Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections

RESEARCH ARTICLE Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections Hoonsik Cho1, Do-Won Jeong1¤a, Qian Liu1¤b, Won-Sik Yeo1, Thomas Vogl2, Eric P. Skaar3, Walter J. Chazin4, Taeok Bae1* a11111 1 Indiana University School of Medicine-Northwest, Gary, Indiana, United States of America, 2 Institute of Immunology, University of Muenster, Muenster, Germany, 3 Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 4 Department of Biochemistry and Chemistry, and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, United States of America ¤a Current address: Department of Food Science and Biotechnology, Shinansan University, Ansan, Republic of Korea ¤b Current address: Department of Clinical Laboratory, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China * OPEN ACCESS Citation: Cho H, Jeong D-W, Liu Q, Yeo W-S, Vogl T, Skaar EP, et al. (2015) Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections. PLoS Pathog 11(7): e1005026. doi:10.1371/journal.ppat.1005026 Editor: Frank R. DeLeo, National Institutes of Health, UNITED STATES Received: December 9, 2014 Accepted: June 16, 2015 Published: July 6, 2015 Copyright: © 2015 Cho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by AI077564 to TB from the National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abstract Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality. Author Summary Staphylococcus aureus is an important human pathogen causing skin infections and a variety of life-threatening diseases such as pneumonia, sepsis, and toxic shock syndrome. Previous study showed that the growth of S. aureus in abscesses is suppressed by the host antimicrobial protein calprotectin, which sequesters Zn and Mn from bacterial usage. During bacterial infection, calprotectin also plays an important role in the production of proinflammatory cytokines. Although the antimicrobial activity of calprotectin has been PLOS Pathogens | DOI:10.1371/journal.ppat.1005026 July 6, 2015 1 / 23 Calprotectin Increases Murine Mortality well defined, it is not known how the proinflammatory property of calprotectin affects staphylococcal infection. In this study, we found that the Zn-binding property of calprotectin increases the pathogenic potential of S. aureus by enhancing the activity of the SaeRS two component system in S. aureus. We also found that, under certain infection conditions, the proinflammatory property of calprotectin is rather detrimental to host survival. Our study illustrates that the important antimicrobial protein can be exploited by S. aureus to render the bacterium a more effective pathogen, and provides an example of the intricate tug-of-war between host and a bacterial pathogen. Introduction S. aureus is an important Gram-positive human pathogen colonizing the skin, anterior nares and other mucosal surfaces in approximately 30% of the human populations, causing a wide variety of diseases [1]. The pathogenesis of S. aureus requires multiple virulence factors, and the expression of those virulence factors is controlled by multiple regulatory systems such as SarA family transcription factors, the agr quorum sensing system, and the SaeRS two component system (TCS) [2,3]. The SaeRS TCS is composed of the sensor kinase SaeS and the response regulator SaeR along with two auxiliary proteins SaeP and SaeQ [4,5,6]. Conserved in all clinical isolates of S. aureus, the SaeRS TCS controls the production of more than 20 virulence factors (e.g., hemolysins, leukocidins, coagulases and immune evasion molecules) and plays an essential role in staphylococcal survival and pathogenesis [7,8,9]. S. aureus appears to use the SaeRS TCS to adapt to hostile host environments such as innate immune responses. The sensor kinase SaeS is activated by human neutrophil peptides (HNPs), small peptides with antimicrobial activity found in the primary granules of human neutrophils [5,10]. In addition, several sae-regulated gene products show anti-neutrophil properties [11,12,13,14]. Neutrophils are the most abundant white blood cells in human blood, consisting of 40–70% of the total white blood cell count. As the first line of defense at the site of bacterial infection, neutrophils phagocytose invading bacteria and kill them using reactive oxygen species, granule proteins (including HNPs), enzymatic intracellular degradation, or via neutrophil extracellular traps (NETs) [15,16]. In addition, the neutrophil cytoplasmic protein, calprotectin (CP), has antimicrobial activity toward various infectious fungi and bacteria including S. aureus [17,18,19]. CP is a heterodimeric S100 class EF-hand Ca-binding protein composed of S100A8 and S100A9 subunits (also called Mrp8/14). In addition to its four Ca binding sites, CP contains two transition metal binding sites S1 and S2 at the subunit interface. S1 can bind to both Zn and Mn whereas S2 binds only Zn [20]. CP is produced primarily by neutrophils and monocytes and released at the sites of inflammation [21,22]. As a main component of NETs and tissue abscesses, its concentration can reach over 1 mg/ml [19]. In tissue abscesses, the sequestration of Zn and Mn by CP suppresses the growth of S. aureus [19] and impairs the activity of Mn-dependent superoxide dismutases, rendering S. aureus more susce (...truncated)