Uncovering the function of insulin receptor substrate in termites' immunity through active immunization.

Insulin receptor substrate (IRS) proteins are key mediators in insulin signaling pathway. In social insect lives, IRS proteins played important roles in caste differentiation and foraging, but there function in disease defenses such as active immunization has not been reported yet. To investigate the issue, we successfully suppressed the IRS gene 3 days after dsRNA injection. Suppressing IRS gene increased the contents of glucose, trehalose, glycogen, and triglyceride and decreased the content of pyruvate in termites, and led to the metabolic disorder of glucose and lipids. IRS suppressing significantly enhanced grooming behaviors of nestmates of fungus-contaminated termites and hence increased the conidial load in the guts of the nestmates. Additionally, IRS suppressing led to significant downregulation of the immune genes Gram-negative bacteria-binding protein2 (GNBP2) and termicin and upregulation of the apoptotic gene caspase8, and hence diminished antifungal activity of nestmates of fungus-contaminated termites. The above abnormal behavioral and physiological responses significantly decreased the survival rate of dsIRS-injected nestmates of the fungus-contaminated termites. These findings suggest that IRS is involved in regulation of active immunization in termites, providing a better understanding of the link between insulin signaling and the social immunity of termites.

Screening of ent-copalyl diphosphate synthase and metabolic engineering to achieve de novo biosynthesis of ent-copalol in Saccharomyces cerevisiae.

The diterpene ent-copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta (Andrographis paniculata). De novo biosynthesis of ent-copalol has not been reported, because the catalytic activity of ent-copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of ent-copalol, Saccharomyces cerevisiae was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of ent-copalol. The strain capable of synthesizing diterpene geranylgeranyl pyrophosphate was constructed by strengthening the mevalonate pathway genes and weakening the competing pathway. Five full-length ApCPSs were screened by transcriptome sequencing of A. paniculata and ApCPS2 had the best activity and produced ent-CPP exclusively. The peak area of ent-copalol was increased after the ApCPS2 saturation mutation and its configuration was determined by NMR and ESI-MS detection. By appropriately optimizing acetyl-CoA supply and fusion-expressing key enzymes, 35.6 mg/L ent-copalol was generated. In this study, de novo biosynthesis and identification of ent-copalol were achieved and the highest titer ever reported. It provides a platform strain for the further pathway analysis of andrographolide and derivatives and provides a reference for the synthesis of other pharmaceutical intermediates.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Roles of selenoprotein T and transglutaminase in active immunization against entomopathogenic fungi in the termite Reticulitermes chinensis.

Active immunization can protect individuals from infectious diseases in social insects. It is well established that trace elements are essential to the host immune system, but the related gene functions in insect social immunity are unknown. Here, we found that the levels of three free elements (Se, Ca and Cr) and selenoprotein T (SELT) expression were significantly decreased in the termite Reticulitermes chinensis Snyder during active immunization against the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin. Thus, we further explored the role of the SELT gene in the active immunization of termites. After SELT was significantly silenced by RNAi, the nestmates of fungus-contaminated termites exhibited reduced antifungal activity and increased mortality, along with increased expression of the immune genes transglutaminase (TG) and transferrin (Tsf), indicating that the active immunization of termites was disrupted by SELT silencing. Moreover, the TG-knockdown nestmates of fungus-contaminated termites significantly decreased grooming behavior, antifungal activity and survival, despite the upregulation of SELT expression, also suggesting that the active immunization of termites was disrupted by the silencing of TG. These findings demonstrated that both SELT gene and TG gene play important roles in driving active immunization against the entomopathogenic fungus M. anisopliae in R. chinensis.

The Mechanisms of Social Immunity Against Fungal Infections in Eusocial Insects.

Entomopathogenic fungus as well as their toxins is a natural threat surrounding social insect colonies. To defend against them, social insects have evolved a series of unique disease defenses at the colony level, which consists of behavioral and physiological adaptations. These colony-level defenses can reduce the infection and poisoning risk and improve the survival of societal members, and is known as social immunity. In this review, we discuss how social immunity enables the insect colony to avoid, resist and tolerate fungal pathogens. To understand the molecular basis of social immunity, we highlight several genetic elements and biochemical factors that drive the colony-level defense, which needs further verification. We discuss the chemosensory genes in regulating social behaviors, the antifungal secretions such as some insect venoms in external defense and the immune priming in internal defense. To conclude, we show the possible driving force of the fungal toxins for the evolution of social immunity. Throughout the review, we propose several questions involved in social immunity extended from some phenomena that have been reported. We hope our review about social 'host-fungal pathogen' interactions will help us further understand the mechanism of social immunity in eusocial insects.