Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana

Abstract

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C 33 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases. INTRODUCTIONUnlike other insect-pathogenic micro-organisms which must be ingested to initiate disease (virus, bacteria, nematodes and protozoa), entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, mostly invade target hosts by penetrating through their cuticle.The outermost insect surface or epicuticle is covered by a lipid-rich layer, usually composed of very long-chain hydrocarbons together with variable amounts of fatty alcohols, fatty acids and wax esters. The epicuticle protects the insect from desiccation, chemical and biological attack, and acts as a platform for host semiochemicals (Blomquist et al., 1987;Figueiras et al., 2009;Juarez, 1994). Entomopathogenic fungi have the ability to degrade insect cuticular lipids with hydrocarbons, the preferred components capable of supporting fungal growth (Napolitano & Juarez, 1997). The first evidence of the complete catabolism of insect-like hydrocarbons by entomopathogenic fungi was obtained from B. bassiana and M. anisopliae (Crespo et al., 2000). In addition, alkane-grown B. bassiana showed enhanced virulence, by both increasing mortality, i.e. a greater proportion of target hosts killed over time (Crespo et al., 2002), and reducing the time to kill its insect hosts (Pedrini et al., 2009). Although several aspects of the biochemistry of the interaction between entomopathogenic Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, AhR ...

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

“…S1). The region surrounding the start codon contained the eukaryotic consensus (G/A)NNATGG sequence, including a purine (A) in position −3, as has been reported for highly expressed genes in Saccharomyces cerevisiae (46) and has also been found in B. bassiana P450 genes (43). Variants of the tripartite signal [5′-TAG... TA(T/A)GT.…”

“…However, entomopathogenic fungi are incapable of lignin degradation, and no information is available regarding the presence and/or function of these enzymes in this group of fungi. However, entomopathogenic fungi are able to assimilate various hydrocarbon and lipid components of the insect cuticle, responding to surface cues on their hosts to initiate programs for infection (41)(42)(43).…”

“…In addition to these two elements, an inverted GC box was found at nucleotide −308 and a stress response element (STRE) was found at position −288. A xenobiotic response element (XRE), also present in the promoters of a set of B. bassiana cytochrome P450 monooxygenases implicated in insect cuticle degradation (43), was identified at positions −235, −267, −326, −539, −599, −606, −616, −1,079, and −1,181 (Fig. S1).…”

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

“…CYPs also play a part in fungal pathogenicity against insects. The insect cuticle comprises a mixture of lipids, including long-chain alkanes, alkenes, wax esters and fatty acids, and recently the involvement of CYPs in breaking down this barrier was shown for Beauveria bassiana [75]. Eight CYPs from seven different CYP families were associated with metabolism of cuticle lipid.…”

“…Candida spp. contain multiple CYP52s with overlapping substrate specificity shown by biochemical and genetic methods [76,77], and it was suggested that the CYPs identified as being induced by cuticle lipids would also show such overlap [75]. Manipulating these activities could increase virulence and has the potential to assist in bio-control of insects using fungal pathogens.…”