A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages Malgorzata Kubica1*, Krzysztof Guzik2, Joanna Koziel1.*, Miroslaw Zarebski3, Walter Richter4, Barbara Gajkowska5, Anna Golda1, Agnieszka Maciag-Gudowska1, Klaudia Brix6, Les Shaw7, Timothy Foster8, Jan Potempa1. 1 Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, 2 Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, 3 Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, 4 Center for Electron Microscopy of the Medical Faculty, Friedrich-Schiller-University Jena, Jena, Germany, 5 Department of Cell Ultrastructure, Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, 6 School of Engineering and Science, Jacobs University Bremen, Bremen, Germany, 7 Department of Biology, University of South Florida, Tampa, Florida, United States of America, 8 Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin, Ireland Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non- professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-c at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in a-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular a-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection. Citation: Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, et al (2008) A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages. PLoS ONE 3(1): e1409. doi:10.1371/journal.pone.0001409 INTRODUCTION S. aureus is a leading etiologic agent of nosocomial and community- acquired infectious diseases. The pathogenesis of staphylococcal disease usually proceeds from a local infection (e.g., wound infection, furuncle and cellulitis) to systemic dissemination (bacteremia) and then to metastatic infections (e.g., endocarditis, osteomyelitis and septic arthritis). Toxinoses (e.g., toxin shock syndrome, scaled skin syndrome, and food-borne gastroenteritis) constitute a separate group of staphylococcal diseases resulting from the local or systemic effects of specific toxins [1,2]. The public health concern regarding staphylococcal infections is magnified by the increasing prevalence of multiply antibiotic resistant strains such as methicillin-resistant S. aureus (MRSA) and glycopeptide-insensitive S. aureus (GISA) [3]. The appearance of vancomycin-resistant strains (VRSA) suggests the possibility of a return to a pre-antibiotic era where 80% of bloodstream infections were fatal. A comprehensive understanding of the pathogenic pathways exerted by S. aureus is urgently needed to aid the design of new therapeutic and preventive treatments. The success of S. aureus as a pathogen is primarily due to its ability to produce a large number of virulence factors. These include several MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that bind fibrinogen, fibronectin, laminin, collagen, vitronectin, and thrombospondin to promote colonization [4]. Host defenses are thwarted by the production of microcapsule, protein A, coagulase, complement inhibitors, fatty acid-metabolizing enzymes and leukocidin while Academic Editor: Robin May, University of Birmingham, United Kingdom Received September 9, 2007; Accepted December 10, 2007; Published January 9, 2008 Copyright: 2008 Kubica 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. Funding: This work was supported by the Commission of the European Communities, specific RTD program "Quality of Life and Management of Living Resources", Project QLRT-2001-01250 "Novel non-antibiotic treatment of staph- ylococcal diseases", grants from the State Committee for Scientific Research (MNiSW, Warsaw, Poland) (KBN 6PO4A 060 19; 2P04A 049 28; 2PO4A 011 26 , 6PO4A 011 27 to KG, JK, MZ and MK respectively) and the European Molecular Biology Organization for a research fellowship (ASTF 103-05) awarded to MK. JP is a recipient of a Subsydium Profesorskie awarded by the Foundation for Polish Science (FNP, Warsaw, Poland). TJF would like to acknowledge support from Science Foundation Ireland, the Health Research Board and The Wellcome Trust. Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: staphaureus@gmail. com (MK); kaniajoanna@gmail.com (JK) . These authors contributed equally to this work. PLoS ONE | www.plosone.org 1 January 2008 | Issue 1 | e1409