Insect Molecular Biology (2000) 9(6), 553–558 © 2000 Blackwell Science Ltd 553 Blackwell Science, Ltd Immune activation upregulates lysozyme gene expression in Aedes aegypti mosquito cell culture Y. Gao and A. M. Fallon Department of Entomology, University of Minnesota, St Paul, MN, USA Abstract After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedes aegypti (Aag-2 cells) secreted an induced protein with a mass of ≈ 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is pro- cessed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anopheles gambiae and Anopheles darlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induc- tion of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12 – 36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells. Keywords: insect immunity, insect cell lines, immunity proteins, RT-PCR, Northern blot, Southern blot. Introduction Lysozymes hydrolyse the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacterial cell walls. The participation of lysozyme-like activities in insect immunity, particularly in lepidopteran species such as Galleria mellonella, Manduca sexta and Hyalophora cecropia was recognized as early as the late 1960s (reviewed by Chadwick & Aston, 1991). More recent molecular analyses have shown that in M. sexta (Mulnix & Dunn, 1994) and H. cecropia (Sun et al., 1991) lysozyme is encoded by a single-copy gene that is constitutively expressed at low levels under normal condi- tions, and upregulated within 2 h of bacterial infection. It has been suggested that lysozyme may protect Lepidoptera against bacteria that could be released into the haemolymph during metamorphosis (Russell & Dunn, 1991). We have previously shown that when Aedes aegypti Aag-2 mosquito cells are induced with heat-killed bacteria, they secrete into the culture medium antibacterial factors that include a 66 kDa mosquito transferrin (Yoshiga et al., 1997) and small peptides identified as cecropin (Y. Gao, unpub- lished) and defensins (Gao et al., 1999). A similar suite of proteins is also secreted by the C7–10 cell line from Aedes albopictus (Hernandez et al., 1994; Yoshiga et al., 1997; Sun et al., 1998, 1999). Because the inducible immunity proteins in Lepidoptera include an ≈ 14 kDa protein that has been identified as lysozyme, we suspected that the inducible p16 common to both mosquito cell lines (Yoshiga et al., 1997) might also be a lysozyme. In this paper, we confirm this hypothesis, and describe the cDNA, deduced protein sequence, and induction of an Ae. aegypti lysozyme. Results When Aag-2 cells were labelled with 35 S-methionine/cysteine as a function of time after treatment with heat-killed bacteria, incorporation of label into a protein measuring ≈ 16 kDa on 15% polyacrylamide sodium dodecyl sulphate (SDS) gels was induced (Fig. 1, Lanes 2–7), relative to that in uninduced control cells (Fig. 1, Lane 1). Incorporation of label into the ≈ 16 kDa band reached a maximum by 36 h after induction (Lane 5; note that Lane 2 is overloaded, relative to the other lanes), and remained high for an addi- tional 36 h. On stained gels (Fig. 2, Lanes 1–5), p16 migrated as the slower of two prominent bands in the 16 kDa region (Fig. 2, Lanes 3–5). On a Western blot, this protein cross- reacted with antibody to chicken egg lysozyme (Fig. 2, Lanes 6–9), and was induced by treatment with Gram- positive Micrococcus luteus, Gram-negative Escherichia coli , or a mixture of both bacteria. Moreover, when this upper Received 4 May 2000; accepted 10 July 2000. Correspondence: Ann M. Fallon, Department of Entomology, University of Minnesota, 1980 Folwell Ave., St Paul, MN 55108, USA. Tel.: 612–625–3728; fax: 612–625–5299; e-mail: fallo002@tc.umn.edu