Silkworm hemolymph inhibits hemolysin production by We purified one factor in the silkworm hemolymph in charge of this inhibitory activity. generates various virulence elements such as for example adhesive elements, exotoxins, and immune system disturbance elements. The manifestation of the virulence elements can be controlled by a genuine amount of transcription elements, including SarA (1), Rot (2), SarZ (3), as well as the DNA-binding protein of two-component systems (4). SaeRS, a two-component program, is necessary for the manifestation SYNS1 of exotoxins, including hemolysins, and is necessary for virulence in mice (5). Manifestation of is triggered by hydrogen peroxide, which kills bacterias in the phagosomes of macrophages, and an antimicrobial peptide, -defensin (6C8). secretes autoinducing peptide, which can be encoded from the gene in the locus and senses the quantity of extracellular autoinducing peptide using the sensor proteins AgrC, leading to activation from the transcription of RNAIII through the P3 promoter (9). RNAIII regulates the manifestation of virulence genes according to cell density (9, 10). Recently, Gresham and co-workers (11, 12) revealed that apolipoprotein B in mammalian blood and peroxides that are produced by macrophages inactivate the quorum-sensing molecule autoinducing peptide and suppress virulence. Invertebrate hemolymph contains antimicrobial peptides that inhibit bacterial growth (13, 14), although the factors that inhibit the bacterial gene expression necessary for virulence have not yet been identified. We previously established an infection model using silkworms and examined the interaction between host animal and pathogenic bacteria (15C22). Silkworms are larvae of the moth hemolysin kills silkworms (23), although deletion mutants of hemolysin genes of do not show attenuated virulence against silkworms.2 These results led us to hypothesize that there is a factor in silkworm hemolymph that suppresses hemolysin production. In the present study, we purified a factor that inhibited production of hemolysin. The factor was apolipophorin (ApoLp),3 a lipid-carrying protein in the silkworm hemolymph. Furthermore, ApoLp inhibited the expression of the virulence regulatory genes and RNAIII and contributed to the defense AS-604850 systems of silkworms against infection. The results serve as an example of a common defense system that suppresses bacterial virulence in both invertebrates and vertebrates. EXPERIMENTAL PROCEDURES Bacterial Strains and Growth Conditions strains were aerobically cultured in tryptic soy broth at 37 C, and 12.5 g of chloramphenicol/ml or 100 g of kanamycin/ml was added to the medium if required. The JM109 strain of AS-604850 was used as a host for pND50, pND50K, and their derivatives. strains transformed with the plasmids were cultured in Luria-Bertani broth containing 50 g/ml kanamycin or 12.5 g/ml chloramphenicol. Details of the bacterial strains and plasmids used in this study are shown in Table AS-604850 1. TABLE 1 List of bacterial strains and plasmids used Measurement of Inhibitory Activity against S. aureus Hemolysin Production An overnight culture of NCTC8325-4 was inoculated into a 100-fold amount of fresh tryptic soy broth and cultured until the culture reached an for 10 min at 4 C, and the supernatant was stored at ?80 C and used in all experiments as silkworm hemolymph. The proteins from 50 ml of hemolymph were precipitated in 70% ammonium sulfate at 4 C and centrifuged at 8000 for AS-604850 30 min. The precipitate was dissolved and dialyzed in buffer A (50 mm MES (pH 6.2), 200 mm NaCl, 2 mm DTT, 5% glycerol). The sample was applied to a phosphocellulose column (bed volume, 47 ml). The proteins were eluted with a linear salt gradient (0.2C0.6 m NaCl). Fractions with inhibitory activity were pooled and dialyzed against 5 liters of buffer B (50 mm MES (pH 6.2), 100 mm NaCl, 2 mm DTT, 5% glycerol) followed by centrifugation at 8000 for 30 min to remove the insoluble materials. The supernatant was applied to a Mono S column (HR5/5; bed volume, 1 ml; GE Healthcare) pre-equilibrated with buffer C (50 mm MES (pH 6.2), 150 mm NaCl, 2 mm DTT, 5% glycerol). The proteins were eluted with a linear salt gradient (0.15C0.6 m NaCl) in a total volume of 30 ml using a fast protein liquid chromatography system. A 200-l aliquot of the pooled fractions was applied to a SuperdexTM 200 (HR10/30; GE Healthcare) column pre-equilibrated with buffer A. The flow rate was 0.5 ml/min, and 0.5 ml was collected in each fraction. Gel filtration chromatography was.