Fungal extracellular ribotoxins as insecticidal agents Miriam Olombrada, Elías Herrero-Galán 1 , Daniel Tello 2 , Mercedes Oñaderra, José G. Gavilanes, Álvaro Martínez-del-Pozo, Lucía García-Ortega * Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain article info Article history: Received 2 July 2012 Received in revised form 24 October 2012 Accepted 25 October 2012 Keywords: Fungal ribotoxin Ribonuclease Ribosome inactivation Insecticide abstract Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen Hirsutella thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and a-sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Microorganisms populate almost any ecological niche and establish complex and sometimes essential relationships with higher organisms. Consequently, a high diversity of interactions can be found, most of them based on biological mutualisms or antag- onisms (Berenbaum and Eisner, 2008). In this regard, fungi constitute a rich source of nitrogen and phosphorous for arthro- pods and therefore share a long evolutionary history with them. Thus, not only fungi are under constant attack by fungivorous animals such as collembolan, mites and insects (Boddy and Jones, 2008; Ruess and Lussenhop, 2005) but also display frequent mutualistic relationships (Scott et al., 2008), as can be exemplified by fungus farming ants (Currie et al., 1999). Underlying these relationships there is a complex interaction network involving preying, defense, and feeding. Elucidation of these interactions can drive to the discovery and understanding of natural products of unforeseen function (Berenbaum and Eisner, 2008). Within this idea, it is noticeable that the fungal genus Hirsutella contains over 50 fungal species which are known to be entomopathogens. Under in vivo conditions, conidia contact the host, attach to the cuticle, germinate, and penetrate through it (Liu et al., 1995). Along the 1990s, crude filtrates of Hirsutella thompsonii, a particular species of this genus, were found to be toxic to a wide variety of arthropods including moth, fly, and mosquito larvae, aphids and mites (Liu et al., 1995, 1996; Omoto and McCoy, 1998; Vey et al., 1993). A toxic protein, Hirsutellin A (HtA), was then isolated from these cultures and proved to show broad pathogenic activity against insects (Krasnoff and Gupta, 1994; Mazet and Vey, 1995). HtA was lethal to Galleria mellonella larvae upon injection (Mazet and Vey, 1995) and caused detectable cytopathic effects on Spodoptera frugiperda cells (Sf9), inhibiting cell growth (Liu et al., 1995), for example. The ribosomal RNA (rRNA) extracted from these cells contained a small fragment of about 500e600 nt (Liu et al., 1996) resembling the a-fragment produced by fungal ribo- toxins upon inactivation of eukaryotic ribosomes (Chan et al., 1983; Endo and Wool, 1982; Endo et al., 1983; Schindler and Davies, 1977). Accordingly, HtA was demonstrated to be a ribotoxin (Herrero- Galán et al., 2008) and subjected to detailed structural and func- tional characterization (Herrero-Galán et al., 2012a, 2012b; Viegas et al., 2009). Fungal ribotoxins are extracellular and highly specific ribonu- cleases which behave as potent inhibitors of protein biosynthesis by being able to inactivate ribosomes from almost any organism (Gasset et al., 1994; Kao et al., 2001; Lacadena et al., 2007; Martínez-Ruiz et al., 2001). They cleave the larger rRNA component at a single phosphodiester bond located within the universally conserved sarcin/ricin loop (SRL) (Chan et al., 1983; Endo and Wool, 1982; Endo et al., 1983; Schindler and Davies, 1977), leading to Abbreviations: HtA, hirsutellin A; RNases, ribonucleases; SRL, sarcin/ricin loop; TLC, thin layer chromatography. * Corresponding author. E-mail address: lucia@bbm1.ucm.es (L. García-Ortega). 1 Present address: Departamento de Estructura de Macromoléculas, Centro Nacional de Biotecnología CNB-CSIC, Darwin 3, 28049 Madrid, Spain. 2 Present address: Servicio de Inmunología, Hospital Universitario de La Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria Princesa (IIS-IP), 28006 Madrid, Spain. Contents lists available at SciVerse ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibmb.2012.10.008 Insect Biochemistry and Molecular Biology 43 (2013) 39e46