ISJ 9: yyy-xxx, 2012 ISJ 9: 163-168, 2012 ISSN 1824-307X MINIREVIEW Utilization of a silkworm model for understanding host-pathogen interactions C Kaito, H Yoshikai, K Sekimizu Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Accepted September 27, 2012 Abstract Studies of the interactions between humans and pathogenic microorganisms require adequate representative animal infection models. Further, the availability of invertebrate models overcomes the ethical and financial issues of studying vertebrate materials. Insects have an innate immune system that is conserved in mammals. The recent utilization of silkworms as an animal infection model led to the identification of novel virulence genes of human pathogenic microorganisms and novel innate immune factors in the silkworm. The silkworm infection model is effective for identifying and evaluating novel factors involved in host-pathogen interactions. Key Words: insect model; innate immune factor; bacteria; fungi; virulence factor Advantages of the silkworm as an animal infection model Invertebrate animals possess an innate immune system, but lack an acquired immune system. Many aspects of the innate immune system of invertebrate animals are conserved in mammals. For example, cationic antimicrobial peptides and Toll receptors recognizing pathogens are found in both invertebrate animals and mammals (Okada and Natori, 1983; Hoffmann, 1995; Natori, 2010). Therefore, studies using invertebrate animals can be performed to develop a better understanding of the host-pathogen interactions in mammals without the ethical and financial issues (Seabra and Bhogal, 2009). Silkworms are larvae of the moth Bombyx mori, a lepidopteran species (Fig. 1). Silkworms form cocoons where they develop into pupae. Humans have used these cocoons as raw materials for silk for over 5000 years (Goldsmith et al., 2005). Bombyx mori is the only domesticated insect species, and the silkworm cannot survive in the natural world, probably due to their ineffective locomotion. In contrast to wild insects, silkworms can barely bite human fingers or escape from a breeding cage. Silkworms typically consume mulberry leaves, but an artificial diet for silkworms has also been established and is commercially available. Thus, rearing silkworms in the laboratory is easy. Studies of host-pathogen interactions require quantitative evaluation of the virulence properties of ___________________________________________________________________________ Corresponding author: Chikara Kaito Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan E-mail: kaito@mol.f.u-tokyo.ac.jp pathogenic microorganisms. To evaluate pathogenic virulence quantitatively, injection of a precise amount of the pathogen solution into model animals is essential. The large body size of the fifth instar silkworm (~ 5 cm) allows for the injection of a very precise amount of the pathogen solution into the silkworm hemolymph using a tuberculin syringe equipped with a 27-gauge needle (Kaito and Sekimizu, 2007), whereas injection of a precise sample amount is more difficult in small body-sized invertebrates such as Drosophila melanogaster and Caenorhabditis elegans. Injection of human pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa into the silkworm hemolymph kills the silkworm (Kaito et al., 2002). S. aureus injected into silkworms proliferates in the hemolymph. The lethal effects of S. aureus injection in silkworms are blocked by the injection of antibiotics. These observations suggest that the lethal effects of S. aureus in silkworms require bacterial proliferation (Kaito et al., 2002). The silkworm-S. aureus infection model allows for the identification of biologic molecules involved in the ability of S. aureus to escape various innate immune factors of the silkworm and to proliferate in the silkworm hemolymph. Importantly, infection experiments using silkworms can be performed at 37 ˚C, the temperature at which most human pathogenic microorganisms exhibit high virulence properties (Kaito et al., 2011). Genetic and biochemical analyses of silkworms are essential for identifying biologic molecules of silkworms that are involved in host-pathogen interactions. The Bombyx mori genome project was recently completed and genome data are now 163 Fig. 1 5th instar larvae of Bombyx mori. Tuberculin syringe equipped with a 27-gauge needle is shown above the silkworm. available on line (Shimomura et al., 2009). In addition, construction of transgenic silkworms is established (Tomita, et al., 2003). For biochemical analysis, biologic molecules from crude silkworm biomaterials must first be purified and identified. A fifth instar silkworm weighs around 2 grams, and thus an adequate amount of silkworm biomaterial can easily be prepared for purifying biologic molecules. Identification of bacterial and fungal virulence factors using silkworms S. aureus is a pathogenic Gram-positive bacterium present in the noses of 30 % of healthy individuals. To identify novel virulence factors of S. aureus, 100 hypothetical genes that are conserved among bacteria were disrupted and examined for lethal activity against silkworms. Gene-disrupted mutants of three novel genes, named cvfA, cvfB, and cvfC (conserved virulence factor A, B, and C), exhibited attenuated lethality in silkworms (Kaito et al., 2005) (Table 1). These gene-disrupted mutants also showed attenuated virulence in mice, indicating that these genes contribute to the virulence of S. aureus not only in insects but also in mammals (Kaito et al., 2005; Matsumoto et al., 2007; Marincola et al., 2012). Streptococcus pyogenes is a human pathogenic Gram-positive bacterium that causes various diseases, including adenoiditis and necrotizing fasciitis. The cvfA gene is also required for the lethality of S. pyogenes in silkworms and mice, and it is involved in the expression of various genes in S. pyogenes (Kaito et al., 2005; Kang et al., 2010; Kang et al., 2012) (Table 1). The cvfA gene is required for hemolysin production in both S. aureus and S. pyogenes. CvfA protein is a cyclic phosphodiesterase that cleaves a 2’,3’-cyclic phosphodiester linkage at the 3’-terminal nucleotide of RNA (Kaito et al., 2005; Nagata et al., 2008). The cvfB gene contributes to S. aureus hemolysin production via a virulence regulatory gene, agr (Matsumoto et al., 2007). Crystal structure analysis revealed that CvfB has a novel L-shaped structure comprising three S1 RNA binding domains and a winged-helix domain (Matsumoto et al., 2010). The cvfC gene contributes to S. aureus resistance to detergents via the expression of thymidylate synthetase (Ikuo et al., 2010). These novel virulence factors are conserved in many human pathogenic bacteria and their molecular functions are different from those of other well-known virulence factors. To determine whether S. aureus virulence factors against mammals contribute to S. aureus lethality in silkworms, S. aureus gene-disrupted mutants of hemolysins, cell wall proteins, and virulence regulators were examined for their attenuated lethality against silkworms (Miyazaki et al., 2012) (Table 1). The results demonstrated that S. aureus hemolysins are not required for virulence in silkworms. In contrast, several cell wall proteins and virulence regulators are required for S. aureus lethality in silkworms. Thus, although not all S. aureus virulence factors against mammals can be evaluated in silkworms, silkworms are useful for evaluating the effects of S. aureus cell wall proteins and virulence regulators. That is, interactions between the host animal and S. aureus cell wall proteins or between the host animal and S. aureus virulence regulators are conserved among invertebrates and vertebrates. The silkworm model is also applicable for evaluating virulence factors of Gram-negative human pathogenic bacteria. Enterohemorrhagic Escherichia coli (EHEC) is a human pathogen that causes encephalopathy and nephropathy. EHEC O157:H7 produces Shiga toxins that are toxic to mammalian cells. The EHEC gene-deleted mutant of Shiga toxin exhibits attenuated virulence in a mouse infection model (Eaton et al., 2008), but not in a silkworm model (Miyashita et al., 2012). In contrast, the EHEC gene-deleted mutant of lipopolysaccharide (LPS) O-antigen synthase showed attenuated lethality in both silkworms and mice (Miyashita et al., 2012) (Table 1). The LPS O-antigen mutant of EHEC is sensitive to both silkworm and porcine antimicrobial factors (Miyashita et al., 2012). Therefore, LPS O-antigen is required for the lethal effects of EHEC in silkworms and mice via conferring resistance against innate immune factors of insects and mammals. A transposon mutant library of Serratia marcescens, a 164 Table 1 Summary of biologic molecules identified in the silkworm infection model Pathogenic microorganism Gene Category Function References Gram-positive bacteria Staphylococcus aureus cvfA regulator 2', 3'-cyclic phosphodiesterase (Kaito et al., 2005) cvfB regulator RNA binding protein (Matsumoto, et al., 2010) cvfC regulator conributing to detergent resistance (Ikuo et al., 2010) sarZ regulator transcription factor (Kaito et al., 2006) agr regulator transcription factor and regulatory RNA (Miyazaki et al., 2012) saeRS regulator a two-component regulatory system (Miyazaki et al., 2012) arlRS regulator a two-component regulatory system (Miyazaki et al., 2012) srtA cell wall protein anchoring proteins to cell wall (Miyazaki et al., 2012) clfB cell wall protein binding mammalian cytokeratins (Miyazaki et al., 2012) fnbB cell wall protein binding mammalian fibronectin (Miyazaki et al., 2012) sdrC cell wall protein adherence to mammalian epithelial cells (Miyazaki et al., 2012) Streptococcus pyogenes cvfA regulator 2', 3'-cyclic phosphodiesterase (Kaito et al., 2005) Gram-negative bacteria Enterohemorrhagic Escherichia coli rfbE lipopolysaccharide lipopolysaccharide O-antigen synthesis (Miyashita et al., 2012) waaL lipopolysaccharide lipopolysaccharide O-antigen ligation (Miyashita et al., 2012) Serratia marcescens wecA lipopolysaccharide lipopolysaccharide O-antigen synthesis (Ishii et al., 2012) flhD flagella flagella synthesis (Ishii et al., 2012) fliR flagella flagella synthesis (Ishii et al., 2012) Pseudomonas aeruginosa toxA toxin exotoxin A (Chieda et al., 2011) exoS toxin type III effector protein (Okuda et al., 2010) sodM stress response manganese-superoxide dismutase (Iiyama et al., 2007) sodB stress response iron-superoxide dismutase (Iiyama et al., 2007) Fungi Cryptococcus neoformans gpa1 regulator G-protein alpha subunit (Matsumoto et al., 2012) pka1 regulator catalytic subunit of protein kinase A (Matsumoto et al., 2012) cna1 regulator catalytic subunit of calcineurin (Matsumoto et al., 2012) Candida albicans cmp1 regulator protein phosphatase (Hanaoka et al., 2008) yvh1 regulator protein phosphatase (Hanaoka et al., 2008) sit4 regulator protein phosphatase (Hanaoka et al., 2008) PTC1 regulator protein phosphatase (Hanaoka et al., 2008) Candida glabrata cyb2p metabolism lactate dehydrogenase (Ueno et al., 2011) Host animal Silkworms apoLp-II/I virulence inhibitor suppressing S. aureus hemolysin production (Hanada et al., 2011) PP cytokine inducing innate immune responses (Ishii, et al., 2010) Silkworm hybrid (Kinshu × Showa) was used in studies of P. aeruginosa (Iiyama et al., 2007; Chieda et al., 2011). Silkworm hybrid (Hu • Yo × Tukuba • Ne) was used in other studies. 165 human pathogenic Gram-negative bacterium, was screened for its attenuated lethality in silkworms, leading to the identification of LPS O-antigen synthase as the factor required for silkworm lethality (Ishii et al., 2012). Exotoxin A, a type III effector protein ExoS, and superoxide dismutase of P. aeruginosa, which are virulence factors in mammals, are also required for killing silkworms (Iiyama et al., 2007; Okuda et al., 2010; Chieda et al., 2011) (Table 1). In contrast, P. aeruginosa pyocyanin, which is a virulence factor in mammals, is not required for killing silkworms (Chieda et al., 2008). Many factors in Gram-negative bacteria are required for virulence in both silkworms and mammals, although some factors are specifically required for virulence in mammals. Several virulence factors of human pathogenic fungi, including Cryptococcus neoformans, Candida glabrata, and Candida albicans, were identified by infecting silkworms with gene-deletion mutants (Hanaoka et al., 2008; Ueno et al., 2011; Matsumoto et al., 2012). Gene-deletion mutants of the virulence factors of C. neoformans and C. albicans in mammals showed attenuated virulence in silkworms (Hanaoka et al., 2008; Matsumoto et al., 2012) (Table 1). Cyb2p of C. glabrata and PTC2 of C. albicans have been identified as virulence factors in silkworms and these genes are also required for virulence in mice (Hanaoka et al., 2008; Ueno et al., 2011) (Table 1). These results suggest that human pathogen virulence factors of Gram-positive bacteria, Gram-negative bacteria, and fungi can be identified and evaluated in a silkworm model by infecting silkworms with gene-disrupted mutants. Identification of innate immune factors in silkworms Injection of S. aureus hemolysins into silkworms kills silkworms (Hossain et al., 2006). In contrast, S. aureus hemolysin gene-deleted mutants did not exhibit attenuated killing ability against silkworms (Miyazaki et al., 2012). These findings suggest that silkworm hemolymph contains a factor that inhibits S. aureus hemolysin production. A lipid carrier protein, apolipophorin (ApoLp), purified from silkworm hemolymph shows inhibitory activity against S. aureus hemolysin production (Hanada et al., 2011) (Table 1). The addition of ApoLp to S. aureus culture decreases the expression of saeRS, which is a positive regulator of S. aureus hemolysin genes. Injection of anti-ApoLp antibodies into silkworms sensitizes silkworms against S. aureus. These findings suggest that ApoLp inactivates S. aureus saeRS and decreases hemolysin expression, leading to silkworm resistance against S. aureus. Mammalian mucin also inhibits S. aureus hemolysin production, indicating that resistance to infection by the inhibition of hemolysin production is conserved among insects and mammals. Most innate immune factors contribute to infection resistance by killing pathogenic microorganisms. Novel innate immune factors that do not inhibit bacterial proliferation and inhibit bacterial virulence are not well understood. In addition to ApoLp, apolipoprotein B in mammalian blood and hydrogen peroxide produced by macrophages inhibit S. aureus virulence (Rothfork et al., 2004; Peterson et al., 2008). ApoLp is the first invertebrate biologic molecule found to inhibit bacterial virulence. Silkworm hemolymph contains a cytokine-like peptide named paralytic peptide (PP) (Ishii et al., 2008) (Table 1). PP is synthesized as an inactive precursor and constitutively exists in silkworm hemolymph. Bacterial peptidoglycans and fungal glucans induce reactive oxygen species (ROS) from silkworm hemocytes and ROS activate serine protease. The activated serine protease digests the PP precursor to produce matured PP. The matured PP activates humoral and cellular immune responses, including phagocytosis by silkworm hemocytes, phosphorylation of p38 mitogen-activated protein kinase, and production of antimicrobial peptides (Ishii et al., 2010). Because injection of the anti-PP antibody into silkworms sensitizes silkworms against S. aureus (Ishii et al., 2008), PP contributes to silkworm resistance against S. aureus. PP was originally identified as a biologic molecule that induces muscle contraction in silkworms (Ha et al., 1999). The biologic significance of the muscle-contracting activity of PP in the innate immune system is unknown. Concluding remarks This minireview describes biologic molecules identified in the silkworm infection model. 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