A novel function of InlB from Listeria monocytogenes: activation of NF-kB in J774 macrophages Ashley Mansell, 1 Laurence Braun, 2 Pascale Cossart 2 and Luke A. J. O'Neill 1 * 1 Department of Biochemistry and Biotechnology Institute, Trinity College, Dublin 2, Ireland. 2 Unite  des Interactions Bacte Âries-Cellules, 25±28 rue du Dr Roux, Institut Pasteur, 75724, Paris, Cedex 15, France. Summary Listeria monocytogenes causes a pro-in¯ammatory response on adhesion to macrophages. Upregulation of in¯ammation genes involves the transcription fac- tor NF-kB. Several components of L. monocytogenes, including lipoteichoic acid (LTA), phospholipases and listeriolysin O (LLO), have since been shown to mediate NF-kB activation. Here, we report that puri- ®ed recombinant InlB, but not internalin (InlA), is a potent activator of NF-kB in the mouse macrophage- like cell line J774. Expression of InlB in Listeria innocua enhances its ability to activate NF-kB, while deletion of InlB from L. monocytogenes marginally decreases its effect on NF-kB, possibly because of the presence of NF-kB activators such as LTA and LLO. The effect cor- relates with the rapid degradation of IkBa, a sustained degradation of IkBb and increases in tumour necrosis factor alpha ( TNF-a) and interleukin (IL) 6 production, two cytokines controlled by NF-kB. Using a series of anti-InlB monoclonal antibodies and domains of InlB, NF-kB activation was shown to be dependent upon the N-terminal 213-amino-acid leucine-rich repeat (LRR) domain of InlB, recently demonstrated to be responsible for InlB-mediated L. monocytogenes invasion and phosphoinositide-3 (PI-3) kinase activa- tion. The effect of InlB was blocked by PI-3 kinase inhi- bitors, indicating the involvement of PI-3 kinase in this response. This report thus illustrates that InlB not only promotes invasion, but also contributes to the macrophage pro-in¯ammatory response. Introduction Listeria monocytogenes is a Gram-positive, food-borne pathogen, which infects both phagocytic and non-phagocytic cell types (Dramsi et al., 1996; Cossart and Lecuit, 1998). Once the bacterium has been internalized into cells, it rapidly replicates within the cytosol. Concomitantly, it becomes coated with actin ®laments, starts moving intra- cellularly and spreads from cell to cell, thus avoiding circulat- ing antibodies or other extracellular bactericidal components (Ireton and Cossart, 1997). Immunity to this bacterium is T-cell mediated and appears to require pro-in¯ammatory cytokines, such as interleukin (IL) 1 and tumour necrosis factor alpha (TNF-a) (Unanue, 1997). Two surface proteins of Listeria, internalin (InlA) and InlB, are crucial in mediating L. monocytogenes cell inva- sion (Ireton and Cossart, 1997; Cossart and Lecuit, 1998). InlA is an 800-amino-acid protein consisting of 15 highly conserved 22-amino-acid leucine-rich repeats (LRRs), an inter-repeat region (IR) and a second repeat region consisting of two repeats of 70 amino acids and a third of 49 amino acids (B repeats). The carboxyl-terminal part contains the LPXTG motif, which is found in more that 50% of Gram-positive surface proteins, and allows a covalent linkage to peptidoglycan (Navarre and Schneewind, 1994). The receptor for InlA is E-cadherin, a transmembrane pro- tein normally involved in cell±cell adhesion (Mengaud et al., 1996a; Lecuit et al., 1998). InlB is a 630-amino-acid protein consisting of eight LRRs, each comprising 22 amino acids with a high degree of homology to InlA LRRs, an inter-repeat region (IR) simi- lar to that of InlA, one B-repeat containing 80 amino acid repeats and a 232-amino-acid carboxyl-terminal region beginning with the sequence GW. InlB is loosely attached to the bacterial surface via the so-called GW repeats and is found in culture supernatants of wild-type L. monocyto- genes (Dramsi et al., 1995; Braun et al., 1997). Puri®ed, recombinant InlB confers invasiveness upon inert latex beads or non-invasive bacteria (Braun et al., 1998). InlB has also been shown to activate phosphoinositide-3 kinase (PI-3 kinase) in Vero cells causing a rapid and tran- sient increase in the lipid products of the PI 3-kinase p85- p110, tyrosine phosphorylation of the mammalian adaptor proteins Gab1, Cbl and Shc and association of these pro- teins with p85 (Ireton et al., 1996; 1999). Recently, it has been reported that the 213-amino-acid LRR region of InlB is suf®cient to promote InlB-mediated invasion, stimulation of PI-3 kinase and membrane ruf¯ing (Braun et al., 1999). A diverse range of bacterial species and products has been shown to be capable of triggering an in¯ammatory Cellular Microbiology (2000) 2(2), 127±136 Q 2000 Blackwell Science Ltd Received 23 August, 1999; revised 14 December, 1999; accepted 8 January, 2000. *For correspondence. E-mail laoneill@tcd.ie; Tel. (353) 1 608 2439; Fax (353) 1 677 2400.