Cellular Microbiology (2005) 7(7), 1009–1017 doi:10.1111/j.1462-5822.2005.00530.x © 2005 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 20057 710091017Original ArticleDownregulation of LL-37 by Neisseria gonorrhoeaeP. Bergman et al. Received 27 September, 2004; revised 25 February, 2005; accepted 28 February, 2005. *For correspondence. E-mail Birgitta.Agerberth @mbb.ki.se; Tel. (+46) 8 5248 7781; Fax (+46) 8 337462. † These authors contributed equally to this work. Neisseria gonorrhoeae downregulates expression of the human antimicrobial peptide LL-37 Peter Bergman, 1† Linda Johansson, 2† Vendela Asp, 2 Laura Plant 2 , Gudmundur H. Gudmundsson, 3 Ann-Beth Jonsson 4 and Birgitta Agerberth 1 * 1 Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm, Sweden. 2 Microbiology and Tumorbiology Center (MTC), Karolinska Institutet, Stockholm, Sweden. 3 Institute of Biology, University of Iceland, Reykjavik, Iceland. 4 Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden. Summary Neisseria gonorrhoeae is a human pathogen causing the sexually transmitted disease gonorrhoeae. The bacteria preferentially attach to and invade epithelial cells of the genital tract. As these cells previously have been shown to express the human cathelicidin LL-37, we wanted to investigate the role of LL-37 dur- ing N. gonorrhoeae infection. The cervical epithelial cell line ME180 was utilized and the expression of LL- 37 was confirmed on both peptide and transcriptional levels. Moreover, LL-37 exhibited potent in vitro activity against N. gonorrhoeae. Interestingly, the transcript and peptide levels of LL-37 were downreg- ulated during infection, according to quantitative real-time polymerase chain reaction (PCR) and immunocyto-chemistry. The downregulation was most prominent with pathogenic strains of Neisseria, while non-pathogenic strains such as Neisseria lac- tamica and Escherichia coli only exhibited moderate effects. Heat-killed N. gonorrhoeae had no impact on the downregulation, emphasizing the importance of live bacteria. The results in this study suggest that pathogenic Neisseria may gain a survival advantage in the female genital tract by downregulating LL-37 expression. Introduction The female genital tract can be divided into two compart- ments: first, the vagina and the cervix, which host a com- mensal flora; second, the uterus and the fallopian tubes, which are sterile. Several defence mechanisms are involved in the protection of the integrity of the upper part of the female genital tract. The epithelial layers of the vagina and cervix produce protective mucus with several important characteristics, such as low pH and large amounts of immunoglobulins (Johansson and Lycke, 2003). In addition, the cervical epithelium synthesizes a number of antimicrobial proteins and peptides such as lactoferrin (Hein et al., 2002), defensins (Svinarich et al., 1997; Quayle et al., 1998) and cathelicidins (Frohm Nils- son et al., 1999). Thus, the cervical epithelium and mucus play important roles in protecting the uterus and the fallo- pian tubes against bacterial invasion. Pathogens, such as Neisseria gonorrhoeae (gonococ- cus) and Chlamydia trachomatis, have developed strate- gies to circumvent the innate defence barrier provided by the cervical epithelium. N. gonorrhoeae employs a recep- tor-mediated strategy that culminates with the attachment and invasion of the human mucosal genital tract (Merz and So, 2000). The initial attachment is mediated by type IV pili, long polymers of protein subunits extending from the bacterial surface (Swanson, 1973). Pili recognize and bind CD46 (Kallstrom et al., 1997), a cell surface glyco- protein expressed on all nucleated cells that protects host tissue from complement activation (Liszewski et al., 1991). The binding of type IV pili to CD46 elicits a Ca 2+ - dependent process in the host cell (Kallstrom et al., 1998) and infection of epithelial cells by gonococci leads to rapid tyrosine phosphorylation of CD46 (Lee et al., 2002). Whether this intracellular signalling is beneficial for the bacteria in order to invade the epithelial cells or is part of a host response is not known. Adherence of the bacterium to host cells is dependent on expression of the bacterial protein PilC, localized in the outer membrane and at the tip of the pilus (Jonsson et al., 1991; Rudel et al., 1995; Rahman et al., 1997). Most strains of Neisseria carry two homologous, but not identical, copies of the pilC gene, pilC1 and pilC2 (Jonsson et al., 1995). A gonococcal mutant in the pilC1 or pilC2 gene results in a piliated adherent bacterium, while a double knockout in both the pilC1 and pilC2 genes results in a non-piliated non-adher- ent phenotype (Jonsson et al., 1991). Upon adherence of gonococci to epithelial cells of the cervicovaginal mucosa, the epithelial cells initiate and coordinate a non-specific immune response, stimulating the production of cytokines and other proinflammatory