Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice

ORIGINAL RESEARCH published: 30 June 2017 doi: 10.3389/fcimb.2017.00300 Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice Masataka Oda 1, 2 , Hisanori Domon 1, 3 , Mie Kurosawa 1 , Toshihito Isono 1 , Tomoki Maekawa 1, 3 , Masaya Yamaguchi 4 , Shigetada Kawabata 4 and Yutaka Terao 1, 3* 1 Division of Microbiology and Infectious Diseases, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan, 2 Department of Microbiology and Infection Control Sciences, Kyoto Pharmaceutical University, Kyoto, Japan, 3 Research Center for Advanced Oral Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan, 4 Department of Oral and Molecular Microbiology, Osaka University, Graduate School of Dentistry, Osaka, Japan Edited by: Yousef Abu Kwaik, University of Louisville, United States Reviewed by: Frank C. Gibson, III, University of Florida, United States Michael L. Vasil, University of Colorado Denver School of Medicine, United States *Correspondence: Yutaka Terao Received: 13 February 2017 Accepted: 19 June 2017 Published: 30 June 2017 Citation: Oda M, Domon H, Kurosawa M, Isono T, Maekawa T, Yamaguchi M, Kawabata S and Terao Y (2017) Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice. Front. Cell. Infect. Microbiol. 7:300. doi: 10.3389/fcimb.2017.00300 The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the 1slaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection. Keywords: S. pyogenes, phospholipase A2 , SlaA, HUVEC, ICAM1, VCAM1 INTRODUCTION Streptococcus pyogenes is a gram-positive bacterium that can cause superficial infections, such as pharyngitis and pyoderma; invasive infections, such as necrotizing fasciitis and streptococcal toxic shock syndrome; and post-infectious diseases, such as rheumatic fever (Cunningham, 2000). S. pyogenes produces many extracellular molecules that contribute to host–pathogen interactions Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 June 2017 | Volume 7 | Article 300 Oda et al. S. pyogenes SlaA and Adhesion Molecules by the Institutional Animal Care and Use Committee of Niigata University. (Musser and Krause, 1998; Cunningham, 2000; Fischetti, 2000; Kurosawa et al., 2016) and most of the extracellular molecules possess enzymatic activities against host tissues and the immune system (Terao et al., 2006, 2008; Honda-Ogawa et al., 2013). Epidemiological studies have shown that M3 serotype strains are the second most common cause of invasive infections in the United States, Canada, Western Europe, Japan, and Israel (Murakami et al., 2002; Terao et al., 2002; Li et al., 2003; Moses et al., 2003; Muller et al., 2003; Nakagawa et al., 2003; Schmitz et al., 2003). Surveillance studies have also revealed that M3 strains cause a higher rate of severe invasive diseases such as necrotizing fasciitis and death than other M-type strains (O’Brien et al., 2002; Sharkawy et al., 2002). S. pyogenes is a diverse species with strain-specific virulence genes derived from multiple prophages (Banks et al., 2002; Beres and Musser, 2007). Genome sequencing analysis of S. pyogenes M1–M6, M12, M18, M28, and M49 serotype strains indicated that these strains have 4–8 prophages or prophagelike elements (Nozawa et al., 2011). We identified that M3 serotype strain SSI-1 possesses the phospholipase A2 (PLA2 ) gene, designated as slaA, on the prophage genome. Beres et al. (2004) reported that the enhanced capacity for invasive infection incurred by M3 correlated with the acquisition of prophage genomes encoding the slaA gene. SlaA has a conserved region of amino acid residues found in several secreted PLA2 enzymes, such as those found in snake venom (Pearson et al., 1993). Sitkiewicz et al. (2007) reported that SlaA enhanced the binding of S. pyogenes to human epithelial cells, such as immortalized pharyngeal cells and human tracheobronchial epithelial cells. However, the pathogenesis due to SlaA in host cells remains unclear. Yokote et al. (1993) reported that endogenous PLA2 from human endothelial cells induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1). In addition, Zhu et al. (1999) reported that cytosolic PLA2 in human eosinophils stimulated the expression of β1- and β2-integrin, resulting in binding to ICAM1 and VCAM1. In this study, we hypothesized that SlaA induces the expression of adhesion molecules. To confirm this hypothesis, we examined the adhesion of human monocytic leukemia cell line (THP-1) cells to human umbilical vein endothelial cells (HUVECs) and the association of this with the expression of adhesion molecules, such as ICAM1, VCAM1, E-selectin, and P-selectin, on HUVECs treated with SlaA. Bacterial Strains S. pyogenes clinical strains were generously provided by Dr. Kawabata of Osaka University (Table 1) (Murakami et al., 2002; Terao et al., 2002). All strains were grown in Todd Hewitt broth (Becton Dickinson, MD, USA) supplemented with 0.2% yeast extract (THY broth) at 37◦ C. Detection of slaA Gene Chromosomal DNA from S. pyogenes was purified with Bactozol (Molecular Research Center, Inc., Cincinnati, OH, USA). The forward primer (5′ -GGGGGATCCGGGATAAATGATAAAATG GAA-3′ ) and reverse primer (5′ -CCCGAATTCTTAACATCCT ATAGAACCTAC-3′ ) were used to amplify the slaA gene of various S. pyogenes strains. Integration Mutagenesis The PCR product of th (...truncated)