The sodium proton exchanger NHE9 regulates phagosome maturation and bactericidal activity in macrophages
2022; Elsevier BV; Volume: 298; Issue: 8 Linguagem: Inglês
10.1016/j.jbc.2022.102150
ISSN1083-351X
AutoresHabiba S. Shamroukh, Nabrah Lone, Muaaz Akhtar, Alhareth Altayib, Shelby Sutliff, Zahraa Kassem, Suvranta K. Tripathy, Kalyan C. Kondapalli,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoAcidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages. Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages. Dynamics of the interactions between bacterial pathogens and the host innate immune system are crucial for determining the trajectory of the infection (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar). Macrophages, along with neutrophils and dendritic cells, are key determinants of the innate immune response (2Steinberg B.E. Huynh K.K. Grinstein S. Phagosomal acidification: measurement, manipulation and functional consequences.Biochem. Soc. Trans. 2007; 35: 1083-1087Crossref PubMed Scopus (31) Google Scholar). Macrophage response is characterized by recognizing the infectious agent through pathogen-associated molecular patterns, proliferation of macrophages into the infected tissue followed by internalizing the bacterial pathogen for degradation, and antigen presentation to T-cells (3Gordon S. The macrophage: past, present and future.Eur. J. Immunol. 2007; 37: S9-S17Crossref PubMed Scopus (448) Google Scholar, 4Murray P.J. Wynn T.A. Protective and pathogenic functions of macrophage subsets.Nat. Rev. Immunol. 2011; 11: 723-737Crossref PubMed Scopus (3561) Google Scholar, 5Wynn T.A. Chawla A. Pollard J.W. 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However, functional link(s) between NHE9 expression and macrophage activity have not been examined. NHE9, encoded by the gene SLC9A9, belongs to an ancient family of evolutionarily conserved transport proteins that regulate cellular and organellar pH (18Brett C.L. Donowitz M. Rao R. Evolutionary origins of eukaryotic sodium/proton exchangers.Am. J. Physiol. Cell Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (451) Google Scholar). Currently, 13 distinct mammalian orthologs of sodium proton exchangers (NHEs) have been recognized, each differing in transport kinetics, cellular localization, tissue distribution, and substrate preferences (18Brett C.L. Donowitz M. Rao R. Evolutionary origins of eukaryotic sodium/proton exchangers.Am. J. Physiol. Cell Physiol. 2005; 288: C223-C239Crossref PubMed Scopus (451) Google Scholar, 19Donowitz M. Ming Tse C. Fuster D. SLC9/NHE gene family, a plasma membrane and organellar family of Na(+)/H(+) exchangers.Mol. 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Cell Neurosci. 2014; 8: 172Crossref PubMed Scopus (69) Google Scholar). NHE9's C-terminal cytoplasmic tail modulates various important cellular signaling pathways via its protein interaction network (23Patak J. Faraone S.V. Zhang-James Y. Sodium hydrogen exchanger 9 NHE9 (SLC9A9) and its emerging roles in neuropsychiatric comorbidity.Am. J. Med. Genet. B Neuropsychiatr. Genet. 2020; 183: 289-305Crossref PubMed Scopus (5) Google Scholar, 24Zhang-James Y. Vaudel M. Mjaavatten O. Berven F.S. Haavik J. Faraone S.V. Effect of disease-associated SLC9A9 mutations on protein-protein interaction networks: implications for molecular mechanisms for ADHD and autism.Atten. Defic. Hyperact. Disord. 2019; 11: 91-105Crossref PubMed Scopus (10) Google Scholar, 25Winklemann I. Matsuoka R. Meier P.F. Shutin D. Zhang C. Orellana L. et al.Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9.EMBO J. 2020; 39e105908PubMed Google Scholar). Though roles in vesicular trafficking, synaptic membrane turnover, and modulation of signaling axes have been recognized for NHE9, its functional profile remains incomplete (25Winklemann I. Matsuoka R. Meier P.F. Shutin D. Zhang C. Orellana L. et al.Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9.EMBO J. 2020; 39e105908PubMed Google Scholar, 26Beydoun R. Hamood M.A. Gomez Zubieta D.M. Kondapalli K.C. Na(+)/H(+) exchanger 9 regulates iron mobilization at the blood-brain barrier in response to iron starvation.J. Biol. Chem. 2017; 292: 4293-4301Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27Kondapalli K.C. Llongueras J.P. Capilla-Gonzalez V. Prasad H. Hack A. Smith C. et al.A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma.Nat. Commun. 2015; 6: 6289Crossref PubMed Scopus (67) Google Scholar, 28Ullman J.C. Yang J. Sullivan M. Bendor J. Levy J. Pham E. et al.A mouse model of autism implicates endosome pH in the regulation of presynaptic calcium entry.Nat. Commun. 2018; 9: 330Crossref PubMed Scopus (19) Google Scholar). Considering these important roles in cellular physiology, it is not surprising that dysfunction of NHE9 is implicated in various diseases. While loss of NHE9 function is associated with familial autism and attention deficit hyperactivity disorder, gain of NHE9 function underlies subsets of cancers such as glioblastoma and esophageal squamous cell carcinoma (23Patak J. Faraone S.V. Zhang-James Y. Sodium hydrogen exchanger 9 NHE9 (SLC9A9) and its emerging roles in neuropsychiatric comorbidity.Am. J. Med. Genet. B Neuropsychiatr. Genet. 2020; 183: 289-305Crossref PubMed Scopus (5) Google Scholar, 27Kondapalli K.C. Llongueras J.P. Capilla-Gonzalez V. Prasad H. Hack A. Smith C. et al.A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma.Nat. 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Hill R.S. et al.Identifying autism loci and genes by tracing recent shared ancestry.Science. 2008; 321: 218-223Crossref PubMed Scopus (566) Google Scholar, 33Zhang-James Y. DasBanerjee T. Sagvolden T. Middleton F.A. Faraone S.V. SLC9A9 mutations, gene expression, and protein-protein interactions in rat models of attention-deficit/hyperactivity disorder.Am. J. Med. Genet. B Neuropsychiatr. Genet. 2011; 156B: 835-843Crossref PubMed Scopus (28) Google Scholar). The factors that determine the phagosomal pH remain poorly understood. The acidity within the phagosome is generated by the V-type H+-ATPase, which transports 3H+ into the lumen by hydrolyzing one ATP molecule (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar). However, without counter-ion fluxes, the electric potential generated by the influx of protons would oppose any significant changes in lumenal pH (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar). As a result of the sequential fusions with endosomal membranes, it is reasonable to hypothesize that H+ leakage on the phagosome is regulated similarly to the endolysosomal system. In this study, we analyzed the functional link between NHE9 and bactericidal activity of macrophages and found that classical activation of macrophages resulted in pronounced downregulation of NHE9 in macrophages. We also examined the importance of NHE9 downregulation on phagocytic ability and bactericidal activity of macrophages by ectopically overexpressing NHE9. Considering the function of NHE9 in serving as a leak pathway for protons, we hypothesized that NHE9 overexpression would impair phagosome acidification and consequently enable bacterial survival within the phagosome. We demonstrate that NHE9 associates with the phagosome early during the maturation process and alkalizes the phagosome lumen to disrupt transport along the microtubules preventing them from reaching the lysosomes. This has direct consequences on the ability of the macrophage to degrade the ingested bacteria. Overall, we present evidence of a novel determinant of phagosomal pH during host–bacteria interaction. Timely regulation of gene expression is essential for macrophage activation and mounting an immune response (34Chen S. Yang J. Wei Y. Wei X. Epigenetic regulation of macrophages: From homeostasis maintenance to host defense.Cell Mol. Immunol. 2020; 17: 36-49Crossref PubMed Scopus (127) Google Scholar). Evaluation of single-cell RNA sequencing data, obtained from the Human Protein Atlas for 192 cell type clusters corresponding to different cell type groups https://www.proteinatlas.org (35Thul P.J. Akesson L. Wiking M. Mahdessian D. Geladaki A. Ait Blal H. et al.A subcellular map of the human proteome.Science. 2017; 356eaal3321Crossref PubMed Scopus (1533) Google Scholar, 36Uhlen M. Fagerberg L. Hallstrom B.M. Lindskog C. Oksvold P. Mardinoglu A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 3471260419Crossref PubMed Scopus (8161) Google Scholar), revealed that NHE9 transcripts are enhanced in immune cells (Fig. 1A), specifically macrophages relative to other cell types (Fig. 1 inset). The high steady-state levels of NHE9 in macrophages are consistent with NHE9's previously identified roles in endocytic trafficking of plasma membrane receptors and transporters (22Kondapalli K.C. Prasad H. Rao R. An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease.Front. Cell Neurosci. 2014; 8: 172Crossref PubMed Scopus (69) Google Scholar, 26Beydoun R. Hamood M.A. Gomez Zubieta D.M. Kondapalli K.C. Na(+)/H(+) exchanger 9 regulates iron mobilization at the blood-brain barrier in response to iron starvation.J. Biol. Chem. 2017; 292: 4293-4301Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27Kondapalli K.C. Llongueras J.P. Capilla-Gonzalez V. Prasad H. Hack A. Smith C. et al.A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma.Nat. Commun. 2015; 6: 6289Crossref PubMed Scopus (67) Google Scholar, 29Kondapalli K.C. Hack A. Schushan M. Landau M. Ben-Tal N. Rao R. Functional evaluation of autism-associated mutations in NHE9.Nat. Commun. 2013; 4: 2510Crossref PubMed Scopus (76) Google Scholar). In the context of macrophages, sorting and turnover of pattern recognition receptors, such as the toll-like receptors, is crucial for recognition of microbial pathogens (37Akira S. Uematsu S. Takeuchi O. Pathogen recognition and innate immunity.Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (9066) Google Scholar, 38Kagan J.C. Medzhitov R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling.Cell. 2006; 125: 943-955Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar). To investigate the role of NHE9 in macrophages, we first analyzed its expression levels after stimulation with toll-like receptor-4 ligand bacterial lipopolysaccharide (LPS), the outer leaflet of the outer membrane of Gram-negative bacteria (39Lou J. Li X. Huang W. Liang J. Zheng M. Xu T. et al.SNX10 promotes phagosome maturation in macrophages and protects mice against Listeria monocytogenes infection.Oncotarget. 2017; 8: 53935-53947Crossref PubMed Scopus (15) Google Scholar). We observed a sharp decrease in total NHE9 protein levels upon LPS activation of RAW 264.7 cells (mouse macrophage cell line derived from BALB/c mice). Densitometric analysis of Western blot data showed a reduction of NHE9 by more than 60% within 3 h of LPS treatment (Fig. 1B and inset). NHE9 protein levels continued to decrease over a 24-h period, with less than 10% of initial NHE9 levels remaining by the 24-h time point (Fig. 1B). Next, we tested if our findings are relevant for bacterial infections by monitoring the expression of NHE9 in RAW 264.7 cells after infection with either a uropathogenic strain of Escherichia coli (gram-negative bacteria) or Staphylococcus aureus (gram-positive bacteria). Consistent with our results from LPS stimulation, we observed a strong decrease in NHE9 transcripts postinfection with gram-positive (Fig. 1C) and gram-negative (Fig. 1D) bacteria. Finally, we confirmed our findings in primary macrophages. Similar to the trend observed in RAW 264.7 cells, in mouse bone marrow–derived macrophages (BMDMs) upon stimulation with LPS, NHE9 transcript levels decreased significantly (Fig. 1E). Overall, we noticed a general trend of NHE9 downregulation in macrophages upon bacterial infection. The process of elimination of bacteria within the macrophage can be broadly divided into two phases: (i) ingestion of bacteria leading to formation of nascent phagosomes and (ii) phagosome maturation and subsequent fusion with the lysosome leading to the death of the trapped microbe (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar). To determine whether NHE9 expression levels affect either or both phases, we engineered stable overexpression of NHE9 in RAW 264.7 cells, henceforth referred to as NHE9+ cells. Quantitative PCR analysis revealed a 12-fold increase in NHE9 transcripts within NHE9+ cells (Fig. 2A). We also confirmed the expression of NHE9 by immunofluorescence (Fig. 2B). We confirmed that NHE9 remain highly expressed in NHE9+ cells even after exposure to bacteria (Fig. S1). After, allowing bacterial phagocytosis to occur for 30 min, we assessed the phagosome formation by comparing the presence of live bacteria immediately after ingestion within the control and NHE9+ cells. We did not observe a statistically significant difference in the pathogen burden, for both E. coli and S. aureus, between control and NHE9+ cells in RAW 264.7 (Fig. 2, C and D) and BMDM (Fig. 2, E and F). Next, we compared the bactericidal activity of NHE9+ and control cells by evaluating the pathogen burden 1 h and 3 h after bacterial uptake. The E. coli burden was ∼40% (1 h after ingestion) and ∼67% (3 h after ingestion) higher in NHE9+ cells compared to control RAW 264.7 cells (Fig. 3A). We noticed a similar trend for S. aureus infection of RAW 264.7 cells (Fig. 3B). To confirm that the reduced bactericidal activity in NHE9+ cells is a consequence of NHE9's ion transport function and not a nonspecific effect of overexpression, we evaluated the effect of overexpressing a loss-of-function mutant of NHE9 (S438P) (29Kondapalli K.C. Hack A. Schushan M. Landau M. Ben-Tal N. Rao R. Functional evaluation of autism-associated mutations in NHE9.Nat. Commun. 2013; 4: 2510Crossref PubMed Scopus (76) Google Scholar). We did not discern a statistically significant difference in bactericidal activity between control and RAW 264.7 cells expressing the functional mutant (Fig. 3, C and D). These data clearly indicate that an increase in NHE9-mediated ion transport resulted in reduced bactericidal activity in RAW 264.7 macrophages. Next, we confirmed the validity of our findings from RAW 264.7 cells in BMDMs (Fig. 3, E and F). Our observations from BMDMs were consistent with the findings from RAW 264.7 cells, indicating that NHE9 expression levels affect phagosome maturation but not phagosome formation.Figure 3NHE9 expression regulates bactericidal activity of macrophages. Surviving E. coli (A) and S. aureus (B) 1 h or 3 h postingestion in RAW 264.7 cells, control, and stably expressing NHE9 (NHE9+). (C) and (D) are the same as (A) and (B) except the bacterial survival in control cells is compared to cells stably expressing a functional mutant of NHE9 (NHE9+ (S438P)). Surviving E. coli (E) and S. aureus (F) 1 h or 3 h postingestion in bone marrow–derived macrophages (BMDMs), control, and ectopically expressing NHE9 (NHE9+). Data are expressed as percent of surviving bacteria, relative to control, determined from colony-forming units as indicated in the methods. Graphs represent mean from three biological replicates. Error bars represent standard deviation (SD). ∗p < 0.05. Statistical analysis was done using Student's t-test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To understand the mechanistic basis of how changes in NHE9 expression impact bactericidal activity of macrophages, we first asked, when does NHE9 associate with the phagosome? Previous studies in several primary cell types and immortalized cell lines have shown that NHE9 localizes to both early and late endosomes (23Patak J. Faraone S.V. Zhang-James Y. Sodium hydrogen exchanger 9 NHE9 (SLC9A9) and its emerging roles in neuropsychiatric comorbidity.Am. J. Med. Genet. B Neuropsychiatr. Genet. 2020; 183: 289-305Crossref PubMed Scopus (5) Google Scholar, 26Beydoun R. Hamood M.A. Gomez Zubieta D.M. Kondapalli K.C. Na(+)/H(+) exchanger 9 regulates iron mobilization at the blood-brain barrier in response to iron starvation.J. Biol. Chem. 2017; 292: 4293-4301Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27Kondapalli K.C. Llongueras J.P. Capilla-Gonzalez V. Prasad H. Hack A. Smith C. et al.A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma.Nat. Commun. 2015; 6: 6289Crossref PubMed Scopus (67) Google Scholar, 28Ullman J.C. Yang J. Sullivan M. Bendor J. Levy J. Pham E. et al.A mouse model of autism implicates endosome pH in the regulation of presynaptic calcium entry.Nat. Commun. 2018; 9: 330Crossref PubMed Scopus (19) Google Scholar, 29Kondapalli K.C. Hack A. Schushan M. Landau M. Ben-Tal N. Rao R. Functional evaluation of autism-associated mutations in NHE9.Nat. Commun. 2013; 4: 2510Crossref PubMed Scopus (76) Google Scholar, 40Hill J.K. Brett C.L. Chyou A. Kallay L.M. Sakaguchi M. Rao R. et al.Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9.J. Neurosci. 2006; 26: 9944-9955Crossref PubMed Scopus (50) Google Scholar). Nascent phagosomes merge with early endosomes to form the early phagosomes, which sequentially fuse with late endosomes that finally merge with the lysosomes (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar, 41Flannagan R.S. Jaumouille V. Grinstein S. The cell biology of phagocytosis.Annu. Rev. Pathol. 2012; 7: 61-98Crossref PubMed Scopus (689) Google Scholar). Consistent with the presence on the early phagosome, we observed colocalization of NHE9 with Rab5- (early endosome marker) and BacLight-stained bacteria (Fig. 4A-top panel). This trend continued with the late phagosome as evident by NHE9's colocalization with Rab7 (late endosome marker) in the bacteria hosting compartment (Fig. 4A-bottom panel). Quantification of colocalization using the Manders' overlap coefficient indicated significantly higher localization of NHE9 with the early phagosome relative to the late phagosome (Fig. 4B). The phagosome undergoes progressive acidification as it matures (1Westman J. Grinstein S. Determinants of phagosomal pH during host-pathogen interactions.Front. Cell Dev. Biol. 2020; 8624958PubMed Google Scholar). Considering the role of NHE9 in transporting protons out of the phagosome lumen, we investigated whether an increase in NHE9 expression results in limiting the acidification of the maturing phagosome. We tracked pH-sensitive fluorescence of pHrodo-green conjugated to S. aureus bioparticles for 2 h in RAW 264.7 cells (Fig. 4C). Differences in uptake were normalized using S. aureus conjugated to pH-insensitive fluorophore (Alexa Fluor 594). Phagosome pH was determined by calibration using buffers of known pH. We found that phagosomes in control cells were consistently more acidic at all the evaluated time points relative to phagosomes in NHE9+ macrophages (Fig. 4D). We did not observe a statistically significant difference in pH between control cells and macrophages expressing NHE9 functional mutant (S438P), ruling out any nonspecific effect of overexpression (Fig. 4E). These data show NHE9 expression prevents the progressive acidification of the lumenal pH in the maturing phagosome. Phagosome maturation is mechanistically dependent on successful centripetal motion of the early and late phagosomes to the perinuclear region for fusion with the lysosomes. Lumenal pH of the phagosome is known to signal changes in lipid–protein architecture on the phagosomal membrane, which in turn regulates motor recruitment and transport (42Rai A. Pathak D. Thak
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