A fungal homologue of neuronal calcium sensor-1, Bbcsa1, regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana

Abstract

A fungal homologue of neuronal calcium sensor-1, Bbcsa1, regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana gene expression and other biological processes. The filamentous fungus Beauveria bassiana is a broad-host-range pathogen of insects that acidifies the extracellular milieu during growth and pathogenesis towards target hosts. A collection of B. bassiana random insertion mutants were screened on pH indicator plates and one mutant was isolated that displayed reduced acidification. The random insertion site was mapped to a gene that displayed homology to the neuronal calcium sensor/frequenin protein family and was designated Bbcsa1. To validate the role of Bbcsa1 in B. bassiana, a targeted gene-knockout was constructed. Data confirmed that Bbcsa1 was not an essential gene and the DBbcsa1 strain displayed delayed acidification of the medium when grown in Czapek-Dox medium, as compared with the wild-type parent. HPLC profiling of secreted metabolites did not detect any major changes in the production of organic acids, although downregulation of the membrane H + pump/ATPase was noted in the mutant. A slight growthdeficient phenotype was observed for the DBbcsa1 strain on Czapek-Dox and potato dextrose media, which was accentuated at high calcium concentrations (500 mM) and 1.5 M sorbitol, but was unaffected by EDTA or SDS. Perturbations in vacuole morphology were also noted for the mutant. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the DBbcsa1 mutant when applied topically, representing the natural route of infection, but no significant effect was observed when fungal cells were directly injected into target hosts. These results suggest that Bbcsa1 participates in pre-penetration or early penetration events, but is dispensable once the insect cuticle has been breached. INTRODUCTIONIntracellular Ca 2+ levels affect many signal transduction cascades that transduce stimuli to appropriate cellular responses. Cellular Ca 2+ levels are carefully controlled by a diverse set of Ca 2+ channels, pumps, transporters and calcium-binding proteins (Cunningham, 2011). Members of the last-named group are often divided into two classes: calcium buffers and calcium sensors. Neuronal calcium sensor-1 (NCS-1)/frequenin (the yeast homologue) represents a subfamily of the calcium sensors that contain three functional calcium-binding EF-hand motifs and show high-affinity calcium binding at concentrations below that which would activate other calcium transducers such as calmodulin (Burgoyne, 2004). Upon calcium binding, these proteins are thought to undergo a conformational change allowing for interactions with target proteins and/ or receptors. Many NCS proteins are N-terminally myristoylated, with the solvent accessibility of this lipid moiety dependent upon Ca 2+ binding. This 'switch' is considered to act as a reversible mechanism for association of NCS proteins with membranes and their components, although in some sy...

Stress response signaling and virulence: insights from entomopathogenic fungi

“…Sequestration of nutrients including Fe 2+ and surface exposure to sunlight, act as nutrient and oxidative stressors, respectively, and penetration through the cuticle and then interaction with immune responses include exposure to further oxidative stress. The nature of membrane carbohydrates, the production of acidic metabolites including oxalate, and use of cytochrome P450s capable of assimilating host hydrocarbons contribute to attachment and degradation of the host epicuticle (Fan et al 2012;Kirkland et al 2005;Pedrini et al 2010;Zhang et al 2011a. Chitinases, protease, and lipases, along with mechanical pressure allow further ingress (Ortiz-Urquiza and Keyhani 2013).…”

Improving mycoinsecticides for insect biological control

“…Targeted deletion of Msn2 resulted in a strain impaired for growth at low pH, showing defects in virulence and sporulation. The ability to acidify the medium, and in particular production of oxalic acid, is known to contribute to the ability of the fungus to penetrate the insect cuticle (Fan et al 2012b;Kirkland et al 2005), and therefore, loss of this ability may in part account for the observed phenotype seen in the Δmsn2 mutant strain. Finally, the evolutionarily conserved transcriptional cofactor, multiprotein bridging factor 1 (MBF1), which acts as a link between TATAbinding protein (TBP) and transcription factors, has been shown to contribute to the regulation of stress responses and virulence in B. bassiana (Ying et al 2014).…”

Bbmsn2 acts as a pH‐dependent negative regulator of secondary metabolite production in the entomopathogenic fungus Beauveria bassiana

“…Construction and preliminary screening of a B. bassiana random insertion (T-DNA) library has already been reported (Luo et al, 2009). Isolation of fungal genomic DNA and use of YADE to clone fragments flanking the T-DNA insertion site as described (Fan et al, 2012a). All PCR products were cloned into the A-T cloning vector pUCm-T (Bio Basic, Canada) and subsequently sequenced.…”