To obtain therapeutically effective new antibiotics, we first searched for bacterial culture supernatants with antimicrobial activity in vitro and then performed a secondary screening using the silkworm infection model. Through further purification of the in vivo activity, we obtained a compound with a previously uncharacterized structure and named it 'lysocin E'. Lysocin E interacted with menaquinone in the bacterial membrane to achieve its potent bactericidal activity, a mode of action distinct from that of any other known antibiotic, indicating that lysocin E comprises a new class of antibiotic. This is to our knowledge the first report of a direct interaction between a small chemical compound and menaquinone that leads to bacterial killing. Furthermore, lysocin E decreased the mortality of infected mice. To our knowledge, lysocin E is the first compound identified and purified by quantitative measurement of therapeutic effects in an invertebrate infection model that exhibits robust in vivo effects in mammals.

The injection of bacteria (Staphylococcus aureus, Stenotrophomonas maltophilia) or true fungi (Candida albicans, Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.

We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450 14DM ) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51, including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.Demethylation inhibitors (DMIs) are widely used as fungicides in agriculture and medicine. In the 1970s, the development of resistance to DMI fungicides under practical conditions was thought to be unlikely (9, 16). However, in practice, DMI-resistant strains occur widely in several important plant and animal pathogens, such as Erysiphe graminis, Sphaerotheca fuliginea, Pyrenophora teres (12), and Candida albicans (11), causing acute problems in crop production and in the treatment of candidiasis of AIDS patients. Determining the mechanism of resistance is, therefore, quite important.We previously showed that an ATP-binding cassette (ABC) transporter gene, PMR1, is involved in DMI resistance in Penicillium digitatum by disrupting the PMR1 gene and concomitantly increasing sensitivity to DMI fungicides (21). PMR1 expression is strongly induced by fungicide treatment in both DMI-sensitive and DMI-resistant strains, but the constitutive expression level of PMR1 in the resistant strain was relatively higher than that in the sensitive strain (21). ABC transportermediated resistance to toxicants in yeast and human cells is a consequence of increased constitutive expression of the ABC transporter gene (3, 7), so we thought that the higher level of constitutive expression of the PMR1 gene could be responsible for the higher DMI resistance of the resistant strains. However, introduction of the PMR1 coding region under the control of a strong constitutive promoter, PgpdA, into a DMIsensitive strain had no observable effect on DMI resistance (H. Hamamoto, O. Nawata, K. Hasegawa, R. Nakaune, Y. J. Lee, Y. Makizumi, K. Akutsu, and T. Hibi, submitted for publication), suggesting that the constitutive expression level of PMR1 is not the determinative factor for DMI resistance. The coding sequences of this gene from three DMI-sensitive and three DMI-resistant strains were also identical. These results sugges...

We report for the ¢rst time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5-cluster) from Fusarium graminearum F15, a strain that produces 3-acetyldeoxynivalenol (3-ADON). A putative tyrosinase and polysaccharide deacetylase gene £ank the Tri5-cluster : the number of pathway genes between them is less than half the total number of steps necessary for 3-ADON biosynthesis. In comparison with partial Tri5-cluster sequences of strains with 15-acetyldeoxynivalenol and 4-acetylnivalenol chemotypes, the Tri5-cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3-ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo-Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C-15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C-3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference. ß

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The insect cytokine paralytic peptide (PP) induces muscle contraction in silkworm larvae. Here we demonstrate that bacterial and fungal cell wall components peptidoglycan and glucan stimulate muscle contraction via activation of PP in the hemolymph. Anti-PP antibody suppressed the muscle contraction induced by PP, peptidoglycan, or glucan. The contraction was also inhibited by free radical scavengers and serine protease inhibitors. Moreover, injecting live silkworms with peptidoglycan or glucan generated the active form of PP. The active form of PP was also produced in vitro when peptidoglycan or glucan was incubated with hemolymph containing the PP precursor. Generation of the active form of PP was suppressed by free radical scavengers and serine protease inhibitors. Furthermore, PP activation in isolated hemolymph was inhibited by potassium cyanide, suggesting that cellular activity is involved. Stimulation by peptidoglycan promoted the generation of reactive oxygen species by silkworm hemocytes. The addition of either the active form of PP or anti-PP antibody to Staphylococcus aureus injected into silkworm larvae delayed or enhanced, respectively, the killing effect of S. aureus, suggesting that activated PP contributes to host resistance to infectious pathogens. These findings suggest that immunologic stimulants such as peptidoglycan or glucan induce reactive oxygen species production from larval hemocytes, followed by the activation of serine protease, which mediates the PP processing reaction and leads to defensive responses.

We have developed a new tobacco mosaic virus (TMV) RNA vector and have used it initially to systemically produce an angiotensin-I-converting enzyme inhibitor peptide (ACEI) in tobacco and tomato plants. This vector incorporates a six base 3' context sequence, which permits readthrough of the stop codon for the TMV 130K protein gene, inserted between the stop codon for the coat protein (CP) gene and ACEI gene. In contrast to previous TMV RNA vectors, the new vector produced both an intact CP and a fused protein consisting of CP and ACEI (CP-ACEI). As a result, the vector could form virus particles and spread systemically from inoculated to non-inoculated leaves. In tomato plants, production of ACEI in fruit was also achieved.

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