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Intracellular Bacterial Infection Lysosomal Biogenesis Limits

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Intracellular Bacterial Infection Lysosomal Biogenesis Limits Published December 14, 2015, doi:10.4049/jimmunol.1501595 The Journal of Immunology Activator of G-Protein Signaling 3–Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection Ali Vural,* Souhaila Al-Khodor,†,1 Gordon Y. C. Cheung,‡ Chong-Shan Shi,* Lalitha Srinivasan,x Travis J. McQuiston,{ Il-Young Hwang,* Anthony J. Yeh,‡ Joe B. Blumer,‖ Volker Briken,x Peter R. Williamson,{ Michael Otto,‡ Iain D. C. Fraser,† and John H. Kehrl* Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Ga subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcrip- tion. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. The Journal of Immunology, 2016, 196: 000–000. athogens, especially those that survive and proliferate in Lysosomes maintain cellular homeostasis by acting as a deg- intracellular niches, have developed sophisticated strate- radative and recycling organelle in the cytosol. They recycle P gies to subvert host defense systems (1). Subject to strong macromolecules, reducing the energy-intensive de novo syn- selective pressures, drug-resistant microorganisms have emerged thesis of raw materials, and dysfunction causes inefficient energy that are unresponsive or poorly responsive to current antimicrobial use, diverting energy to synthesis pathways at the expense of by guest on October 1, 2021. Copyright 2015 Pageant Media Ltd. agents (2). This provides an incentive to better understand host– energy-storage depots. Lysosomes degrade substrates and met- pathogen interfaces: on the pathogen side to unravel the mecha- abolic waste products by fusing with endocytic, autophagic, and nisms by which pathogens target and hijack host factors for their phagocytic vesicles (4). Both lysosomal mass and function are own benefit and on the host side to delineate how host cells subject to regulation by the cellular physiologic state and in re- marshal intracellular defense systems to combat infections. Such sponse to pathologic conditions, including bacterial infections, understanding may lead to alternative strategies to improve host neurodegenerative disorders, and lysosomal storage diseases defense and enhance cellular resistance (3). Lysosomes occupy a (LSD) (5). The coordinated upregulation of lysosomal volume and crucial position in host–pathogen interactions, by being both tar- function is governed by the transcriptional factor EB (TFEB), geted by pathogens and serving as a major mechanism for killing which controls lysosomal biogenesis by enhancing the expression http://classic.jimmunol.org intracellular invaders. of the CLEAR gene network. Among the induced genes are ly- *B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National A.V. and J.H.K. designed and analyzed the experiments; A.V. did the experimental Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, work; S.A.-K. performed Burkholderia cenocepacia J2315 infection experiments; MD 20892; †Signaling Systems Unit, Laboratory of Systems Biology, National Institute G.Y.C.C. and A.J.Y. performed methicillin-resistant Staphylococcus aureus infection of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; experiments; L.S. performed Mycobacterium tuberculosis infection experiments; ‡Pathogen Molecular Genetics Section, Laboratory of Human Bacterial Pathogenesis, T.J.M. performed the autophagic proteolysis experiments; I.-Y.H. performed flow Downloaded from National Institute of Allergy and Infectious Diseases, National Institutes of Health, cytometry experiments; J.B.B., C.-S.S., P.R.W., I.D.C.F., M.O., and V.B. provided Bethesda, MD 20892; xDepartment of Cell Biology and Molecular Genetics, University expert advice; and A.V. and J.H.K. wrote and edited the manuscript. of Maryland, College Park, MD 20742; {Translational Mycology Unit, Laboratory of Address correspondence and reprint requests to Dr. John H. Kehrl, B-Cell Molecular Clinical Infectious Disease, National Institute of Allergy and Infectious Diseases, Na- ‖ Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy tional Institutes of Health, Bethesda, MD 20892; and Department of Cell and Molecular and Infectious Diseases, National Institutes of Health, 10 Center Drive, MSC Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 1876, Building 10, Room 11N212, Bethesda, MD 20892-1876. E-mail address: Charleston, SC 29425 [email protected] 1Current address: Infectious Disease Unit, Division of Translational Medicine, Sidra Abbreviations used in this article: AGS3, activator of G-protein signaling 3; BMDM, Medical and Research Center, Doha, Qatar. bone marrow–derived macrophage; GDI, guanine nucleotide dissociation inhibitor; ORCIDs: 0000-0003-1796-6058 (G.Y.C.C.); 0000-0001-7826-8101 (T.J.M.); 0000-0002- GPCR, G-protein–coupled receptor; iBMDM, immortalized BMDM; LDH, lactate 0020-3815 (J.B.B.); 0000-0002-2222-4115 (M.O.); 0000-0002-6526-159X (J.H.K.). dehydrogenase; LSD, lysosomal storage disease; MOI, multiplicity of infection; MRSA, methicillin-resistant S. aureus; mTOR, mammalian target of rapamycin; Received for publication July 15, 2015. Accepted for publication November 14, mTORC1, mTOR complex 1; siRNA, small interfering RNA; S6K1, S6 kinase 1; 2015. TFEB, transcriptional factor EB; TSC, tuberous sclerosis complex; WT, wild-type. This work was supported by the intramural program of the National Institute of Allergy and Infectious Diseases. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501595 2 MACROPHAGE AGS3 EXPRESSION ENHANCES LYSOSOMAL BIOGENESIS sosomal hydrolases, lysosomal membrane proteins, and the com- fected into THP-1 cells, as previously described (29), using the human ponents of vacuolar H+-ATPase, which causes lysosomal acidifi- monocyte nucleofector kit (VPA-1007; Lonza). The cells were selected cation (6). Recently, TFEB was shown to be rapidly activated in with G418 (100 mg/ml) and later sorted on the basis of GFP expression into AGS3lo and AGS3hi cells using a FACSAria flow cytometer (Becton murine macrophages upon Staphylococcus aureus infection and Dickinson). The AGS3-GFP stable HeLa cell lines were generated by required for the proper transcriptional induction of several transfection of the AGS3-GFP expression plasmid using Lipofectamine proinflammatory cytokines and chemokines (7). Yet, the precise 2000. The cells were selected with 1000 mg/ml G418 and sorted based on lo hi hi signaling pathways that govern TFEB activity and lysosome GFP expression into HeLa AGS3 and AGS3 cell lines. The AGS3 cells were periodically resorted to maintain high levels of GFP expression. biogenesis in macrophages are largely unknown. In this article, we show the level of activator of G-protein sig- Burkholderia cenocepacia infection naling 3 (AGS3), also referred to as G-protein signaling modulator B. cenocepacia J2315 bacteria were grown on blood agar plates at 37˚C for (Gpsm1), in macrophages helps to govern lysosomal biogenesis 3 d. Single colonies were picked and grown overnight at 37˚C in 10 ml and function by affecting the translocation of TFEB into the nu- Luria-Bertani broth while shaking. The OD of bacterial suspensions was cleus through the regulation of the mammalian target of rapa- measured at an absorbance of 600 nm, and the CFU/ml was calculated 3 8 mycin (mTOR) pathway. AGS3 was originally identified as a based on the previously established equation: CFU/ml/OD600 = 2.7 10 . guanine nucleotide dissociation inhibitor (GDI) in a yeast-based In addition, formalin-killed bacteria were prepared using 3.65% parafor- maldehyde in PBS solution for 30 min and then washed with 150 mM functional screen of mammalian cDNA libraries for proteins that NaCl. The infections were performed at a multiplicity of infection (MOI) activate G-protein signaling independent of G-protein–coupled of 1 into THP-1 and BMDM cell cultures, which were seeded at a density receptors (GPCR) (8). As a GDI, AGS3 binds to the Gi/Go/ of 2.0 3 105 cells/ml 2 d earlier. The plates were centrifuged at 1200 rpm transducin family of G proteins via its G-protein regulatory for 5 min to synchronize the infection, and this step corresponded to the zero time point. Next, the infected cells were incubated for an additional (GoLoco)
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