Summary: Silkworms are killed by injection of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pyogenes, into the haemolymph. Gene disruption mutants of S. aureus whose open reading frames were previously uncharacterized and that are conserved among bacteria were examined for their virulence in silkworms. Of these 100 genes, three genes named cvfA, cvfB, and cvfC were required for full virulence of S. aureus in silkworms. Haemolysin production was decreased in these mutants. The cvfA and cvfC mutants also had attenuated virulence in mice. S. pyogenes cvfA‐disrupted mutants produced less exotoxin and had attenuated virulence in both silkworms and mice. These results indicate that the silkworm‐infection model is useful for identifying bacterial virulence genes.

The intestinal resident Candida glabrata opportunistically infects humans. However few genetic factors for adaptation in the intestine are identified in this fungus. Here we describe the C. glabrata CYB2 gene encoding lactate dehydrogenase as an adaptation factor for survival in the intestine. CYB2 was identified as a virulence factor by a silkworm infection study. To determine the function of CYB2, we analysed in vitro phenotypes of the mutant Δcyb2. The Δcyb2 mutant grew well in glucose medium under aerobic and anaerobic conditions, was not supersensitive to nitric oxide which has fungicidal-effect in phagocytes, and had normal levels of general virulence factors protease, lipase and adherence activities. A previous report suggested that Cyb2p is responsible for lactate assimilation. Additionally, it was speculated that lactate assimilation was required for Candida virulence because Candida must synthesize glucose via gluconeogenesis under glucose-limited conditions such as in the host. Indeed, the Δcyb2 mutant could not grow on lactate medium in which lactate is the sole carbon source in the absence of glucose, indicating that Cyb2p plays a role in lactate assimilation. We hypothesized that Cyb2p-mediated lactate assimilation is necessary for proliferation in the intestinal tract, as the intestine is rich in lactate produced by bacteria flora, but not glucose. The Δcyb2 mutant showed 100-fold decreased adaptation and few cells of Saccharomyces cerevisiae can adapt in mouse ceca. Interestingly, C. glabrata could assimilate lactate under hypoxic conditions, dependent on CYB2, but not yeast S. cerevisiae. Because accessible oxygen is limited in the intestine, the ability for lactate assimilation in hypoxic conditions may provide an advantage for a pathogenic yeast. From those results, we conclude that Cyb2p-mediated lactate assimilation is an intestinal adaptation factor of C. glabrata.

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals.

Summary: We previously reported that the cvfA gene is a virulence regulatory gene in Staphylococcus aureus. Here, we identified a novel gene named sarZ that acts as a multicopy suppressor of decreased haemolysin production in the cvfA deletion mutant. The amount of sarZ transcripts was decreased in the cvfA mutant. The sarZ‐deletion mutant produced less haemolysin and attenuated virulence in a silkworm‐infection model and a mouse‐infection model. The amino acid sequence of the sarZ gene product had 19% identity with the transcription factor MarR in Escherichia coli, and the internal region contained a winged helix–turn–helix motif (wHTH), a known DNA binding domain. Purified recombinant SarZ protein had binding affinity for the promoter region of the hla gene that encodes α‐haemolysin. SarZ mutant proteins with an amino acid substitution in the N‐terminal region or in the wHTH motif had significantly decreased DNA binding. The mutated sarZ genes encoding SarZ mutant proteins with a low affinity for DNA did not complement the decreased haemolysin production or the attenuated killing ability against silkworms in the sarZ mutant. These results suggest that the DNA binding activity of the SarZ protein is required for virulence in S. aureus.

Silkworm, Bombyx mori, has various advantages as an experimental animal, such as the low cost for rearing and fewer ethical problems. Models utilizing silkworms of infection with pathogenic bacteria have been established for identification of genes encoding virulence factors by large‐scale in vivo screening. In this review, we describe recent progress in the study of silkworm infection models for elucidating the mechanisms of fungi infection. Silkworm infection models have been established for Candida albicans, Candida tropicalis, Candida glabrata and Cryptococcus neoformans, which are yeast type fungi, and Aspergillus fumigatus, Arthroderma vanbreuseghemii, Arthroderma benhamiae, Microsporum canis, Trichophyton rubrum, and Rhizopus oryzae, which are filamentous fungi. Novel genes encoding virulence factors in C. albicans and C. glabrata have been identified by using the silkworm infection models. We here outline the benefits of using silkworm infection models and a strategy for identifying the genes responsible for pathogenicity of microorganisms such as fungi. © 2019 The Authors. Microbiology and Immunology Published by The Societies and John Wiley & Sons Australia, Ltd.

Aims: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. Methods and Results: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD₅₀ value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. Conclusions: The silkworm–C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. Significance and Impact of the Study: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.