Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana

Abstract

Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

Proteomics as a tool for tapping potential of entomopathogens as microbial insecticides

“…Proteomics of EPF have been applied to elucidate insect host response to EPF toxic secondary metabolites, and profiling secreted proteins (secretome) during the EPF infection (Beys‐da‐Silva et al, ; Dionisio, Kryger, & Steenberg, ; J. Fan, Han, Chen, Hu, & Ye, ; Murad et al, ; Murad, Laumann, Mehta, Noronha, & Franco, ).…”

“…Secretome analysis of B. bassiana and M. anisopliae against the range of insect cuticles and intact cadavers reported several secreted proteins that are involved in cuticular degradation and defense against insect immunity responses (Beys‐da‐Silva et al, ; Dionisio et al, ; Murad et al, , ). Murad et al (, ) had performed the two‐dimensional gel electrophoresis (2‐DE) proteomic analysis of the secretome of B. bassiana and M. anisopliae , respectively, in response to the presence of cowpea weevil's ( Callosobruchus maculatus ) cuticles.…”

“…A more recent secretome analysis used the DDA shotgun‐based gel‐free proteomics in characterizing B. bassiana and M. anisopliae secretome (Beys‐da‐Silva et al, ; Dionisio et al, ; Kislinger & Emili, ). Furthermore, both studies harnessed the availability of annotated genome sequence of both EPF which aided protein identification immensely.…”

“…Furthermore, both studies harnessed the availability of annotated genome sequence of both EPF which aided protein identification immensely. Secretome analysis on B. bassiana during infection on American cockroach ( Periplaneta americiana ) had identified two Pr1 subtilisins (Pr1A and Pr1B), several GH18 class chitinases and chitosanases, which are involved in chitin degradation, several peptidases pertinent for deriving nutrients from insect cuticles and expression of autolysins are implicated in perturbing chitin‐trigged insect immunity (Dionisio et al, ).…”

“…In contrast, M. anisopliae expressed different combinations of Pr1 subtilisins (Pr1A, Pr1B, Pr1I, Pr1J, and Pr1C), antioxidants (catalases and superoxide dismutase), and immune suppressing‐related protein, tyrosine phosphatase (Beys‐da‐Silva et al, ). Interestingly, though B. bassiana and M. anispoliae studies both reported upregulation of Pr1A and Pr1B, M. anisopliae was found to overexpress more Pr1B proteins compared with Pr1B whereas B. bassiana appears to relatively more Pr1A instead (Beys‐da‐Silva et al, ; Dionisio et al, ). Pr1 works together with other cuticle‐degrading enzymes in achieving cuticular penetration (Butt et al, ).…”