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Article Cite This: ACS Omega 2019, 4, 1601−1610
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Polysialic Acid Modulates Only the Antimicrobial Properties of Distinct Histones Kristina Zlatina and Sebastian P. Galuska* Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
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S Supporting Information *
ABSTRACT: Histones are not only the organizing and regulatory elements of the genome, the five histonesH1, H2A, H2B, H3, and H4also fulfill many tasks outside the cell. Their release in the extracellular fluid can accidentally occur by necrosis or be actively triggered during apoptosis or NETosis. The latter serves as a mechanism of the immune system to combat pathogens. In this defense mechanism, neutrophils spread out their chromatin, including histones. These small proteins are antimicrobial; however, they are also able to disrupt the membranes of endogenous cells. High concentrations of extracellular histones have been described as triggering different organ dysfunctions. Interestingly, the carbohydrate polysialic acid (polySia) is known to reduce the negative outcomes of all five histones. In the present study, we investigated to which extent polySia influences the antimicrobial properties of histones against bacteria. All applied histones showed antimicrobial characteristics in the absence of polySia. However, histone H3’s antimicrobial properties were completely inhibited by polySia. In contrast, the histones H1, H2A, and H2B all exhibited antibacterial properties in the presence of polySia. Remarkably, H2A and H2B represent the main fraction of all histones in NET with ∼70%. Thus, the results suggest that polySia might be a tool to decrease the histone-mediated cell damage of eukaryotic cells without losing the antimicrobial activity of distinct histones.
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necrosis.8 Extracellular histones are procoagulant and proinflammatory and have cytotoxic properties. The cationic character allows the disruption of cell membranes after binding to phospholipids and mediates histones’ cytotoxicity. This membrane perforation leads to calcium influx, which is associated with further cytokine release and immune system activation.9 Thus, besides the valuable functions of histones like the organization and regulation of DNA, high concentrations of histones in the extracellular fluid may mediate thrombosis,10−12 sepsis,13 organ dysfunction like acute lung injury,9,14 and kidney injury.15 Because of these negative effects, histones have gained interest as a therapeutic target. In several studies, different approaches for antihistone antibodies were tested. In some cases, specific antibodies were indeed helpful for different diseases.13,15−18 Recombinant activated protein C was also a promising approach in therapy of sepsis, but due to missing effects in sepsis shock therapy, it was withdrawn from the market in 2011.19,20 Until today, the most promising approach seems to be heparin, which neutralizes histone-mediated cytotoxicity, reduces thrombosis,21,22 and increases the survival
INTRODUCTION Histones seem to be older than eukaryotic cells. Even some archaea possess DNA-binding proteins with a similarity of more than 30% to today’s known eukaryotic histones.1−3 These proteins enable the packaging of genome information within cells. Besides their role in the organization of DNA, histones are involved in the access and regulation of genes by other proteins and enzymes. Histones can be grouped into core (H2A, H2B, H3, and H4) and linkage histones (H1) or, due to their amino acid composition, into lysine- (H1, H2A, H2B) and arginine-rich histones (H3, H4).4 Individual histones share a similar structure of a long helix in the center flanked by one or two shorter helices connected by loops on each end. These motifs are highly conserved between species. Aside from their role in the nucleus, histones fulfill many different tasks in the cytosol, at the cell surface and beyond the cell membrane in the extracellular fluid. For example, histone H1 is a mediator for the binding of thyroglobulin on mouse macrophages.5 Moreover, cytosolic H2B binds viral double stranded DNA and mediates the subsequent innate antiviral immune response.6 In this study, we focused on the antimicrobial role of histones. The release of histones may be intentionally triggered to the cell surface during apoptosis or to the extracellular fluid during NETosis.7 It may also occur accidently during © 2019 American Chemical Society
Received: August 30, 2018 Accepted: November 29, 2018 Published: January 18, 2019 1601
DOI: 10.1021/acsomega.8b02222 ACS Omega 2019, 4, 1601−1610
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Figure 1. Antimicrobial effects of calf thymus histones (CTHs) on E. coli. (A) Growth-curves of bacterial survival in percent of CTH over the time were recorded. All results are from four independent experiments. (B) In addition, endpoint measurement of bacterial survival at 210 min were analyzed. Untreated E. coli was set to 100%. Boxplots show the median as well as the min and max whiskers with smallest and largest values. All results are from five independent experiments. For reasons of clarity, the mean values as well as standard deviation of growth-curves are summarized in Table S1 in the Supporting Information. The statistics were performed by one-way analysis of variance (ANOVA) including Tukey’s test: ns, not significant; **p < 0.01 and ***p < 0.001.
Figure 2. Influence of polySia on bacterial survival. (A) Bacterial survival is displayed in the presence of polySia at different concentrations (10− 100 μg/mL) over the time as growth-curves on top (n = 4) and as endpoint at the bottom (n = 5). (B) Coincubation of CTH (H) (60 μg/mL) and different polySia (P) concentrations (10−100 μg/mL) with bacteria; growth-curves on top (n = 4) and as endpoint at the bottom (n = 5). Boxplots show the median as well as the min and max whiskers with smallest and larges values. Untreated E. coli was set to 100%. Mean values as well as standard deviation of growth-curves are summarized in Table S2A,B in the Supporting Information. The statistics were performed by oneway ANOVA including Tukey’s test: ns, not significant; ***p < 0.001.
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DOI: 10.1021/acsomega.8b02222 ACS Omega 2019, 4, 1601−1610
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Table 1. Overview of PolySia’s Influence on CTHa
Given are quotients after division of CTH + polySia and CTH. Quotients >1 → polySia has an effect, the higher the number, the higher the effect of polySia (colored in dark red). Quotients 1 or 1), the more the polySia reduced CTH’s antimicrobial effects. The highest ratios are colored in dark red, whereas brighter colors indicate a decreasing polySia effect. The nonshaded ratios of 1 or
