OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

RESEARCH ARTICLE OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii Ming-Feng Lin1, Pei-Wen Tsai2, Jeng-Yi Chen2, Yun-You Lin2, Chung-Yu Lan2,3* 1 Department of Medicine, National Taiwan University Hospital Chu-Tung Branch, Hsin-Chu County, Taiwan, 2 Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsin-Chu City, Taiwan, 3 Department of Life Science, National Tsing Hua University, Hsin-Chu City, Taiwan * Abstract OPEN ACCESS Citation: Lin M-F, Tsai P-W, Chen J-Y, Lin Y-Y, Lan C-Y (2015) OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii. PLoS ONE 10(10): e0141107. doi:10.1371/journal.pone.0141107 Editor: Surajit Bhattacharjya, Nanyang Technological University, SINGAPORE Received: August 6, 2015 Accepted: October 5, 2015 Published: October 20, 2015 Copyright: © 2015 Lin 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. Funding: This work was supported by grant MOST104-2622-B-007-001-CC2 (to CYL) from the Ministry of Science and Technology (Taiwan). Competing Interests: The authors have declared that no competing interests exist. Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding. Introduction Antimicrobial peptides (AMPs) are generated by a wide variety of organisms as a part of the host defense. In humans, AMPs can be produced by various cells and tissues and play a critical role in innate immunity [1,2]. AMPs are generally short (10–100 amino acids), positively charged (normally +2 to +9) and amphiphilic [3]. AMPs can be divided into three major classes based on their gross amino acid composition and certain structural features, including PLOS ONE | DOI:10.1371/journal.pone.0141107 October 20, 2015 1 / 19 OmpA Binding-Dependent Effect of LL-37 on A. baumannii linear alpha-helical peptides (without cysteines), cysteine-containing peptides linked by disulfide bonds and peptides with a high ratio of specific amino acids [2]. For example, human defensins belong to the second class, and histatins are members of the third class. hCAP-18 (the only member of the cathelicidin AMP family in humans) contains an N-terminal domain, a cathelin domain and a C-terminal LL-37 domain [4]. LL-37 is extracellularly cleaved from hCAP-18 by proteinase 3 and belongs to the class of linear alpha peptides. LL-37 owes its name to the fact that it consists of 37 amino acids that begin with two leucine residues [5]. Different types of AMPs use different mechanisms to disrupt bacterial structures or inhibit cell growth [6,7]. For example, the amphipathic conformation change can help an AMP gain access or insert into the plasma membrane of bacteria to disrupt the cells [7]. However, AMPs not only attack membranes but also inhibit cell wall biosynthesis, protein folding, enzyme activity and even protein synthesis through DNA binding [6]. In addition to the direct killing of bacteria, AMPs also play an important role in immunomodulation [8]. AMPs activate the adaptive immune system by stimulating gene transcription to activate macrophages, inducing interleukin-8 in airway epithelial cells to recruit neutrophils, promoting histamine release to increase blood vessel permeability, activating fibroblast growth to facilitate wound healing and presenting chemotactic activity to recruit monocytes [1,9]. These multi-functional responses induced by AMP make it a promising candidate adjuvant therapeutic agent, especially against multidrug-resistant pathogens. Human LL-37 is able to defend against various bacterial and fungal pathogens [10–12]. Recently, Acinetobacter baumannii has emerged as an important pathogen in nosocomial infections [13]. Infections and outbreaks caused by multidrug-resistant A. baumannii (MDRAB) are rapidly increasing [14]. Resistance to the last resort antibiotics for carbapenemresistant A. baumannii, including tigecycline and colistin, has been reported [15,16]. A previous study reported a lipopolysaccharide (LPS)-deficient, colistin-resistant A. baumannii strain that showed reduced viability even at a low concentration of LL-37 [17]. Moreover, LL-37 and its fragments possess both antimicrobial and antibiofilm activities against MDRAB [18]. Therefore, human antimicrobial peptides (especially LL-37) may function as potential therapeutic alternatives or adjuvants to antibiotics. The OmpA outer membrane protein of Escherichia coli and other enterobacteria is a multifaceted protein, which functions as an adhesin and invasin, participates in biofilm formation, acts as both an immune target and evasin, and serves as bacteriophage receptor [19]. The A. baumannii outer membrane protein A (AbOmpA) is a trimeric porin that is involved in solute transport and virulence [20]. The contributions of AbOmpA to pathogenesis include apoptosis, immunomodulation, cell adherence and invasion, biofilm formation and serum resistance. AbOmpA can induce dendritic cell death via targeting to the mitochondria [21]. Interaction of laryngeal epithelial cells with AbOmpA has a significant impact on the induction of innate immunity during the early stages of A. baumannii infection [22]. AbOmpA also plays a role in (...truncated)