Downloaded from www.microbiologyresearch.org by IP: 54.91.150.243 On: Fri, 02 Dec 2016 17:16:51 A novel human antimicrobial factor targets Pseudomonas aeruginosa through its type III secretion system Fareeha Mahmood, 1 Arnavaz Hakimiyan, 2 Vijayakumar Jayaraman, 1 Stephen Wood, 1 Gayathri Sivaramakrishnan, 1 Tooba Rehman, 1 Bradley L. Reuhs, 2 Susanna Chubinskaya 3 and Sasha H. Shafikhani 1 Correspondence Sasha H. Shafikhani Sasha_Shafikhani@rush.edu Received 16 August 2012 Accepted 2 January 2013 1 Department of Immunology/Microbiology, Rush University Medical Center, Chicago, IL 60612, USA 2 Department of Food Sciences, Purdue University, West Lafayette, IN 47907, USA 3 Department of Biochemistry, Rush University Medical Center, Chicago, IL 60612, USA Pseudomonas aeruginosa is an important opportunistic bacterial pathogen. Despite its metabolic and virulence versatility, it has not been shown to infect articular joints, which are areas that are rarely infected with bacteria in general. We hypothesized that articular joints possess antimicrobial activity that limits bacterial survival in these environments. We report that cartilages secrete a novel antimicrobial factor, henceforth referred to as the cartilage-associated antimicrobial factor (CA-AMF), with potent antimicrobial activity. Importantly, CA-AMF exhibited significantly more antimicrobial activity against P. aeruginosa strains with a functional type III secretion system (T3SS). We propose that CA-AMF represents a new class of human antimicrobial factors in innate immunity, one which has evolved to selectively target pathogenic bacteria among the beneficial and commensal microflora. The T3SS is the first example, to the best of our knowledge, of a pathogen-specific molecular target in this antimicrobial defence system. INTRODUCTION Pseudomonas aeruginosa is an important opportunistic bacterial pathogen. Despite aggressive antibiotic therapy, the morbidity and mortality rates associated with P. aeruginosa infection are extremely high due to the intrinsic resistance of P. aeruginosa to many antibiotics and the emergence of multi-drug resistant strains (Koch, 2002; Engel, 2003). P. aeruginosa is a highly versatile pathogen, capable of infecting various human tissues so long as the tissue defences are compromised in some manner (Yamaguchi & Yamada, 1991; Zahm et al., 1991; Tsang et al., 1994; de Bentzmann et al., 1996; Engel, 2003; Daas et al., 2009; Lin et al., 2009; Shigemura et al., 2009). Articular joints, however, are rarely infected with bacterial pathogens, including P. aeruginosa. We hypothesized that articular cartilages possess antimicrobial activity that limits P. aeruginosa survival within joint environments. Using an ex vivo explant culture model (Pascual Garrido et al., 2009), we examined the capacity of P. aeruginosa to proliferate in the presence of human articular cartilages obtained from asymptomatic organ and tissue donors. We report that cartilages from both ankle and knee joints secrete a novel antimicrobial factor(s) referred to as the cartilage-associated antimicrobial factor (CA-AMF), with potent microbicidal activity against P. aeruginosa. Importantly, CA-AMF required P. aeruginosa to possess a functional type III secretion system (T3SS) in order to exert its cytotoxicity, indicating that the T3SS played a crucial role in pathogen recognition by CA-AMF and/or its antimicrobial activity. The T3SS is a highly conserved needle-like secretion apparatus that is absent in commensal bacteria but is present in many important animal and plant Gram-negative bacterial pathogens and is essential for their pathogenesis (Engel, 2003; Stavrinides et al., 2008; Hauser, 2009; Veesenmeyer et al., 2009). We postulate that CA- AMF may represent a new class of human antimicrobial factors in innate immunity, one which has evolved to selectively target pathogenic bacteria amongst the beneficial and commensal microflora. METHODS Articular cartilage culture and conditioned media preparation. We have approval from the Rush University Medical Center Abbreviations: CA-AMF, cartilage-associated antimicrobial factor; hBD-2, human b-defensin 2; IF, immunofluorescent; SF, synovial fluid; T3SS, type III secretion system; DT, DexoT; DU, DexoU. Three supplementary figures, one supplementary table and three video clips are available with the online version of this paper. Journal of Medical Microbiology (2013), 62, 531–539 DOI 10.1099/jmm.0.051227-0 051227 G 2013 SGM Printed in Great Britain 531