Toll family members bind multiple Spätzle proteins and activate antimicrobial peptide gene expression in Drosophila

Abstract

The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes. The canonical Toll receptor (Toll-1) is activated by the cytokine Spätzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well-understood. Here, we conducted in vitro assays in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine whether they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin. All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation. We found that the Toll-1 and Toll-7 ectodomains bind Spz-1,-2, and-5, and also vesicular stomatitis virus (VSV) virions, and that Spz-1,-2,-5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7. In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species (Enterococcus faecalis and Pseudomonas aeruginosa), the opportunistic fungal pathogen Candida albicans, and VSV with different survival times in adult females and males compared with WT fly survival. Our results suggest that all Toll family members can activate several AMP genes. Our results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but they differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression.

A Toll-Spätzle Pathway in the Immune Response of Bombyx mori

“…The yeast two-hybrid assay results showed that BmToll11 and BmToll9-1 could interact with BmSpz2 and activate the expression of AMP genes (Figures 2 and 4). Additionally, the results indicated that one BmSpz could combine with multiple BmTolls, which is consistent with the reported binding of Spzs to multiple Tolls in D. melanogaster [6]. In our study, we also found that other Tolls, except for BmToll9-1 and BmToll11, Yeast two-hybrid assay for determination of BmToll ecto 11 and BmToll ecto 9-1 with BmSpz2.…”

“…The mechanism that the activation of the Toll signaling pathway in D. melanogaster and M. sexta is relatively clear. In Drosophila, the Toll pathway is activated after the activated Spz binding with the Toll receptor [6]. We demonstrated that BmToll11 and BmToll9-1 genes function in innate immune responses in silkworms.…”

TmSpz4 Plays an Important Role in Regulating the Production of Antimicrobial Peptides in Response to Escherichia coli and Candida albicans Infections

“…Thus, the molecular functions of spätzle proteins in Toll receptor activation and the subsequent activation of AMPs have been well studied in various insects. For example, in Drosophila, Spz-1, -2, and -5 bind to Toll-1 and Toll-7 to produce drosomycin and several other AMPs [16,29]. Additionally, in B. mori, spätzle-1 binds to the Toll receptor to activate the production of attacin-1, cecropin-6, and moricin [30].…”

“…In Drosophila, six spätzle homologues (Spz1-6) have been identified, including the first identified spätzle gene, spz-1, which encodes a protein containing neurotrophin-like cystine-knot domains [15]. The study of the interaction of spätzle family proteins with each other and with Toll receptors during the activation of AMP gene expression showed that the binding of Spz-1, -2, and -5 to the Toll-1 and Toll-7 ectodomains promotes the activation of drosomycin and several other AMP genes [16][17][18].…”

Transcriptomic analyses reveal comprehensive responses of insect hemocytes to mycopathogen Beauveria bassiana, and fungal virulence-related cell wall protein assists pathogen to evade host cellular defense

“…At the late stage, Toll-like receptor signaling pathway is activated. In Drosophila species, spätzle protein functions as the ligand for the Toll-1 receptor and activates the expression of AMP genes [41]. This result suggests that G. mellonella hemocytes sequentially perform transcriptional regulation of Toll pathway.…”