Filamentous fungi produce a variety of secondary metabolites, many of which are involved in virulence [23, 24] . The ability of an entomopathogenic fungus to infect its insect host is thought to depend on the coordinated activity of these virulence molecules together with mechanical pressure of hyphae on the exoskeleton and/or gut peritrophic membrane. In support of this model, we detected several transcripts involved in the production of A. apis hydrolytic enzymes, listed in Additional file 1: Table S1. For example, we identified genes encoding chitinases, proteases and esterases, degrading enzymes that could be involved in both host invasion and escape processes. We have also identified several genes encoding homologs of a cutinase transcription factor, Ctf1, indicating the ability of this fungus to utilize different nutrient substrates, including plant cutin, a variety of lipids as well as synthetic triacylglycerols and esters .
Furthermore, our updated genome annotation reveals a number of genes encoding homologs of well-known toxins and a diverse family of enzymes, some of them involved in the Aflotoxin (AF)-Sterigmatocystin (ST) biosynthesis pathway (AflR
SteW, OmtA, OrdA), HC-toxin biosynthesis (Tox A, ToxG, ToxD, ToxF and Hts1), and a super killer protein 3 (Ski3) (Additional file 1: Table S1). In addition to genes involved in toxin biosynthesis, our gene set includes a large variety of genes involved in transcription, translation and biosynthesis of enzymes that have also been implicated in virulence. The list includes an extracellular glucoamylase, 3 chitinases, 16 amidases, 30 esterases, 42 proteases, 24 lipases and others (Additional file 1: Table S1). A large number of these enzymes were found to be expressed in the infected host, which may implicate their role in host pathogenesis. Among them, a member of the metallopeptidase family M28 containing a transferrin receptor-like dimerisation domain (IPR007365) and a protease-associated PA domain (IPR003137) similar to a metallopeptidase found in C. immitis as well as vacuolar endopeptidase Pep2, a broad specificity secreted hydrolase that has been implicated in Aspergillus fumigatus virulence . Another interesting example is that of the phospholipases (plcA, plcB, plcC and plcD) previously identified in Mycobacterium tuberculosis. The A. apis genome encodes multiple genes homologous to those found in M. tuberculosis, Neosartorya ficheri and Phytophtora infestans (Additional file 1: Table S1).
Many virulence factors produced by A. apis have been previously identified using enzymatic activity assays and electrophoresis. The list includes galactosidases, glucosidases, catalases, phoshphatases, DNAses, and RNAses [27, 28]. Among those, 12 enzymes and 285 isozymes were found in A. apis and closely related fungi, Ascosphaera aggregata and Ascosphaera proliperda[27, 29]. Enzymatic activity of 11 enzymes (i.e. protease, β-N-acetylglucosaminidase, alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucosidase and α-mannosidase) has been characterized by . However, it is very difficult to compare our findings to those previously reported since earlier investigations mostly focused on the enzymatic activity, and therefore lacked gene and/or protein related data [27, 28]. Furthermore, some of the previously published data is not supported by our findings. For example, the lack of chitinase activity in A. apis has been determined experimentally by many investigators, that led to a conclusion that A. apis does not digest insect chitin [27, 30–32]. In the absence of chitinase activity, the N-acetyl-β-glucosaminidase in conjunction with hyphal pressure has been suspected in aiming penetration of the peritrophic matrices lining the gut epithelium . However, our gene predictions identified at least three different glycosyl hydrolases with the GH18 Pfam domain characteristic of class III and class V chitinases (http://www.cazy.org) (Additional file 1: Table S1). GH family 18 chitinases are frequently implicated in the mycoparasitic activity by the degradation of exogenous chitin  and thus their presence in A. apis revives the possibility that these enzymes are involved in host pathogenesis. We detected four and two sequence reads from this gene in axenic culture and infection, respectively, indicating that it is not exclusively expressed during infection. However, additional work is needed to definitively determine whether any of these chitinases are involved in penetrating the peritrophic matrix of the honey bee larva. It is noteworthy that GH18 chitinases are highly sensitive to allosamidin (β-D-allopyranoside), a potent chitinase inhibitor (http://www.reference.md) and therefore may be a potential target for fungal control.