role in the pathogenesis of Mycobacterium tuberculosis infection. J Intern Med 2017; https://doi.org/10.1111/joim.12714 Background. Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome–lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. Objective. The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. Results. We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked
Introduction Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is a highly successful human pathogen that causes more deaths than any other single infectious agent [1]. Although previously thought to be nearly eradicated in developed countries, there has been a dramatic resurgence of TB worldwide, with 10.4 million new active disease cases and 1.4 million deaths each *These authors contributed equally to this study.
reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTBinfected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. Conclusion. These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB. Keywords: autophagy, cystamine, LC3, transglutaminase type 2.
year [2]. This represents a major health problem, which is further exacerbated by the emergence of strains resistant to the most commonly used antiTB drugs [3]. It is estimated that one-third of the global population has latent TB infection, with no overt signs or symptoms of the disease but with MTB residing in different cells and tissues and contained by the host immune response [4–6]. Failure to eradicate an initial MTB infection, or to contain replication of bacilli, is a consequence of complex mechanisms employed by MTB to evade
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innate and adaptive host immune responses, which promote MTB survival and replication inside macrophages [7]. MTB is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome–lysosome fusion [8–10] by proteins actively secreted through specialized secretion systems, the most important of which is ESX-1 [11]. Blockade of phagosome–lysosome fusion can be overcome by activation of cellular autophagy, reversing the usual acidification defect observed in mycobacteria-containing phagosomes, and resulting in their colocalization with autophagosomes and lysosomes [12, 13]. This in turn leads to decreased viability of intracellular mycobacteria. Supporting evidence has been provided by the observation that induction of autophagy in both Mycobacterium bovis (BCG)- and MTB-infected macrophages by serum starvation, rapamycin treatment or interferon-c activation leads to killing of intracellular bacteria as a result of trafficking bacteria to the lysosome [14]. Autophagy is an ancient evolutionarily conserved cellular process responsible for the degradation of long-lived proteins and organelles at low levels under normal conditions and plays an essential role in cellular and energy homeostasis in all eukaryotic cells [14]. Under physiological conditions, autophagy clears misfolded/mutated proteins and damaged organelles [15]. Under stressful cellular conditions, such as nutrient and energy starvation, it recycles cytoplasmic components to supply cells with amino acids and fatty acids, thus contributing to maintain metabolism compatible with cell survival [16]. In addition, autophagy has a protective role through its various actions in immunity, including the elimination of invasive microbes and participation in antigen presentation [16–18]. Indeed, colocalization of MTB with LC3 on autophagosomes was observed following physiological and pharmacological stimulation of autophagy [19] and it has been shown that MTB employs various mechanisms to avoid autophagic degradation [14, 20, 21]. However, the molecular basis of these mechanisms remains to be elucidated and recent evidence obtained in genetically manipulated mice, in which several key genes involved in the autophagic pathway were inactivated, questioned the role of autophagy during MTB infection [22]. Thus, defining whether modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance is of considerable interest. Consistent with this possibility, transglutaminase type 2 (TG2) has been of interest as a potential 2
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therapeutic target for treatment of autophagyrelated diseases and fibrosis. TG2 is a unique pleiotropic enzyme belonging to the transglutaminase family which catalyses post-translational modifications of proteins through Ca2+-dependent reactions including protein–protein cross-linking, incorporation of primary amines into proteins and glutamine deamination [23]. TG2 is predominantly a cytosolic protein but is also present in mitochondria, in the nucleus, on the plasma membrane and under pathological settings in the extracellular matrix [23–25]. Under steady-state conditions, TG2 exists in a compacted conformation; upon stress stimulation, Ca2+-dependent activation causes TG2 to undergo a conformational change and become catalytically active as a transamidating enzyme [24, 26]. In addition to protein transamidation, TG2 displays other enzymatic activities which do not require Ca2+ such as GTPase, protein kinase and protein disulphide isomerase activity [23, 26]. TG2 is involved in the regulation of numerous cell functions, including cell death/survival and autophagy [23, 27, 28]. We considered that investigating the role of TG2 during MTB infection would be of importance, given that a hallmark of MTB infection is the inhibition of phagosome–lysosome fusion. So far, data on the involvement of TG2 in mycobacterial infection are limited; only one study has been conducted suggesting that TG2 cross-linking activity is essential for formation of the apoptotic envelope during infection with the avirulent strain H37Ra, but not with the virulent H37Rv strain in macrophages [29]. The aim of this study was to determine whether TG2 is involved in MTB pathogenesis in vivo. To this purpose, we have used genetically engineered mice lacking TG2, and examined the course of infections with BCG and with the virulent MTB, both in vitro and in vivo. Finally, we investigated whether pharmacological modulation of TG2 function could be beneficial for inhibiting MTB infection. Material and methods Antibodies and reagents Anti-p62/SQSTM1 (PM045; MBL international corporation, Woburn, MA, USA), anti-LC3 (NB1002331; Novus Biologicals, Littleton, CO, USA), antiBAG3 (Proteintech, Rosemont, IL, USA) and antiactin (2066; Sigma, St Louis, MO, USA) primary
TG2 in MTB infection / I. Palucci et al.
antibodies were used for immunoblotting analysis. HRP-conjugated secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, California). For pharmacological inhibition of TG2 cross-linking activity, Z-DON (Zedira, Darmstadtn, Germany) and cystamine (Sigma) were added to macrophage cultures at concentrations of 40 and 400 l mol L 1, respectively; rapamycin (Enzo Life Sciences, Farmingdale, NY, USA) was added at a concentration of 1 l mol L 1 and capreomycin (Sigma) at 4 lg mL 1. Mycobacterial strains and growth conditions MTB H37Rv and BCG Pasteur were used in this study and, when required, transformed with plasmid expressing the mCherry red fluorescent protein (RFP), which was kindly provided by Wilbert Bitter [30]. Transformed colonies were selected on 7H11 agar plates (Becton Dickinson Difco, Detroit, MI, USA) containing 10% Middlebrook OADC enrichment (Microbiol, Cagliari Italy) and 50 lg mL 1 hygromycin B (Sigma). MTB H37Rv and BCG were grown in Middlebrook 7H9 medium (Becton Dickinson Difco, Sparks, MD, USA) supplemented with ADC (Microbiol), glycerol (0.5% vol/vol) and 0.05% Tween 80. During the late log phase, mycobacteria were harvested, resuspended in 10% glycerol and stored at 80 °C until use. Before infection, bacterial concentrations were determined by plating serial dilutions in 7H11 agar supplemented with OADC. Animals Male mice, 8–10 weeks old, were used in all experiments. Wild-type (wt) C57BL/6 mice and TG2 / mice on a C57BL/6 background were obtained from the Laboratory of G. Melino (University of Rome ‘Tor Vergata’). TG2 / GFP-LC3 mice were generated by crossing C57Bl/6 TG2 / mice with C57Bl/6 GFP-LC3 transgenic mice as previously described [27]. All animals used in the experiments were maintained in a pathogen-free facility at the University of Rome ‘Tor Vergata’ and the Catholic University of the Sacred Heart with a natural light–dark cycle at 20 °C and given food and tap water ad libitum. Mice were kept and handled in accordance with standard guidelines and ethically approved protocols, and experiments were performed in compliance with the legislative decree of the Italian Government 27 January 1992, n. 116 and the Health Minister memorandum 14 May 2001, n. 6.
In vivo MTB infection in wt and TG2
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mice
For in vivo MTB infections, wt and TG2 / GFPLC3+/+C57BL/6 mice were infected with approximately 100 colony-forming units (CFUs) of MTB Erdman (TMC107) by aerosol. On day 1 or 28, postinfection mice were sacrificed and lung and spleen tissues were homogenized and bacterial counts determined as previously described [31]. All in vivo animal experiments were performed strictly in accordance with protocols approved by the Catholic University of the Sacred Heart Ethical Committee (n. O21/2009). Macrophage isolation and cell cultures Peritoneal macrophages (pMM0s) were obtained from wt and TG2 / C57BL/6 mice (9–14 weeks old) previously immunized by intraperitoneal injection of 4% thioglycolate (2 mL per mouse) [32]. Bone marrow-derived macrophages (BMM0s) were isolated and differentiated from wt and TG2 / GFP-LC3 C57Bl/6 transgenic mice as indicated previously [33]. Experiments were approved by the Ethical Committee of the Catholic University of the Sacred Heart (no. T21/2011). On day 7, differentiated BMM0s were infected with BCG and MTB H37Rv strains. J774, and primary macrophages were infected with MTB [multiplicity of infection (MOI) 1 : 1], and at various time-points (4 h, 2, 3 and/or 6 days), cells were washed twice with sterile phosphate-buffered saline (PBS) to remove extracellular bacteria, lysed in 0.01% Triton-9100 (Sigma) and intracellular bacterial loads (in CFUs) determined as previously described [32]. Western blotting Cells were pelleted and lysed using ice-cold cell lytic buffer (Sigma-Aldrich) with inhibitors of protease (diluted 1 : 200). Protein extracts were heated for 10 min at 97 °C in Laemmli buffer and loaded on a 10% SDS polyacrylamide gel, which was run in Tris-glycine buffer at 100 V and transferred to a Whatman nitrocellulose membrane (Sigma-Aldrich). Membranes were incubated with 5% milk in PBS-Tween to prevent nonspecific binding of the monoclonal antibody. Membranes were then incubated with anti-p62, anti-LC3, antiBAG3 and anti-actin primary antibodies overnight at 4 °C. After several washes with PBS-Tween, membranes were incubated with the appropriate secondary antibodies for 1 h. Following a final washing step, the protein signal was detected by ª 2017 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine
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chemiluminescence (Amersham/GE HealthCare, Buckinghamshire, UK). BAG3/actin, LC3-II/actin and p62/actin ratios were quantified by densitometric analysis using ImageJ software. Transmission electron microscopy For ultrastructural analysis, pMM0s from C57/ BL6 wt and TG2 / mice were infected with MTB (MOI 1 : 1). Cell cultures were fixed with 2.5% glutaraldehyde in 0.1 mol L 1 cacodylate buffer for 1 h at 4 °C, and postfixed in 1% osmium tetroxide in 0.1 mol L 1 cacodylate buffer for 1 h. The cells were then dehydrated in graded ethanol and embedded in Epon resin. Ultrathin sections were stained with 2% uranyl acetate and observed under a Zeiss (Oberkochen, Germany) EM900 transmission electron microscope. Images were captured digitally with a Mega View II digital camera (Silicon Integrated Systems Corp, SiS, Hsinchu City, Taiwan). Confocal microscopy For confocal microscopy experiments, pMM0s from C57/BL6 wt and TG2 / mice were seeded onto glass chamber slides (Thermo Scientific Nunc, Rochester, NY, USA) at a density of 5 9 105 cells per slide. Cells were infected with mycobacteria as described above. At different time-points (4 h, 1 day and 3 days), cells were fixed in ice-cold ethanol/acetone (1 : 1) at 20 °C for 10 min. Samples were briefly rinsed in PBS and then mounted in SlowFade Antifade (Invitrogen, Life Technologies). Fluorescence was analyzed with a TCS SP2 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence images obtained separately in both channels through a 639 objective (magnification 92) were acquired with Leica Confocal Software. Green (for LC3-GFP), and red (for mycobacteria RFP) images were merged to reveal colocalization that appeared yellow. Ex vivo two-photon microscopy analysis of wt and TG2 lungs
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The lobes of the left lung isolated at day 28 from wt and TG2 / GFP-LC3+/+C57BL/6-infected mice were prepared for fluorescence observation as previously described [34]. In particular, organs were perfused and fixed with 10% paraformaldehyde in PBS and GFP-LC3 intensity images were obtained with an inverted confocal microscope 4
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(DMIRE2, Leica Microsystems) using a 639 oil immersion objective (NA 1.4) and excitation at 800 nm with a mode-locked titanium–sapphire laser (Chamaleon, Coherent, Santa Clara, CA, USA). Internal photon multiplier tubes collected images in eight-bit, unsigned images at a 400 Hz scan speed. Intensity images were recorded with emission in the range of 560–600 nm for GFP-LC3. Imaging was performed at room temperature. Successive focal planes were imaged in 2 lm steps for a total depth of 30 lm more than sufficient to encompass the entire depth of the lung tissue. The interval between planes was chosen for optimum reconstruction of the cell volume without gaps [35]. Images of 512 9 512 pixels were acquired at approximately 4/s with a pixel dwell time of 1.6 ls. Four scans were acquired and averaged to produce a single image. Lungs derived from wt nontransgenic C57BL/6 mice infected with the same MTB strain and extracted in the same manner were used as negative controls to eliminate the intrinsic fluorescence error. Background values of GFP-LC3 fluorescence images (defined as intensities below 7% of the maximum intensity) were set to zero in image-J. Fluorescence values recovered from images were integrated for a fixed depth for each sample (30 lm) and averaged over multiple regions of interest [36]. Photomultiplier and excitation intensity were not changed during the measurement session. Statistical analysis All values obtained were means of at least three independent experiments performed in duplicate or triplicate. Results are presented as mean value SD. Control and treated groups were analyzed by two-way ANOVA followed by Bonferroni posttest. In all analyses, a P-value of
