Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner Verónica Regueiro, 1,2† David Moranta, 1,2† Christian G. Frank, 1,2† Eider Larrarte, 3 Javier Margareto, 3 Catalina March, 1,2 Junkal Garmendia 1,2,4 and José A. Bengoechea 1,2,5 * 1 Program Infection and Immunity, Fundació Caubet-CIMERA Illes Balears, Bunyola, Spain. 2 Program Host–Pathogen interactions, Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), Bunyola, Spain. 3 Unidad de Genómica, LEIA-Salud, Miñano, Spain. 4 Instituto de Agrobiotecnología, UPNA-CSIC, Mutilva Baja, Spain. 5 Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. Summary Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for patho- gen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro- inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-kB via the deubiquitinase CYLD and blocks the phospho- rylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-kB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1b-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1b-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenu- ate inflammation. Introduction Activation of innate immune responses, particularly inflam- mation, is a key event in the host defence against patho- gens. Innate responses rely on the activation of signalling pathways resulting in the production of antimicrobial mol- ecules, the expression of co-stimulatory molecules and the release of cytokines and chemokines (Mogensen, 2009). These signalling pathways converge on a limited set of transcriptional activators including NF-kB and mitogen- activated protein kinases (MAPKs) (Medzhitov, 2007; Kumar et al., 2009a; Mogensen, 2009). To launch these responses, the host recognizes conserved molecules uniquely expressed by pathogens, the so-called pathogen- associated molecular patterns (PAMPs), through a set of germ line-encoded receptors referred to as pattern- recognition receptors (PRRs) (Medzhitov, 2007; Kumar et al., 2009a). The best-characterized PRRs belong to the Toll-like receptors (TLRs) and nucleotide binding and oli- gomerization domain-like receptors (NLRs) families (Cha- maillard et al., 2003; Inohara et al., 2005; Kumar et al., 2009b). Among TLRs, most of the studies focus on TLR4, mainly involved in the detection of lipopolysaccharide (LPS), and on TLR2 which responds to a variety of Gram- positive PAMPs (Medzhitov, 2007; Kumar et al., 2009a; Mogensen, 2009). Among NLRs, NOD1 receives increas- ing attention. NOD1 is located intracellularly and recog- nizes a peptidoglycan (PGN) motif, g-D-glutamyl-meso- diaminopimelic acid from Gram-negative bacteria (Chamaillard et al., 2003; Inohara et al., 2005). Subversion of the fast-acting inflammatory response is considered important for pathogen survival during the early stages of infection. Such subversion allows pathogens to avoid immediate elimination by the host defence and Received 20 July, 2010; revised 3 September, 2010; accepted 6 September, 2010. *For correspondence. E-mail bengoechea@ caubet-cimera.es; Tel. (+34) 971 011780; Fax (+34) 971 011797. † These authors have contributed equally to this work. Cellular Microbiology (2011) 13(1), 135–153 doi:10.1111/j.1462-5822.2010.01526.x First published online 14 October 2010 © 2010 Blackwell Publishing Ltd cellular microbiology