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Weiner JM, Lee WH, Nolan EM, Oglesby AG. Calprotectin elicits aberrant iron starvation responses in Pseudomonas aeruginosa under anaerobic conditions. J Bacteriol 2025; 207:e0002925. [PMID: 40135923 PMCID: PMC12004955 DOI: 10.1128/jb.00029-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2025] [Accepted: 01/31/2025] [Indexed: 03/27/2025] Open
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that uses several mechanisms to survive in the iron-limiting host environment. The innate immune protein calprotectin (CP) sequesters ferrous iron [Fe(II)], among other divalent transition metal ions, to limit its availability to pathogens. CP levels are increased in individuals with cystic fibrosis (CF), a hereditary disease that leads to chronic pulmonary infection by P. aeruginosa. We previously showed that aerobic CP treatment of P. aeruginosa induces a multi-metal starvation response that alters expression of several virulence properties. However, the CF lung is a hypoxic environment due to the growth of P. aeruginosa in dense biofilms. Here, we report that anaerobic CP treatment of P. aeruginosa induces many processes associated with an aerobic iron starvation response, including decreased phenazine production and increased expression of the PrrF small regulatory RNAs (sRNAs). However, the iron starvation response elicited by CP in anaerobic conditions shows characteristics that are distinct from responses observed in aerobic growth, including a lack of siderophore production and increased induction of genes for the FeoAB Fe(II) and Phu heme uptake systems. Also distinct from aerobic conditions, CP treatment induces expression of genes for predicted manganese transporters MntH1 and MntH2 during anaerobic growth while eliciting a less robust zinc starvation response compared to aerobic conditions. Induction of mntH2 is dependent on the PrrF sRNAs, suggesting a novel example of metal regulatory cross-talk. Thus, anaerobic CP treatment results in a multi-metal starvation response with key distinctions from aerobic conditions, revealing differences in P. aeruginosa metal homeostasis during anaerobic growth.IMPORTANCEIron is critical for most microbial pathogens, and the innate immune system sequesters this metal to limit microbial growth. Pathogens must overcome iron sequestration to survive during infection. For many pathogens, iron homeostasis has primarily been studied in aerobic conditions. Nevertheless, some host environments are hypoxic, including chronic lung infection sites in individuals with cystic fibrosis (CF). Here, we use the innate immune protein calprotectin, which sequesters divalent metal ions including Fe(II), to study the anaerobic iron starvation response of a common CF lung pathogen, Pseudomonas aeruginosa. We report several distinctions of this response during anaerobiosis, highlighting the importance of carefully considering the host environment when investigating the role of nutritional immunity in host-pathogen interactions.
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Affiliation(s)
- Jacob M. Weiner
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, USA
| | - Wei Hao Lee
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | - Elizabeth M. Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | - Amanda G. Oglesby
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, USA
- Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, Maryland, USA
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2
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Pampuscenko K, Jankeviciute S, Morkuniene R, Sulskis D, Smirnovas V, Brown GC, Borutaite V. S100A9 protein activates microglia and stimulates phagocytosis, resulting in synaptic and neuronal loss. Neurobiol Dis 2025; 206:106817. [PMID: 39884585 DOI: 10.1016/j.nbd.2025.106817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Revised: 01/24/2025] [Accepted: 01/27/2025] [Indexed: 02/01/2025] Open
Abstract
S100 calcium-binding protein A9 (S100A9, also known as calgranulin B) is expressed and secreted by myeloid cells under inflammatory conditions, and S100A9 can amplify inflammation. There is a large increase in S100A9 expression in the brains of patients with neurodegenerative diseases, such as Alzheimer's disease, and S100A9 has been suggested to contribute to neurodegeneration, but the mechanisms are unclear. Here we investigated the effects of extracellular recombinant S100A9 protein on microglia, neurons and synapses in primary rat brain neuronal-glial cell cultures. Incubation of cell cultures with 250-500 nM S100A9 caused neuronal loss without signs of apoptosis or necrosis, but accompanied by exposure of the "eat-me" signal - phosphatidylserine on neurons. S100A9 caused activation of microglial inflammation as evidenced by an increase in the microglial number, morphological changes, release of pro-inflammatory cytokines, and increased phagocytic activity. At lower concentrations, 10-100 nM S100A9 induced synaptic loss in the cultures. Depletion of microglia from the cultures prevented S100A9-induced neuronal and synaptic loss, indicating that neuronal and synaptic loss was mediated by microglia. These results suggest that extracellular S100A9 may contribute to neurodegeneration by activating microglial inflammation and phagocytosis, resulting in loss of synapses and neurons. This further suggests the possibility that neurodegeneration may be reduced by targeting S100A9 or microglia.
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Affiliation(s)
- Katryna Pampuscenko
- Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.
| | - Silvija Jankeviciute
- Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.
| | - Ramune Morkuniene
- Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.
| | - Darius Sulskis
- Life Sciences Center, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania.
| | - Vytautas Smirnovas
- Life Sciences Center, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania.
| | - Guy C Brown
- Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
| | - Vilmante Borutaite
- Neuroscience Institute, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.
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Van Linthout S. Shared Mechanisms in Cancer and Cardiovascular Disease: S100A8/9 and the NLRP3 Inflammasome: JACC: CardioOncology State-of-the-Art Review. JACC CardioOncol 2024:S2666-0873(24)00370-3. [PMID: 40260700 DOI: 10.1016/j.jaccao.2024.10.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 09/23/2024] [Accepted: 10/01/2024] [Indexed: 04/24/2025] Open
Abstract
Inflammation and a dysregulated immune system are common denominators in cancer and cardiovascular disease (CVD). The Canakinumab Anti-Inflammatory Thrombosis Outcome Study (CANTOS) highlighted the convergence of interleukin (IL)-1β biology in cancer and CVD, and the potential of anti-IL-1β drugs for the treatment of both disease entities. Accumulating evidence further supports the role of the innate immunity members and IL-1β activators, S100A8/9 and the NLRP3 inflammasome, in both cancer and CVD. This review outlines the common involvement of S100A8/9 and the NLRP3 inflammasome, in cancer and CVD. Specifically, their time-, cell-, and context-dependent actions and hereto-related dichotomous role in different cancers and CVD are addressed, highlighting the need for further insights to allow tailored therapies.
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Affiliation(s)
- Sophie Van Linthout
- Berlin Institute of Health at Charité-Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT), Berlin, Germany; German Centre for Cardiovascular Research (DZHK), partner site Berlin, Berlin, Germany.
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4
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Chen JM, He J, Qiu JM, Yang GG, Wang D, Shen Z. Netrin-1-CD146 and netrin-1-S100A9 are associated with early stage of lymph node metastasis in colorectal cancer. BMC Gastroenterol 2024; 24:308. [PMID: 39261771 PMCID: PMC11389491 DOI: 10.1186/s12876-024-03401-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Accepted: 09/03/2024] [Indexed: 09/13/2024] Open
Abstract
BACKGROUND The netrin-1/CD146 pathway regulates colorectal cancer (CRC) liver metastasis, angiogenesis, and vascular development. However, few investigations have yet examined the biological function of netrin-1/CD146 complex in CRC. In this work, we investigated the relationship between the netrin-1/CD146 axis and S100 proteins in sentinel lymph node, and revealed a possible new clue for vascular metastasis of CRC. METHODS The expression levels of netrin-1 and CD146 proteins in CRC, as well as S100A8 and S100A9 proteins in the sentinel lymph nodes were determined by immunohistochemistry. Using GEPIA and UALCAN, we analyzed netrin-1 and CD146 gene expression in CRC, their association with CRC stage, and their expression levels and prognosis in CRC patients. RESULTS The expression level of netrin-1 in N1a+1b (CRC lymphatic metastasis groups, exculded N1c) was positively increased with N0 (p = 0.012). The level of netrin-1 protein was positively correlated with CD146 protein (p < 0.05). The level of S100A9 protein was positively correlated with CD146 protein (r = 0.492, p = 0.007). Moreover, netrin-1 expression was obviously correlated with S100A9 expression in the N1 stage (r = 0.867, p = 0.000). CD146 level was correlated with S100A9 level in the N2 stage (r = 0.731, p = 0.039). CD146 mRNA expression was higher in normal colorectal tissues than in CRC (p < 0.05). Netrin-1 and CD146 expression were not significantly associated with the tumor stages and prognosis of patients with CRC (p > 0.05). CONCLUSIONS The netrin-1/CD146 and netrin-1/S100A9 axis in CRC tissues might related with early stage of lymph node metastasis, thus providing potential novel channels for blocking lymphatic metastasis and guiding biomarker discovery in CRC patients.
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Affiliation(s)
- Jin-Ming Chen
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China.
| | - Jun He
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China
| | - Jian-Ming Qiu
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China
| | - Guan-Gen Yang
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China
| | - Dong Wang
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China
| | - Zhong Shen
- Department of Anorectal Surgery, the Third People's Hospital of Hangzhou, 38 West Lake Avenue, 310009, Hangzhou, People's Republic of China.
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Chen F, He Z, Wang C, Si J, Chen Z, Guo Y. Advances in the study of S100A9 in cardiovascular diseases. Cell Prolif 2024; 57:e13636. [PMID: 38504474 PMCID: PMC11294427 DOI: 10.1111/cpr.13636] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Revised: 03/08/2024] [Accepted: 03/13/2024] [Indexed: 03/21/2024] Open
Abstract
Cardiovascular disease (CVD) is a group of diseases that primarily affect the heart or blood vessels, with high disability and mortality rates, posing a serious threat to human health. The causative factors, pathogenesis, and characteristics of common CVD differ, but they all involve common pathological processes such as inflammation, oxidative stress, and fibrosis. S100A9 belongs to the S100 family of calcium-binding proteins, which are mainly secreted by myeloid cells and bind to the Toll-like receptor 4 and receptor for advanced glycation end products and is involved in regulating pathological processes such as inflammatory response, fibrosis, vascular calcification, and endothelial barrier function in CVD. The latest research has found that S100A9 is a key biomarker for diagnosing and predicting various CVD. Therefore, this article reviews the latest research progress on the diagnostic and predictive, and therapeutic value of S100A9 in inflammatory-related CVD such as atherosclerosis, myocardial infarction, and arterial aneurysm and summarizes its molecular mechanisms in the progression of CVD, aiming to explore new predictive methods and to identify potential intervention targets for CVD in clinical practice.
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Affiliation(s)
- Fengling Chen
- Hengyang Medical SchoolUniversity of South ChinaHengyangHunanChina
- Department of Cardiovascular Medicine, Zhuzhou Hospital Affiliated to Xiangya School of MedicineCentral South UniversityZhuzhouHunanChina
| | - Ziyu He
- Department of Cardiovascular Medicine, Zhuzhou Hospital Affiliated to Xiangya School of MedicineCentral South UniversityZhuzhouHunanChina
| | - Chengming Wang
- Department of Cardiovascular Medicine, Zhuzhou Hospital Affiliated to Xiangya School of MedicineCentral South UniversityZhuzhouHunanChina
| | - Jiajia Si
- Hunan Key Laboratory of Biomedical Nanomaterials and DevicesHunan University of TechnologyZhuzhouChina
| | - Zhu Chen
- Hengyang Medical SchoolUniversity of South ChinaHengyangHunanChina
- Hunan Key Laboratory of Biomedical Nanomaterials and DevicesHunan University of TechnologyZhuzhouChina
| | - Yuan Guo
- Hengyang Medical SchoolUniversity of South ChinaHengyangHunanChina
- Department of Cardiovascular Medicine, Zhuzhou Hospital Affiliated to Xiangya School of MedicineCentral South UniversityZhuzhouHunanChina
- Hunan Key Laboratory of Biomedical Nanomaterials and DevicesHunan University of TechnologyZhuzhouChina
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Brackman LC, Jung MS, Ogaga EI, Joshi N, Wroblewski LE, Piazuelo MB, Peek RM, Choksi YA, Algood HMS. IL-17RA-Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection. Immunohorizons 2024; 8:339-353. [PMID: 38639570 PMCID: PMC11066722 DOI: 10.4049/immunohorizons.2300078] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Accepted: 03/21/2024] [Indexed: 04/20/2024] Open
Abstract
Helicobacter pylori is a Gram-negative pathogen that colonizes the stomach, induces inflammation, and drives pathological changes in the stomach tissue, including gastric cancer. As the principal cytokine produced by Th17 cells, IL-17 mediates protective immunity against pathogens by inducing the activation and mobilization of neutrophils. Whereas IL-17A is largely produced by lymphocytes, the IL-17 receptor is expressed in epithelial cells, fibroblasts, and hematopoietic cells. Loss of the IL-17RA in mice results in impaired antimicrobial responses to extracellular bacteria. In the context of H. pylori infection, this is compounded by extensive inflammation in Il17ra-/- mice. In this study, Foxa3creIl17rafl/fl (Il17raΔGI-Epi) and Il17rafl/fl (control) mice were used to test the hypothesis that IL-17RA signaling, specifically in epithelial cells, protects against severe inflammation after H. pylori infection. The data indicate that Il17raΔGI-Epi mice develop increased inflammation compared with controls. Despite reduced Pigr expression, levels of IgA increased in the gastric wash, suggesting significant increase in Ag-specific activation of the T follicular helper/B cell axis. Gene expression analysis of stomach tissues indicate that both acute and chronic responses are significantly increased in Il17raΔGI-Epi mice compared with controls. These data suggest that a deficiency of IL-17RA in epithelial cells is sufficient to drive chronic inflammation and hyperactivation of the Th17/T follicular helper/B cell axis but is not required for recruitment of polymorphonuclear neutrophils. Furthermore, the data suggest that fibroblasts can produce chemokines in response to IL-17 and may contribute to H. pylori-induced inflammation through this pathway.
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Affiliation(s)
- Lee C. Brackman
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
- Division of Infectious Disease, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Matthew S. Jung
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
- Division of Infectious Disease, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Eseoghene I. Ogaga
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
| | - Nikhita Joshi
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
- Vanderbilt University, School of Biological Sciences, Nashville, TN
| | - Lydia E. Wroblewski
- Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - M. Blanca Piazuelo
- Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Richard M. Peek
- Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Yash A. Choksi
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
- Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Holly M. Scott Algood
- Tennessee Valley Healthcare System, Department of Veterans Affairs, Nashville, TN
- Division of Infectious Disease, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN
- Vanderbilt Institute of Infection, Immunity, and Inflammation, Vanderbilt University Medical Center, Nashville, TN
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7
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Xia P, Ji X, Yan L, Lian S, Chen Z, Luo Y. Roles of S100A8, S100A9 and S100A12 in infection, inflammation and immunity. Immunology 2024; 171:365-376. [PMID: 38013255 DOI: 10.1111/imm.13722] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Accepted: 11/16/2023] [Indexed: 11/29/2023] Open
Abstract
S100 proteins are small proteins that are only expressed in vertebrates. They are widely expressed in many different cell types and are involved in the regulation of calcium homeostasis, glucose metabolism, cell proliferation, apoptosis, inflammation and tumorigenesis. As members of the S100 protein subfamily of myeloid-related proteins, S100A8, S100A9 and S100A12 play a crucial role in resisting microbial infection and maintaining immune homeostasis. These proteins chelate the necessary metal nutrients of pathogens invading the host by means of 'nutritional immunity' and directly inhibit the growth of pathogens in the host. They interact with receptors on the cell surface to initiate inflammatory signal transduction, induce cytokine expression and participate in the inflammatory response and immune regulation. Furthermore, the increased content of these proteins during the pathological process makes them useful as disease markers for screening and detecting related diseases. This article summarizes the structure and function of the proteins S100A8, S100A9 and S100A12 and lays the foundation for further understanding their roles in infection, immunity and inflammation, as well as their potential applications in the prevention and treatment of infectious diseases.
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Affiliation(s)
- Pengpeng Xia
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
| | - Xingduo Ji
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
| | - Li Yan
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
| | - Siqi Lian
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
| | - Ziyue Chen
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
| | - Yi Luo
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China
- International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou, China
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8
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Peet JJY, Phan AD, Oglesby AG, Nolan EM. Iron Sequestration by Murine Calprotectin Induces Starvation Responses in Pseudomonas aeruginosa. ACS Infect Dis 2024; 10:688-700. [PMID: 38261753 PMCID: PMC11273327 DOI: 10.1021/acsinfecdis.3c00539] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2024]
Abstract
Pathogen sensing by the mammalian host induces a pro-inflammatory response that involves release of the antimicrobial metal-sequestering protein calprotectin (CP, S100A8/S100A9 heterooligomer, MRP8/MRP14 heterooligomer) from neutrophils. Biochemical investigations on human CP (hCP) have informed the molecular basis of how this protein sequesters metal ions. Murine models of infection have provided invaluable insights into the ability of murine CP (mCP) to compete with bacterial pathogens for essential metal nutrients. Despite this extensive work, our knowledge of how mCP sequesters metals from bacterial pathogens and its impacts on bacterial physiology is limited. Moreover, whether mCP sequesters iron and induces iron-starvation responses in bacterial pathogens has not been evaluated. Here, we examine the ability of mCP to withhold iron from Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen that causes severe infections in immunocompromised individuals and cystic fibrosis patients. We demonstrate that mCP prevents iron uptake and induces iron-starvation responses in P. aeruginosa laboratory strains PA14 and PAO1 and the JSRI-1 clinical isolate from a cystic fibrosis patient. We also show that mCP prevents iron uptake and induces an iron-starvation response in the Gram-positive bacterial pathogen Staphylococcus aureus. The His6 site of mCP is the iron-sequestering site; it exhibits Ca(II)-dependent Fe(II) affinity and binds Fe(II) with subpicomolar affinity in the presence of excess Ca(II) ions. This work is important for understanding the structure, function, and physiological consequences of mCP and how the mammalian host and bacterial pathogens compete for essential metal nutrients.
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Affiliation(s)
- Janet J. Y. Peet
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Angelica D. Phan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Amanda G. Oglesby
- School of Pharmacy, Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD, 21201, USA
- School of Medicine, Department of Microbiology and Immunology, University of Maryland, Baltimore, MD, 21021, USA
| | - Elizabeth M. Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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9
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Killian MM, Brophy MB, Nolan EM, Brunold TC. Spectroscopic and computational investigations of Cobalt(II) binding to the innate immune protein human calprotectin. J Biol Inorg Chem 2024; 29:127-137. [PMID: 38233645 DOI: 10.1007/s00775-023-02034-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Accepted: 10/30/2023] [Indexed: 01/19/2024]
Abstract
Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8 kDa) and S100A9 (β subunit, 13.2 kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His3Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His3Asp and His6 sites of CP were further characterized by utilizing Co2+ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co2+-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His3Asp and His6 sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.
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Affiliation(s)
- Michelle M Killian
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
| | - Megan B Brophy
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Elizabeth M Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
| | - Thomas C Brunold
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
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Lewin GR, Kapur A, Cornforth DM, Duncan RP, Diggle FL, Moustafa DA, Harrison SA, Skaar EP, Chazin WJ, Goldberg JB, Bomberger JM, Whiteley M. Application of a quantitative framework to improve the accuracy of a bacterial infection model. Proc Natl Acad Sci U S A 2023; 120:e2221542120. [PMID: 37126703 PMCID: PMC10175807 DOI: 10.1073/pnas.2221542120] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Accepted: 04/07/2023] [Indexed: 05/03/2023] Open
Abstract
Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.
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Affiliation(s)
- Gina R. Lewin
- School of Biological Sciences and Center for Microbial Dynamics and Infection, Georgia Institute of Technology, Atlanta, GA30332
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
| | - Ananya Kapur
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Daniel M. Cornforth
- School of Biological Sciences and Center for Microbial Dynamics and Infection, Georgia Institute of Technology, Atlanta, GA30332
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
| | - Rebecca P. Duncan
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
- Department of Pediatrics, Division of Pulmonary, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, GA30322
| | - Frances L. Diggle
- School of Biological Sciences and Center for Microbial Dynamics and Infection, Georgia Institute of Technology, Atlanta, GA30332
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
| | - Dina A. Moustafa
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
- Department of Pediatrics, Division of Pulmonary, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, GA30322
| | - Simone A. Harrison
- Department of Biochemistry, Vanderbilt University, Nashville, TN37232
- Department of Chemistry, Vanderbilt University, Nashville, TN37232
- Center for Structural Biology, Vanderbilt University, Nashville, TN37232
| | - Eric P. Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN37232
| | - Walter J. Chazin
- Department of Biochemistry, Vanderbilt University, Nashville, TN37232
- Department of Chemistry, Vanderbilt University, Nashville, TN37232
- Center for Structural Biology, Vanderbilt University, Nashville, TN37232
| | - Joanna B. Goldberg
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
- Department of Pediatrics, Division of Pulmonary, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, GA30322
| | - Jennifer M. Bomberger
- Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA15219
| | - Marvin Whiteley
- School of Biological Sciences and Center for Microbial Dynamics and Infection, Georgia Institute of Technology, Atlanta, GA30332
- Emory-Children’s Cystic Fibrosis Center, Atlanta, GA30332
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11
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Pro-inflammatory protein S100A9 alters membrane organization by dispersing ordered domains. BIOCHIMICA ET BIOPHYSICA ACTA. BIOMEMBRANES 2023; 1865:184113. [PMID: 36567033 DOI: 10.1016/j.bbamem.2022.184113] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 12/06/2022] [Accepted: 12/16/2022] [Indexed: 12/24/2022]
Abstract
Pro-inflammatory, calcium-binding protein S100A9 is localized in the cytoplasm of many cells and regulates several intracellular and extracellular processes. S100A9 is involved in neuroinflammation associated with the pathogenesis of Alzheimer's disease (AD). The number of studies on the impact of S100A9 in co-aggregation processes with amyloid-like proteins is increasing. However, there is still a lack of data on how this protein interacts with lipid membranes. We employed atomic force microscopy (AFM), dynamic light scattering (DLS), and fluorescence measurements (Laurdan and Thioflavin-T) to study the interaction between protein and the membrane surface. We used lipid vesicles in bulk and planar tethered lipid bilayers as biomimetic membrane models. We demonstrated that the protein accumulates on negatively charged lipid bilayers but with no further loss of the bilayer's integrity. The most important result is that the initial adsorption and accumulation of apo-form of S100A9 on the lipid membrane surface is lipid phase-sensitive. The breaking down of raft-like and disappearance of gel-like domains indicate that protein incorporates into the hydrophobic part of the lipid bilayer. We observed the most noticeable loss of integrity in lipid bilayers constructed from a lipid mixture (brain total lipid extract). Understanding the function and interactions of these proteins in cellular environments might expand the development of new diagnostic and therapeutic approaches for AD or other related diseases.
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12
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Post-translational modifications on the metal-sequestering protein calprotectin. Biometals 2023:10.1007/s10534-023-00493-x. [PMID: 36826733 PMCID: PMC10393864 DOI: 10.1007/s10534-023-00493-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Accepted: 01/19/2023] [Indexed: 02/25/2023]
Abstract
Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant neutrophil protein that contributes to innate immunity by sequestering nutrient metal ions in the extracellular space. This process starves invading microbial pathogens of essential metal nutrients, which can inhibit growth and colonization. Over the past decade, fundamental and clinical studies have revealed that the S100A8 and S100A9 subunits of CP exhibit a variety of post-translational modifications (PTMs). This review summarizes PTMs on the CP subunits that have been detected and highlights two recent studies that evaluated the structural and functional consequences of methionine and cysteine oxidation on CP. Collectively, these investigations indicate that the molecular speciation of extracellular CP is complex and composed of multiple proteoforms. Moreover, PTMs may impact biological function and the lifetime of the protein. It is therefore important that post-translationally modified CP species receive consideration and integration into the current working model for how CP functions in nutritional immunity.
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13
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RAGE Inhibitors for Targeted Therapy of Cancer: A Comprehensive Review. Int J Mol Sci 2022; 24:ijms24010266. [PMID: 36613714 PMCID: PMC9820344 DOI: 10.3390/ijms24010266] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2022] [Revised: 10/28/2022] [Accepted: 12/16/2022] [Indexed: 12/28/2022] Open
Abstract
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin family that is overexpressed in several cancers. RAGE is highly expressed in the lung, and its expression increases proportionally at the site of inflammation. This receptor can bind a variety of ligands, including advanced glycation end products, high mobility group box 1, S100 proteins, adhesion molecules, complement components, advanced lipoxidation end products, lipopolysaccharides, and other molecules that mediate cellular responses related to acute and chronic inflammation. RAGE serves as an important node for the initiation and stimulation of cell stress and growth signaling mechanisms that promote carcinogenesis, tumor propagation, and metastatic potential. In this review, we discuss different aspects of RAGE and its prominent ligands implicated in cancer pathogenesis and describe current findings that provide insights into the significant role played by RAGE in cancer. Cancer development can be hindered by inhibiting the interaction of RAGE with its ligands, and this could provide an effective strategy for cancer treatment.
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14
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Chung YH, Volckaert BA, Steinmetz NF. Metastatic Colon Cancer Treatment Using S100A9-Targeted Cowpea Mosaic Virus Nanoparticles. Biomacromolecules 2022; 23:5127-5136. [PMID: 36375170 PMCID: PMC9772157 DOI: 10.1021/acs.biomac.2c00873] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Peritoneal metastases (PMs) occur due to the metastasis of gynecological and gastrointestinal cancers such as ovarian, colon, pancreatic, or gastric tumors. PM outgrowth is often fatal, and patients with PMs have a median survival of 6 months. Cowpea mosaic virus (CPMV) has been shown, when injected intratumorally, to act as an immunomodulator reversing the immunosuppressive tumor microenvironment, therefore turning cold tumors hot and priming systemic antitumor immunity. However, not all tumors are injectable, and PMs especially will require targeted treatments to direct CPMV toward the disseminated tumor nodules. Toward this goal, we designed and tested a CPMV nanoparticle targeted to S100A9, a key immune mediator for many cancer types indicated in cancer growth, invasiveness, and metastasis. Here, we chose to use an intraperitoneal (IP) colon cancer model, and analysis of IP gavage fluid demonstrates that S100A9 is upregulated following IP challenge. S100A9-targeted CPMV particles displaying peptide ligands specific for S100A9 homed to IP-disseminated tumors, and treatment led to improved survival and decreased tumor burden. Targeting CPMV to S100A9 improves preclinical outcomes and harbors the potential of utilizing CPMV for the treatment of IP-disseminated diseases.
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Affiliation(s)
- Young Hun Chung
- Department of Bioengineering and Moores Cancer Center, University of California, San Diego, La Jolla, California 92093-0021, United States
| | - Britney A. Volckaert
- Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093-0021, United States
| | - Nicole F. Steinmetz
- Corresponding Author: Nicole F. Steinmetz – Department of Bioengineering, Moores Cancer Center, Department of NanoEngineering, Department of Radiology, Institute for Materials Discovery and Design, Center for Nano-Immuno Engineering, and Center for Engineering in Cancer, University of California, San Diego, La Jolla, California 92093-0021, United States;
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15
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Interactions between S100A9 and Alpha-Synuclein: Insight from NMR Spectroscopy. Int J Mol Sci 2022; 23:ijms23126781. [PMID: 35743221 PMCID: PMC9224231 DOI: 10.3390/ijms23126781] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 06/14/2022] [Accepted: 06/15/2022] [Indexed: 12/14/2022] Open
Abstract
S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (α-syn), which is involved in Parkinson's disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution 19F and 2D 15N-1H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that α-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using 19F NMR spectroscopy.
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16
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Garcia V, Perera YR, Chazin WJ. A Structural Perspective on Calprotectin as a Ligand of Receptors Mediating Inflammation and Potential Drug Target. Biomolecules 2022; 12:519. [PMID: 35454108 PMCID: PMC9026754 DOI: 10.3390/biom12040519] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Revised: 03/21/2022] [Accepted: 03/25/2022] [Indexed: 01/11/2023] Open
Abstract
Calprotectin, a heterodimer of S100A8 and S100A9 EF-hand calcium-binding proteins, is an integral part of the innate immune response. Calprotectin (CP) serves as a ligand for several pattern recognition cell surface receptors including the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and cluster of differentiation 33 (CD33). The receptors initiate kinase signaling cascades that activate inflammation through the NF-kB pathway. Receptor activation by CP leads to upregulation of both receptor and ligand, a positive feedback loop associated with specific chronic inflammatory syndromes. Hence, CP and its two constituent homodimers have been viewed as potential targets to suppress certain chronic inflammation pathologies. A variety of inhibitors of CP and other S100 proteins have been investigated for more than 30 years, but no candidates have advanced significantly into clinical trials. Here, current knowledge of the interactions of CP with its receptors is reviewed along with recent progress towards the development of CP-directed chemotherapeutics.
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Affiliation(s)
- Velia Garcia
- Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA;
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37240, USA;
| | - Yasiru Randika Perera
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37240, USA;
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37240, USA
| | - Walter Jacob Chazin
- Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA;
- Center for Structural Biology, Vanderbilt University, Nashville, TN 37240, USA;
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37240, USA
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17
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TdfH selectively binds metal-loaded tetrameric calprotectin for zinc import. Commun Biol 2022; 5:103. [PMID: 35102276 PMCID: PMC8803948 DOI: 10.1038/s42003-022-03039-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2021] [Accepted: 01/06/2022] [Indexed: 01/31/2023] Open
Abstract
To combat nutritional immunity, N. gonorrhoeae has evolved systems to hijack zinc and other metals directly from host metal-binding proteins such as calprotectin (CP). Here, we report the 6.1 Å cryoEM structure of the gonococcal surface receptor TdfH in complex with a zinc-bound CP tetramer. We further show that TdfH can also interact with CP in the presence of copper and manganese, but not with cobalt. The human defense mechanism against pathogens involves limiting essential nutrients, such as zinc which is countered by surface receptor TdfH pirating zinc from human calprotectin. The TdfH cryo-EM structure from N. gonorrhoeae in complex with a calprotectin tetramer provides insights into the protein-protein interaction and the role of metal ions.
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18
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Monteith AJ, Miller JM, Williams JM, Voss K, Rathmell JC, Crofford LJ, Skaar EP. Altered Mitochondrial Homeostasis during Systemic Lupus Erythematosus Impairs Neutrophil Extracellular Trap Formation Rendering Neutrophils Ineffective at Combating Staphylococcus aureus. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2022; 208:454-463. [PMID: 34930781 PMCID: PMC8761356 DOI: 10.4049/jimmunol.2100752] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Accepted: 11/09/2021] [Indexed: 01/17/2023]
Abstract
Inflammation involves a delicate balance between pathogen clearance and limiting host tissue damage, and perturbations in this equilibrium promote disease. Patients suffering from autoimmune diseases, such as systemic lupus erythematosus (SLE), have higher levels of serum S100A9 protein and increased risk for infection. S100A9 is highly abundant within neutrophils and modulates antimicrobial activity in response to bacterial pathogens. We reasoned that increased serum S100A9 in SLE patients reflects accumulation of S100A9 protein in neutrophils and may indicate altered neutrophil function. In this study, we demonstrate elevated S100A9 protein within neutrophils from SLE patients, and MRL/lpr mice associates with lower mitochondrial superoxide, decreased suicidal neutrophil extracellular trap formation, and increased susceptibility to Staphylococcus aureus infection. Furthermore, increasing mitochondrial superoxide production restored the antibacterial activity of MRL/lpr neutrophils in response to S. aureus These results demonstrate that accumulation of intracellular S100A9 associates with impaired mitochondrial homeostasis, thereby rendering SLE neutrophils inherently less bactericidal.
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Affiliation(s)
- Andrew J Monteith
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN
| | - Jeanette M Miller
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN
| | - Jonathan M Williams
- Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN
- Division of Rheumatology and Immunology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN; and
| | - Kelsey Voss
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN
| | - Jeffrey C Rathmell
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN
- Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN
- Division of Rheumatology and Immunology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN; and
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN
| | - Leslie J Crofford
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN
- Division of Rheumatology and Immunology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN; and
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN
| | - Eric P Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN;
- Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN
- Division of Rheumatology and Immunology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN; and
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN
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19
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Metal sequestration by S100 proteins in chemically diverse environments. Trends Microbiol 2022; 30:654-664. [DOI: 10.1016/j.tim.2021.12.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Revised: 12/07/2021] [Accepted: 12/08/2021] [Indexed: 12/22/2022]
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20
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Mitochondrial calcium uniporter affects neutrophil bactericidal activity during Staphylococcus aureus infection. Infect Immun 2021; 90:e0055121. [PMID: 34871043 DOI: 10.1128/iai.00551-21] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1-/-) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1-/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus. Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1-/- neutrophils alone are not more antibacterial towards S. aureus, but rather enhanced suicidal NETosis by MICU1-/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1-/- neutrophils in the heart produced higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.
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21
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Silvers R, Stephan JR, Griffin RG, Nolan EM. Molecular Basis of Ca(II)-Induced Tetramerization and Transition-Metal Sequestration in Human Calprotectin. J Am Chem Soc 2021; 143:18073-18090. [PMID: 34699194 PMCID: PMC8643164 DOI: 10.1021/jacs.1c06402] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ)2 heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or "inactive" state and a tetramerization-competent or "active" state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ)2 heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His3Asp and His6 metal-binding sites. Ca(II) binding to the noncanonical EF-hand of S100A9 positions (A9)D30 and organizes the His3Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix αIV of S100A9, which stabilize the α-helicity at positions H91 and H95 and thereby organize the functionally versatile His6 site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.
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Affiliation(s)
- Robert Silvers
- Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306, USA
- Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA
| | - Jules R. Stephan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Robert G. Griffin
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
- Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Elizabeth M. Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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22
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Chung YH, Park J, Cai H, Steinmetz NF. S100A9-Targeted Cowpea Mosaic Virus as a Prophylactic and Therapeutic Immunotherapy against Metastatic Breast Cancer and Melanoma. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:e2101796. [PMID: 34519180 PMCID: PMC8564454 DOI: 10.1002/advs.202101796] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/29/2021] [Revised: 07/05/2021] [Indexed: 05/05/2023]
Abstract
Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease.
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Affiliation(s)
- Young Hun Chung
- Department of BioengineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
| | - Jooneon Park
- Department of NanoengineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
| | - Hui Cai
- Department of NanoengineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
| | - Nicole F. Steinmetz
- Department of BioengineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
- Department of NanoengineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
- Department of RadiologyUniversity of CaliforniaLa JollaSan DiegoCAUSA
- Institute for Materials Discovery and DesignUniversity of CaliforniaLa JollaSan DiegoCAUSA
- Center for Nano‐ImmunoEngineeringUniversity of CaliforniaLa JollaSan DiegoCAUSA
- Moores Cancer CenterUniversity of CaliforniaLa JollaSan DiegoCAUSA
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23
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Singh S, Diwaker A, Singh BP, Singh RK. Nutritional Immunity, Zinc Sufficiency, and COVID-19 Mortality in Socially Similar European Populations. Front Immunol 2021; 12:699389. [PMID: 34603280 PMCID: PMC8484327 DOI: 10.3389/fimmu.2021.699389] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Accepted: 08/18/2021] [Indexed: 11/13/2022] Open
Abstract
The impact of zinc (Zn) sufficiency/supplementation on COVID-19-associated mortality and incidence (SARS-CoV-2 infections) remains unknown. During an infection, the levels of free Zn are reduced as part of “nutritional immunity” to limit the growth and replication of pathogen and the ensuing inflammatory damage. Considering its key role in immune competency and frequently recorded deficiency in large sections of different populations, Zn has been prescribed for both prophylactic and therapeutic purposes in COVID-19 without any corroborating evidence for its protective role. Multiple trials are underway evaluating the effect of Zn supplementation on COVID-19 outcome in patients getting standard of care treatment. However, the trial designs presumably lack the power to identify negative effects of Zn supplementation, especially in the vulnerable groups of elderly and patients with comorbidities (contributing 9 out of 10 deaths; up to >8,000-fold higher mortality). In this study, we have analyzed COVID-19 mortality and incidence (case) data from 23 socially similar European populations with comparable confounders (population: 522.47 million; experiencing up to >150-fold difference in death rates) and at the matching stage of the pandemic (March 12 to June 26, 2020; first wave of COVID-19 incidence and mortality). Our results suggest a positive correlation between populations’ Zn-sufficiency status and COVID-19 mortality [r (23): 0.7893–0.6849, p-value < 0.0003] as well as incidence [r (23):0.8084–0.5658; p-value < 0.005]. The observed association is contrary to what would be expected if Zn sufficiency was protective in COVID-19. Thus, controlled trials or retrospective analyses of the adverse event patients’ data should be undertaken to correctly guide the practice of Zn supplementation in COVID-19.
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Affiliation(s)
- Samer Singh
- Centre of Experimental Medicine & Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
| | - Amita Diwaker
- Department of Obstetrics and Gynecology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
| | - Brijesh P Singh
- Department of Statistics, Institute of Science, Banaras Hindu University, Varanasi, India
| | - Rakesh K Singh
- Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India
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24
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Jukic A, Bakiri L, Wagner EF, Tilg H, Adolph TE. Calprotectin: from biomarker to biological function. Gut 2021; 70:1978-1988. [PMID: 34145045 PMCID: PMC8458070 DOI: 10.1136/gutjnl-2021-324855] [Citation(s) in RCA: 255] [Impact Index Per Article: 63.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Accepted: 06/02/2021] [Indexed: 12/15/2022]
Abstract
The incidence of inflammatory bowel diseases (IBD) emerged with Westernisation of dietary habits worldwide. Crohn's disease and ulcerative colitis are chronic debilitating conditions that afflict individuals with substantial morbidity and challenge healthcare systems across the globe. Since identification and characterisation of calprotectin (CP) in the 1980s, faecal CP emerged as significantly validated, non-invasive biomarker that allows evaluation of gut inflammation. Faecal CP discriminates between inflammatory and non-inflammatory diseases of the gut and portraits the disease course of human IBD. Recent studies revealed insights into biological functions of the CP subunits S100A8 and S100A9 during orchestration of an inflammatory response at mucosal surfaces across organ systems. In this review, we summarise longitudinal evidence for the evolution of CP from biomarker to rheostat of mucosal inflammation and suggest an algorithm for the interpretation of faecal CP in daily clinical practice. We propose that mechanistic insights into the biological function of CP in the gut and beyond may facilitate interpretation of current assays and guide patient-tailored medical therapy in IBD, a concept warranting controlled clinical trials.
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Affiliation(s)
- Almina Jukic
- Department of Internal Medicine I, Gastroenterology, Hepatology, Endocrinology & Metabolism, Medical University of Innsbruck, Innsbruck, Austria
| | - Latifa Bakiri
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
| | - Erwin F Wagner
- Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
- Department of Dermatology, Medical University of Vienna, Vienna, Austria
| | - Herbert Tilg
- Department of Internal Medicine I, Gastroenterology, Hepatology, Endocrinology & Metabolism, Medical University of Innsbruck, Innsbruck, Austria
| | - Timon E Adolph
- Department of Internal Medicine I, Gastroenterology, Hepatology, Endocrinology & Metabolism, Medical University of Innsbruck, Innsbruck, Austria
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25
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Monteith AJ, Miller JM, Maxwell CN, Chazin WJ, Skaar EP. Neutrophil extracellular traps enhance macrophage killing of bacterial pathogens. SCIENCE ADVANCES 2021; 7:eabj2101. [PMID: 34516771 PMCID: PMC8442908 DOI: 10.1126/sciadv.abj2101] [Citation(s) in RCA: 86] [Impact Index Per Article: 21.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Accepted: 07/20/2021] [Indexed: 05/16/2023]
Abstract
Neutrophils and macrophages are critical to the innate immune response, but cooperative mechanisms used by these cells to combat extracellular pathogens are not well understood. This study reveals that S100A9-deficient neutrophils produce higher levels of mitochondrial superoxide in response to Staphylococcus aureus and, as a result, form neutrophil extracellular traps (suicidal NETosis). Increased suicidal NETosis does not improve neutrophil killing of S. aureus in isolation but augments macrophage killing. NET formation enhances antibacterial activity by increasing phagocytosis by macrophages and by transferring neutrophil-specific antimicrobial peptides to them. Similar results were observed in response to other phylogenetically distinct bacterial pathogens including Streptococcus pneumoniae and Pseudomonas aeruginosa, implicating this as an immune defense mechanism that broadly enhances antibacterial activity. These results demonstrate that achieving maximal bactericidal activity through NET formation requires macrophages and that accelerated and more robust suicidal NETosis makes neutrophils adept at increasing antibacterial activity, especially when A9 deficient.
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Affiliation(s)
- Andrew J. Monteith
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jeanette M. Miller
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
| | - C. Noel Maxwell
- Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
- Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
| | - Walter J. Chazin
- Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
- Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
- Department of Chemistry, Vanderbilt University, Nashville, TN, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Eric P. Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA
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S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation. Int J Mol Sci 2021; 22:ijms22157972. [PMID: 34360737 PMCID: PMC8348003 DOI: 10.3390/ijms22157972] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Revised: 07/18/2021] [Accepted: 07/19/2021] [Indexed: 12/13/2022] Open
Abstract
The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson's disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution's ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.
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Singh H, Rai V, Agrawal DK. Discerning the promising binding sites of S100/calgranulins and their therapeutic potential in atherosclerosis. Expert Opin Ther Pat 2021; 31:1045-1057. [PMID: 34056993 DOI: 10.1080/13543776.2021.1937122] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
INTRODUCTION Atherosclerosis is a chronic inflammatory disease in which the members of S100 family proteins (calgranulins) bind with their receptors, particularly receptor for advanced glycation end products (RAGE) and toll-like receptor-4 (TLR-4) and play a key role in the pathogenesis and progression of disease. Thus, these proteins could be considered as potential biomarkers and therapeutic targets in the treatment of atherosclerotic inflammation. AREAS COVERED This review summarizes the pathology of S100A8, S100A9, and S100A12 in the development of atherosclerosis and reveals key structural features of these proteins which are potentially critical in their pathological effects. This article focuses on the translational significance of antagonizing these proteins by using small molecules in patent literature, clinical and preclinical studies and also discusses future approaches that could be employed to block these proteins in the treatment of atherosclerosis. EXPERT OPINION Based on the critical role of S100/calgranulins in the regulation of atherosclerosis, these proteins are potential targets to develop better therapeutic options in the treatment of inflammatory diseases. However, further research is still needed to clarify their exact molecular mechanism by analyzing their detailed structural features that can expedite future research to develop novel therapeutics against these proteins to treat atherosclerotic inflammation.
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Affiliation(s)
- Harbinder Singh
- Department of Translational Research, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, California, USA
| | - Vikrant Rai
- Department of Translational Research, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, California, USA
| | - Devendra K Agrawal
- Department of Translational Research, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, California, USA
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28
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Park J, Wen AM, Gao H, Shin MD, Simon DI, Wang Y, Steinmetz NF. Designing S100A9-Targeted Plant Virus Nanoparticles to Target Deep Vein Thrombosis. Biomacromolecules 2021; 22:2582-2594. [PMID: 34060817 DOI: 10.1021/acs.biomac.1c00303] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Thromboembolic conditions are a leading cause of death worldwide, and deep vein thrombosis (DVT), or occlusive venous clot formation, is a critical and rising problem that contributes to damage of vital organs, long-term complications, and life-threatening conditions such as pulmonary embolism. Early diagnosis and treatment are correlated to better prognosis. However, current technologies in these areas, such as ultrasonography for diagnostics and anticoagulants for treatment, are limited in terms of their accuracy and therapeutic windows. In this work, we investigated targeting myeloid related protein 14 (MRP-14, also known as S100A9) using plant virus-based nanoparticle carriers as a means to achieve tissue specificity aiding prognosis and therapeutic intervention. We used a combinatorial peptide library screen to identify peptide ligands that bind MRP-14. Candidates were selected and formulated as nanoparticles by using cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV). Intravascular delivery of our MRP-14-targeted nanoparticles in a murine model of DVT resulted in enhanced accumulation in the thrombi and reduced thrombus size, suggesting application of nanoparticles for molecular targeting of MRP-14 could be a promising direction for improving DVT diagnostics, therapeutics, and therefore prognosis.
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Affiliation(s)
- Jooneon Park
- Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093, United States
| | - Amy M Wen
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Huiyun Gao
- Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Matthew D Shin
- Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093, United States
| | - Daniel I Simon
- Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Yunmei Wang
- Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States
| | - Nicole F Steinmetz
- Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093, United States.,Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, United States.,Department of Radiology, University of California, San Diego, La Jolla, California 92093, United States.,Center for Nano-ImmunoEngineering, University of California, San Diego, La Jolla, California 92093, United States.,Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, United States.,Institute for Materials Discovery and Design, University of California, San Diego, La Jolla, California 92093, United States
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29
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Divalent cations influence the dimerization mode of murine S100A9 protein by modulating its disulfide bond pattern. J Struct Biol 2020; 213:107689. [PMID: 33359632 DOI: 10.1016/j.jsb.2020.107689] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 12/08/2020] [Accepted: 12/14/2020] [Indexed: 12/19/2022]
Abstract
S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro- and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein.
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30
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Zhong X, Xie F, Chen L, Liu Z, Wang Q. S100A8 and S100A9 promote endothelial cell activation through the RAGE‑mediated mammalian target of rapamycin complex 2 pathway. Mol Med Rep 2020; 22:5293-5303. [PMID: 33174028 PMCID: PMC7646991 DOI: 10.3892/mmr.2020.11595] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Accepted: 08/21/2020] [Indexed: 02/06/2023] Open
Abstract
S100 calcium binding protein A8 (S100A8) and A9 (S100A9) belong to the S100 family of calcium-binding proteins and have important roles in inflammation. They increase endothelial cell proliferation, thereby affecting inflammation, angiogenesis and tumorigenesis. However, the mechanism of action of S100A8/9 in endothelial cells needs further study. Therefore, the present study sought to investigate the effects of S100A8/9 on the proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and their mechanism of action. The viability of HUVECs was determined through a Cell Counting Kit-8 assay. The effect of S100A8/9 on the proliferation of HUVECs was detected by flow cytometry. Migration was evaluated by a Transwell migration assay. Apoptosis was evaluated by Annexin V-FITC and PI staining via flow cytometry. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were performed to evaluate the activation of the phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mTOR pathway and mTOR complex 2 (mTORC2). We previously confirmed that S100A8/9 were consistently overexpressed at 1 and 7 days post-surgery in a rabbit vein graft model, which is the period when apoptosis changes to proliferation in neointimal hyperplasia. In the present study, proliferation, viability and migration were increased after treating HUVECs with S100A8/9. S100A8/9 stimulated the PI3K/Akt/mTOR pathway and mTORC2, which was significantly suppressed by a receptor for advanced glycation end products (RAGE)-blocking antibody. Furthermore, depleting expression of RAGE or mTORC2 protein components (rapamycin-insensitive companion of mTOR) by small interfering RNA was found to reduce the cell viability, migration and angiogenesis of S100A8/9-treated HUVECs. The development of neointimal hyperplasia is a complex process initiated by damage to endothelial cells. In conclusion, S100A8/9 has an important role in intimal hyperplasia by promoting cell growth and angiogenesis via RAGE signaling and activation of mTORC2.
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Affiliation(s)
- Xiang Zhong
- Department of Cardiac Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
| | - Fengwen Xie
- Department of Cardiac Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
| | - Li Chen
- Department of Ultrasound, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
| | - Zhixing Liu
- Department of Ultrasound, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
| | - Qun Wang
- Department of Cardiac Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
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31
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Adhikari J, Stephan JR, Rempel DL, Nolan EM, Gross ML. Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry. J Am Chem Soc 2020; 142:13372-13383. [PMID: 32589841 DOI: 10.1021/jacs.9b11950] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.
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Affiliation(s)
- Jagat Adhikari
- Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States
| | - Jules R Stephan
- Department of Chemistry, Massachusetts Institute of Technology, Boston, Massachusetts 02139, United States
| | - Don L Rempel
- Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States
| | - Elizabeth M Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Boston, Massachusetts 02139, United States
| | - Michael L Gross
- Department of Chemistry, Washington University at St. Louis, St. Louis, Missouri 63130, United States
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32
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Li K, Chen G, Luo H, Li J, Liu A, Yang C, Wang J, Xu J, Gao S, Chen P, Jiang Y. MRP8/14 mediates macrophage efferocytosis through RAGE and Gas6/MFG-E8, and induces polarization via TLR4-dependent pathway. J Cell Physiol 2020; 236:1375-1390. [PMID: 33128793 DOI: 10.1002/jcp.29944] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2020] [Revised: 05/25/2020] [Accepted: 07/01/2020] [Indexed: 12/16/2022]
Abstract
Myeloid-related protein 8/14 (MRP8/14) participates in various inflammatory responses, however, its effect on macrophage efferocytosis remains unclear. Here, we demonstrate that MRP8/14 significantly inhibits the efferocytosis of apoptotic thymocytes by mouse bone marrow-derived macrophages (BMDMs), which later proves to be associated with the receptor for advanced glycation end products (RAGE) or for reducing the expression of growth arrest-specific protein 6 and milk fat globule epidermal growth factor 8, independent of RAGE. Furthermore, MRP8/14 promotes polarization of BMDMs from the M2 - to M1 -like phenotype by upregulating expression of M1 -related surface receptor proteins and signature M1 -marker genes and by downregulating signature M2 -marker gene expression, which depends on Toll-like receptor 4 and p38 mitogen-activated protein kinase/nuclear factor κB pathways. Thus, we report a significant inhibitory effect of MRP8/14 on macrophage efferocytosis and MRP8/14-mediated phenotypic polarization, which may be helpful in developing novel therapeutic strategies leading to inflammation resolution.
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Affiliation(s)
- Kangxin Li
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Guiming Chen
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Haihua Luo
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Jianhang Li
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Aihua Liu
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Chen Yang
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Juan Wang
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Jia Xu
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Shenghan Gao
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Peng Chen
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Yong Jiang
- Guangdong Provincial Key Laboratory of Proteomics, State Key Laboratory of Organ Failure Research, Department of Pathophysiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
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33
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Rosen T, Nolan EM. Metal Sequestration and Antimicrobial Activity of Human Calprotectin Are pH-Dependent. Biochemistry 2020; 59:2468-2478. [PMID: 32491853 DOI: 10.1021/acs.biochem.0c00359] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Human calprotectin (CP, S100A8/S100A9 oligomer) is an abundant innate immune protein that sequesters transition metal ions in the extracellular space to limit nutrient availability and the growth of invading microbial pathogens. Our current understanding of the metal-sequestering ability of CP is based on biochemical and functional studies performed at neutral or near-neutral pH. Nevertheless, CP can be present throughout the human body and is expressed at infection and inflammation sites that tend to be acidic. Here, we evaluate the metal binding and antimicrobial properties of CP in the pH range of 5.0-7.0. We show that Ca(II)-induced tetramerization, an important process for the extracellular functions of CP, is perturbed by acidic conditions. Moreover, a low pH impairs the antimicrobial activity of CP against some bacterial pathogens, including Staphylococcus aureus and Salmonella enterica serovar Typhimurium. At a mildly acidic pH, CP loses the ability to deplete Mn from microbial growth medium, indicating that Mn(II) sequestration is attenuated under acidic conditions. Evaluation of the Mn(II) binding properties of CP at pH 5.0-7.0 indicates that mildly acidic conditions decrease the Mn(II) binding affinity of the His6 site. Lastly, CP is less effective at preventing capture of Mn(II) by the bacterial solute-binding proteins MntC and PsaA at low pH. These results indicate that acidic conditions compromise the ability of CP to sequester Mn(II) and starve microbial pathogens of this nutrient. This work highlights the importance of considering the local pH of biological sites when describing the interplay between CP and microbes in host-pathogen interactions.
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Affiliation(s)
- Tomer Rosen
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
| | - Elizabeth M Nolan
- Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
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34
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Samonig L, Loipetzberger A, Blöchl C, Rurik M, Kohlbacher O, Aberger F, Huber CG. Proteins and Molecular Pathways Relevant for the Malignant Properties of Tumor-Initiating Pancreatic Cancer Cells. Cells 2020; 9:E1397. [PMID: 32503348 PMCID: PMC7349116 DOI: 10.3390/cells9061397] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 05/26/2020] [Accepted: 05/30/2020] [Indexed: 12/29/2022] Open
Abstract
Cancer stem cells (CSCs), a small subset of the tumor bulk with highly malignant properties, are deemed responsible for tumor initiation, growth, metastasis, and relapse. In order to reveal molecular markers and determinants of their tumor-initiating properties, we enriched rare stem-like pancreatic tumor-initiating cells (TICs) by harnessing their clonogenic growth capacity in three-dimensional multicellular spheroid cultures. We compared pancreatic TICs isolated from three-dimensional tumor spheroid cultures with nontumor-initiating cells (non-TICs) enriched in planar cultures. Employing differential proteomics (PTX), we identified more than 400 proteins with significantly different expression in pancreatic TICs and the non-TIC population. By combining the unbiased PTX with mRNA expression analysis and literature-based predictions of pro-malignant functions, we nominated the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14) as well as galactin-3-binding protein LGALS3BP (MAC-2-BP) as putative determinants of pancreatic TICs. In silico pathway analysis followed by candidate-based RNA interference mediated loss-of-function analysis revealed a critical role of S100A8, S100A9, and LGALS3BP as molecular determinants of TIC proliferation, migration, and in vivo tumor growth. Our study highlights the power of combining unbiased proteomics with focused gene expression and functional analyses for the identification of novel key regulators of TICs, an approach that warrants further application to identify proteins and pathways amenable to drug targeting.
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Affiliation(s)
- Lisa Samonig
- Department of Biosciences, Bioanalytical Research Labs, University of Salzburg, A-5020 Salzburg, Austria; (L.S.); (C.B.)
| | - Andrea Loipetzberger
- Department of Biosciences, Cancer Cluster Salzburg, Molecular Cancer and Stem Cell Research, University of Salzburg, A-5020 Salzburg, Austria;
| | - Constantin Blöchl
- Department of Biosciences, Bioanalytical Research Labs, University of Salzburg, A-5020 Salzburg, Austria; (L.S.); (C.B.)
| | - Marc Rurik
- Institute for Bioinformatics and Medical Informatics, University of Tübingen, Sand 14, 72076 Tübingen, Germany; (M.R.); (O.K.)
| | - Oliver Kohlbacher
- Institute for Bioinformatics and Medical Informatics, University of Tübingen, Sand 14, 72076 Tübingen, Germany; (M.R.); (O.K.)
- Biomolecular Interactions, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tübingen, Germany
- Institute for Translational Bioinformatics, University Hospital Tübingen, Hoppe-Seyler-Str. 9, 72076 Tübingen, Germany
- Quantitative Biology Center, University of Tübingen, Auf der Morgenstelle 10, 72076 Tübingen, Germany
| | - Fritz Aberger
- Department of Biosciences, Cancer Cluster Salzburg, Molecular Cancer and Stem Cell Research, University of Salzburg, A-5020 Salzburg, Austria;
- Department of Biosciences, Cancer Cluster Salzburg, University of Salzburg, A-5020 Salzburg, Austria
| | - Christian G. Huber
- Department of Biosciences, Bioanalytical Research Labs, University of Salzburg, A-5020 Salzburg, Austria; (L.S.); (C.B.)
- Department of Biosciences, Cancer Cluster Salzburg, University of Salzburg, A-5020 Salzburg, Austria
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Grim KP, Radin JN, Solórzano PKP, Morey JR, Frye KA, Ganio K, Neville SL, McDevitt CA, Kehl-Fie TE. Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity. J Bacteriol 2020; 202:e00014-20. [PMID: 32071094 PMCID: PMC7148132 DOI: 10.1128/jb.00014-20] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2020] [Accepted: 02/10/2020] [Indexed: 02/07/2023] Open
Abstract
The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant.IMPORTANCEStaphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.
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Affiliation(s)
- Kyle P Grim
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Jana N Radin
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Paola K Párraga Solórzano
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
- Departmento de Ciencias de la Vida, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador
| | - Jacqueline R Morey
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Katie A Frye
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
| | - Katherine Ganio
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Stephanie L Neville
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Christopher A McDevitt
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
| | - Thomas E Kehl-Fie
- Department of Microbiology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA
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36
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S100 proteins in atherosclerosis. Clin Chim Acta 2020; 502:293-304. [DOI: 10.1016/j.cca.2019.11.019] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2019] [Revised: 11/11/2019] [Accepted: 11/14/2019] [Indexed: 02/07/2023]
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37
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Dubois C, Payen D, Simon S, Junot C, Fenaille F, Morel N, Becher F. Top-Down and Bottom-Up Proteomics of Circulating S100A8/S100A9 in Plasma of Septic Shock Patients. J Proteome Res 2020; 19:914-925. [PMID: 31913637 DOI: 10.1021/acs.jproteome.9b00690] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Well-characterized prognostic biomarkers and reliable quantitative methods are key in sepsis management. Among damage-associated molecular patterns, S100A8/S100A9 complexes are reported to be markers for injured cells and to improve the prediction of death in septic shock patients. In view of the structural diversity observed for the intracellular forms, insight into circulating complexes and proteoforms is required to establish prognostic biomarkers. Here, we developed top-down and bottom-up proteomics to characterize the association of S100A8 and S100A9 in complexes and major circulating proteoforms. An antibody-free method was developed for absolute quantification of S100A8/S100A9 in a cohort of 49 patients to evaluate the prognostic value on the first day after admission for septic shock. The predominant circulating forms identified by top-down proteomics were S100A8, mono-oxidized S100A8, truncated acetylated S100A9, and S-nitrosylated S100A9. S100A8, truncated acetylated S100A9, and mono-oxidized S100A8 discriminated between survivors and nonsurvivors, along with total S100A8/S100A9 measured by the antibody-free bottom-up method. Overall, new insights into circulating S100A8/S100A9 and confirmation of its prognostic value in septic shock are crucial in qualification of this biomarker. Also, the simple antibody-free assay would support the harmonization of S100A8/S100A9 measurements.
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Affiliation(s)
- Christelle Dubois
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
| | - Didier Payen
- Université Paris 7 Cité Sorbonne, UMR INSERM 1160 , 110 Avenue de Verdun , Paris 75010 , France.,Department of Anesthesiology & Critical Care , Hôpital Lariboisière, Assistance Publique-Hôpitaux de Paris (AP-HP) , Paris 75010 , France
| | - Stéphanie Simon
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
| | - Christophe Junot
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
| | - François Fenaille
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
| | - Nathalie Morel
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
| | - François Becher
- Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France
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Ishizuka K, Fujii W, Azuma N, Mizobuchi H, Morimoto A, Sanjoba C, Matsumoto Y, Goto Y. Pathological roles of MRP14 in anemia and splenomegaly during experimental visceral leishmaniasis. PLoS Negl Trop Dis 2020; 14:e0008020. [PMID: 31961866 PMCID: PMC6994150 DOI: 10.1371/journal.pntd.0008020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Revised: 01/31/2020] [Accepted: 01/01/2020] [Indexed: 01/03/2023] Open
Abstract
Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL.
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Affiliation(s)
- Kanna Ishizuka
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Wataru Fujii
- Laboratory of Applied Genetics, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Natsuho Azuma
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Haruka Mizobuchi
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Ayako Morimoto
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Chizu Sanjoba
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Yoshitsugu Matsumoto
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
| | - Yasuyuki Goto
- Laboratory of Molecular Immunology, Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
- * E-mail:
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Lonergan ZR, Skaar EP. Nutrient Zinc at the Host-Pathogen Interface. Trends Biochem Sci 2019; 44:1041-1056. [PMID: 31326221 PMCID: PMC6864270 DOI: 10.1016/j.tibs.2019.06.010] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Revised: 06/13/2019] [Accepted: 06/25/2019] [Indexed: 12/12/2022]
Abstract
Zinc is an essential cofactor required for life and, as such, mechanisms exist for its homeostatic maintenance in biological systems. Despite the evolutionary distance between vertebrates and microbial life, there are parallel mechanisms to balance the essentiality of zinc with its inherent toxicity. Vertebrates regulate zinc homeostasis through a complex network of metal transporters and buffering systems that respond to changes in nutritional zinc availability or inflammation. Fine-tuning of this network becomes crucial during infections, where host nutritional immunity attempts to limit zinc availability to pathogens. However, accumulating evidence demonstrates that pathogens have evolved mechanisms to subvert host-mediated zinc withholding, and these metal homeostasis systems are important for survival within the host. We discuss here the mechanisms of vertebrate and bacterial zinc homeostasis and mobilization, as well as recent developments in our understanding of microbial zinc acquisition.
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Affiliation(s)
- Zachery R Lonergan
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Microbe-Host Interactions Training Program, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Eric P Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA.
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Lopez CA, Beavers WN, Weiss A, Knippel RJ, Zackular JP, Chazin W, Skaar EP. The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation. mBio 2019; 10:e02289-19. [PMID: 31744916 PMCID: PMC6867894 DOI: 10.1128/mbio.02289-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2019] [Accepted: 10/09/2019] [Indexed: 02/06/2023] Open
Abstract
The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.
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Affiliation(s)
- Christopher A Lopez
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - William N Beavers
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Andy Weiss
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Reece J Knippel
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Joseph P Zackular
- Department of Pathology and Laboratory Medicine, Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
- Division of Protective Immunity, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Walter Chazin
- Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
- Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA
| | - Eric P Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
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Hadley RC, Gagnon DM, Ozarowski A, Britt RD, Nolan EM. Murine Calprotectin Coordinates Mn(II) at a Hexahistidine Site with Ca(II)-Dependent Affinity. Inorg Chem 2019; 58:13578-13590. [PMID: 31145609 DOI: 10.1021/acs.inorgchem.9b00763] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Manganese is an essential metal ion that bacterial pathogens need to acquire from the vertebrate host during infection. In the mammalian nutritional immunity strategy to combat bacterial infection, the host restricts bacterial access to Mn(II) by sequestering this metal nutrient using the protein calprotectin (CP). The role of murine calprotectin (mCP) in Mn(II) sequestration has been demonstrated in vivo, but the molecular basis of this function has not been evaluated. Herein, biochemical assays and electron paramagnetic resonance (EPR) spectroscopy are employed to characterize the Mn(II) binding properties of mCP. We report that mCP has one high-affinity Mn(II) binding site. This site is a His6 site composed of His17 and His27 of mS100A8 and His92, His97, His105, and His107 of mS100A9. Similar to the human ortholog (hCP), Ca(II) binding to the EF-hand domains of mCP enhances the Mn(II) affinity of the protein; however, this effect requires ≈10-fold more Ca(II) than was previously observed for hCP. Mn(II) coordination to the His6 site also promotes self-association of two mCP heterodimers to form a heterotetramer. Low-temperature X-band EPR spectroscopy revealed a nearly octahedral Mn(II) coordination sphere for the Mn(II)-His6 site characterized by the zero-field splitting parameters D = 525 MHz and E/D = 0.3. Further electron-nuclear double resonance studies with globally 15N-labeled mCP provided hyperfine couplings from the coordinating ε-nitrogen atoms of the His ligands (aiso = 4.3 MHz) as well as the distal δ-nitrogen atoms (aiso = 0.25 MHz). Mn(II) competition assays between mCP and two bacterial Mn(II) solute-binding proteins, staphylococcal MntC and streptococcal PsaA, showed that mCP outcompetes both proteins for Mn(II) under conditions of excess Ca(II). In total, this work provides the first coordination chemistry study of mCP and reveals striking similarities in the Mn(II) coordination sphere as well as notable differences in the Ca(II) sensitivity and oligomerization behavior between hCP and mCP.
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Affiliation(s)
- Rose C Hadley
- Department of Chemistry , Massachusetts Institute of Technology (MIT) , Cambridge , Massachusetts 02139 , United States
| | - Derek M Gagnon
- Department of Chemistry , University of California, Davis , Davis , California 95616 , United States
| | - Andrew Ozarowski
- National High Magnetic Field Laboratory , Florida State University , Tallahassee , Florida 32310 , United States
| | - R David Britt
- Department of Chemistry , University of California, Davis , Davis , California 95616 , United States
| | - Elizabeth M Nolan
- Department of Chemistry , Massachusetts Institute of Technology (MIT) , Cambridge , Massachusetts 02139 , United States
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Garcia V, Chazin WJ. A new approach to discovery of S100 protein heterodimers. FEBS J 2019; 286:1838-1840. [PMID: 31050191 DOI: 10.1111/febs.14851] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2019] [Accepted: 04/11/2019] [Indexed: 11/29/2022]
Abstract
S100 proteins are a sub-family of EF-hand calcium binding proteins that form very similar, unique dimer structures. Although the possibility has been known for many years, no systematic study of the formation of S100 protein heterodimers has been reported. Shaw and coworkers demonstrate the application of a new approach that provides an in-depth evaluation of the distribution of S100 protein homo- and heterodimer and show that the prevalence of heterodimers may be much higher than previously anticipated.
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Affiliation(s)
- Velia Garcia
- Departments of Biochemistry and Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
| | - Walter J Chazin
- Departments of Biochemistry and Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, TN, USA
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Hoskin TS, Crowther JM, Cheung J, Epton MJ, Sly PD, Elder PA, Dobson RCJ, Kettle AJ, Dickerhof N. Oxidative cross-linking of calprotectin occurs in vivo, altering its structure and susceptibility to proteolysis. Redox Biol 2019; 24:101202. [PMID: 31015146 PMCID: PMC6477633 DOI: 10.1016/j.redox.2019.101202] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2019] [Revised: 04/09/2019] [Accepted: 04/11/2019] [Indexed: 01/27/2023] Open
Abstract
Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9)2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function.
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Affiliation(s)
- Teagan S Hoskin
- Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand.
| | - Jennifer M Crowther
- Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
| | - Jeanette Cheung
- Canterbury Respiratory Research Group, Respiratory Services, Christchurch Hospital, Canterbury District Health Board, New Zealand
| | - Michael J Epton
- Canterbury Respiratory Research Group, Respiratory Services, Christchurch Hospital, Canterbury District Health Board, New Zealand
| | - Peter D Sly
- Child Health Research Centre, University of Queensland, Brisbane, Australia
| | - Peter A Elder
- Endocrinology and Steroid Laboratory, Canterbury Health Laboratories, New Zealand
| | - Renwick C J Dobson
- Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; Bio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
| | - Anthony J Kettle
- Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand
| | - Nina Dickerhof
- Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand
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Manganese Detoxification by MntE Is Critical for Resistance to Oxidative Stress and Virulence of Staphylococcus aureus. mBio 2019; 10:mBio.02915-18. [PMID: 30808698 PMCID: PMC6391924 DOI: 10.1128/mbio.02915-18] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate Staphylococcus aureus utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of mntE leads to a significant reduction in S. aureus resistance to oxidative stress and S. aureus-mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and S. aureus virulence. Therefore, this establishes MntE as a potential target for development of anti-S. aureus therapeutics. Manganese (Mn) is an essential micronutrient critical for the pathogenesis of Staphylococcus aureus, a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in S. aureus, MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of mntE transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the mntABC Mn uptake system. Inactivation of mntE or mntR leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of mntE results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for mntE are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, mntE and mntR are required for full virulence of S. aureus during infection, suggesting S. aureus experiences Mn toxicity in vivo. Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of mntABC and induction of mntE, both of which are critical for S. aureus pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis.
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Spratt DE, Barber KR, Marlatt NM, Ngo V, Macklin JA, Xiao Y, Konermann L, Duennwald ML, Shaw GS. A subset of calcium-binding S100 proteins show preferential heterodimerization. FEBS J 2019; 286:1859-1876. [PMID: 30719832 DOI: 10.1111/febs.14775] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Revised: 12/19/2018] [Accepted: 01/31/2019] [Indexed: 12/22/2022]
Abstract
The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co-expression methods in order to identify and quantify the distribution of homo- and heterodimers for four specific pairs of S100 proteins in their calcium-free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo- and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells.
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Affiliation(s)
- Donald E Spratt
- Department of Biochemistry, The University of Western Ontario, London, Canada
| | - Kathryn R Barber
- Department of Biochemistry, The University of Western Ontario, London, Canada
| | - Nicole M Marlatt
- Department of Biochemistry, The University of Western Ontario, London, Canada
| | - Vy Ngo
- Department of Pathology and Laboratory Medicine, The University of Western Ontario, London, Canada
| | - Jillian A Macklin
- Department of Biochemistry, The University of Western Ontario, London, Canada
| | - Yiming Xiao
- Department of Chemistry, The University of Western Ontario, London, Canada
| | - Lars Konermann
- Department of Biochemistry, The University of Western Ontario, London, Canada.,Department of Chemistry, The University of Western Ontario, London, Canada
| | - Martin L Duennwald
- Department of Pathology and Laboratory Medicine, The University of Western Ontario, London, Canada
| | - Gary S Shaw
- Department of Biochemistry, The University of Western Ontario, London, Canada
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Lonergan ZR, Nairn BL, Wang J, Hsu YP, Hesse LE, Beavers WN, Chazin WJ, Trinidad JC, VanNieuwenhze MS, Giedroc DP, Skaar EP. An Acinetobacter baumannii, Zinc-Regulated Peptidase Maintains Cell Wall Integrity during Immune-Mediated Nutrient Sequestration. Cell Rep 2019; 26:2009-2018.e6. [PMID: 30784584 PMCID: PMC6441547 DOI: 10.1016/j.celrep.2019.01.089] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Revised: 12/21/2018] [Accepted: 01/24/2019] [Indexed: 01/10/2023] Open
Abstract
Acinetobacter baumannii is an important nosocomial pathogen capable of causing wound infections, pneumonia, and bacteremia. During infection, A. baumannii must acquire Zn to survive and colonize the host. Vertebrates have evolved mechanisms to sequester Zn from invading pathogens by a process termed nutritional immunity. One of the most upregulated genes during Zn starvation encodes a putative cell wall-modifying enzyme which we named ZrlA. We found that inactivation of zrlA diminished growth of A. baumannii during Zn starvation. Additionally, this mutant strain displays increased cell envelope permeability, decreased membrane barrier function, and aberrant peptidoglycan muropeptide abundances. This altered envelope increases antibiotic efficacy both in vitro and in an animal model of A. baumannii pneumonia. These results establish ZrlA as a crucial link between nutrient metal uptake and cell envelope homeostasis during A. baumannii pathogenesis, which could be targeted for therapeutic development.
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Affiliation(s)
- Zachery R Lonergan
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Microbe-Host Interactions Training Program, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Brittany L Nairn
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jiefei Wang
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA; Department of Chemistry, Indiana University, Bloomington, IN, USA
| | - Yen-Pang Hsu
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA
| | - Laura E Hesse
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Microbe-Host Interactions Training Program, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - William N Beavers
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Walter J Chazin
- Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Chemistry, Vanderbilt University, Nashville, TN, USA; Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jonathan C Trinidad
- Department of Chemistry, Indiana University, Bloomington, IN, USA; Laboratory for Biological Mass Spectrometry, Department of Chemistry, Indiana University, Bloomington, IN, USA
| | - Michael S VanNieuwenhze
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA; Department of Chemistry, Indiana University, Bloomington, IN, USA
| | - David P Giedroc
- Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA; Department of Chemistry, Indiana University, Bloomington, IN, USA
| | - Eric P Skaar
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN, USA.
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Hadley RC, Nolan EM. Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin. Methods Mol Biol 2019; 1929:397-415. [PMID: 30710287 DOI: 10.1007/978-1-4939-9030-6_25] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Calprotectin (CP, S100A8/S100A9 heterooligomer) is an abundant metal-sequestering host-defense protein expressed by neutrophils, other white blood cells, and epithelial cells. The apoprotein is a S100A8/S100A9 heterodimer that contains two sites for transition metal binding at the S100A8/S100A9 interface: a His3Asp motif (site 1) and a His6 motif (site 2). In this chapter, we provide a step-by-step protocol for the overexpression and purification of the human and murine orthologues of CP that affords each apo heterodimer in high yield and purity. In these procedures, the S100A8 and S100A9 subunits are overexpressed in Escherichia coli BL21(DE3), and each apo heterodimer is obtained following cell lysis, folding, column chromatography, and dialysis against Chelex resin to reduce metal contamination. Recent studies demonstrated that human CP coordinates Fe(II) and that the protein affects the redox speciation of Fe in solution. An Fe redox speciation assay employing ferrozine is described that demonstrates the ability of both the human and murine orthologues of CP to shift the redox speciation of Fe from the ferric to the ferrous oxidation state over time.
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Affiliation(s)
- Rose C Hadley
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Elizabeth M Nolan
- Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA.
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Abstract
S100 proteins are distinct dimeric EF-hand Ca2+-binding proteins that can bind Zn2+, Mn2+, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a "nutritional immunity" mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.
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Stephan JR, Yu F, Costello RM, Bleier BS, Nolan EM. Oxidative Post-translational Modifications Accelerate Proteolytic Degradation of Calprotectin. J Am Chem Soc 2018; 140:17444-17455. [PMID: 30380834 PMCID: PMC6534964 DOI: 10.1021/jacs.8b06354] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Oxidative post-translational modifications affect the structure and function of many biomolecules. Herein we examine the biophysical and functional consequences of oxidative post-translational modifications to human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer, calgranulins A/B oligomer). This abundant metal-sequestering protein contributes to innate immunity by starving invading microbial pathogens of transition metal nutrients in the extracellular space. It also participates in the inflammatory response. Despite many decades of study, little is known about the fate of CP at sites of infection and inflammation. We present compelling evidence for methionine oxidation of CP in vivo, supported by using 15N-labeled CP-Ser (S100A8(C42S)/S100A9(C3S)) to monitor for adventitious oxidation following human sample collection. To elucidate the biochemical and functional consequences of oxidative post-translational modifications, we examine recombinant CP-Ser with methionine sulfoxide modifications generated by exposing the protein to hydrogen peroxide. These oxidized species coordinate transition metal ions and exert antibacterial activity. Nevertheless, oxidation of M81 in the S100A9 subunit disrupts Ca(II)-induced tetramerization and, in the absence of a transition metal ion bound at the His6 site, accelerates proteolytic degradation of CP. We demonstrate that native CP, which contains one Cys residue in each full-length subunit, forms disulfide bonds within and between S100A8/S100A9 heterodimers when exposed to hydrogen peroxide. Remarkably, disulfide bond formation accelerates proteolytic degradation of CP. We propose a new extension to the working model for extracellular CP where post-translational oxidation by reactive oxygen species generated during the neutrophil oxidative burst modulates its lifetime in the extracellular space.
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Affiliation(s)
- Jules R Stephan
- Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States
| | - Fangting Yu
- Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States
| | - Rebekah M Costello
- Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States
| | - Benjamin S Bleier
- Department of Otolaryngology , Massachusetts Eye and Ear Infirmary, Harvard Medical School , Boston , Massachusetts 02115 , United States
| | - Elizabeth M Nolan
- Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States
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Aranda CJ, Ocón B, Arredondo‐Amador M, Suárez MD, Zarzuelo A, Chazin WJ, Martínez‐Augustin O, Sánchez de Medina F. Calprotectin protects against experimental colonic inflammation in mice. Br J Pharmacol 2018; 175:3797-3812. [PMID: 30007036 PMCID: PMC6135788 DOI: 10.1111/bph.14449] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2017] [Revised: 03/23/2018] [Accepted: 03/24/2018] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND AND PURPOSE Calprotectin is a heterodimer composed of two myeloid-related proteins, S100A8 and S100A9, that is abundant in neutrophils and monocytes/macrophages. Faecal levels of calprotectin are used routinely to monitor inflammatory bowel disease activity. EXPERIMENTAL APPROACH We aimed to assess the role of calprotectin in intestinal inflammation, using the dextran sulfate sodium model of colitis in mice. Calprotectin was administered (50 or 100 μg·day-1 ) by the intrarectal or by i.p. injection (50 μg·day-1 only). The condition of the mice was characterized by morphological and biochemical methods. KEY RESULTS Intrarectal calprotectin protected significantly against colitis, as shown by lower levels of macroscopic and microscopic damage, colonic myeloperoxidase activity and decreased expression of TNFα and toll-like receptor 4. IL-17 production by spleen and mesenteric lymph node cells was reduced. Calprotectin had no effect on body weight loss or colonic thickening. There were no effects of calprotectin after i.p. injection. Calprotectin had virtually no effects in control, non-colitic mice. Calprotectin had almost no effect on the colonic microbiota but enhanced barrier function. Treatment of rat IEC18 intestinal epithelial cells in vitro with calprotectin induced output of the chemokines CXL1 and CCL2, involving the receptor for advanced glycation end products- and NFκB. CONCLUSION AND IMPLICATIONS Calprotectin exerted protective effects in experimental colitis when given by the intrarectal route, by actions that appear to involve effects on the epithelium.
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Affiliation(s)
- Carlos J Aranda
- Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - Borja Ocón
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - María Arredondo‐Amador
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - María Dolores Suárez
- Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - Antonio Zarzuelo
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - Walter J Chazin
- Department of Biochemistry and Chemistry, Center for Structural BiologyVanderbilt UniversityNashvilleTNUSA
| | - Olga Martínez‐Augustin
- Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
| | - Fermín Sánchez de Medina
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADAUniversity of GranadaGranadaSpain
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