Short-Form Bomanins Mediate Humoral Immunity in Drosophila

Abstract

The Bomanins (Boms) are a family of a dozen secreted peptides that mediate the innate immune response governed by the Drosophila Toll receptor. We recently showed that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. Here, we characterize the activity of individual Bom family members. We provide evidence that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. We further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, we find that the level of antifungal activity both in vivo and in vitro is linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) drosomycin and metchnikowin, we find no evidence that Boms act by modifying the expression of the mature forms of these antifungal AMPs.

Keywords: Antifungal activity; Antimicrobial peptides; Bomanins; Drosophila; Effector; Host defense; NF-κB; Toll receptor.

Fig. 1

Organization of the Bom family cluster at cytogenetic map position 55C. This figure reflects a renaming of the Bom family members, with the previously used CG or IM prefixes replaced with the Bom prefix, while retaining the gene number for both DIMs (1 or 2 digits) and CGs (last 3 digits) for continuity with prior literature [9, 17]. Accordingly, the 2 remaining Bom genes, located outside the 55C region, are now Bom778 and Bom791. For the 10 genes in the drawing, arrowhead direction denotes gene orientation. The color indicates the form of the encoded Bom peptide: short (red), tailed (yellow), or bicipital (orange). The bracket at the top encompasses the Bom genes in the 55C-Right genomic construct and the dotted lines below illustrate the extent of the chromosomal deletions in Bom∆^55C and Bom∆^left.

Fig. 2

A single Bom gene rescues the Bom∆^55C immunodeficiency toward C. glabrata. The graph illustrates the survival of transgenic adults at indicated intervals after infection with C. glabrata. Each transgene encodes either the 4 Bom peptides of 55C-Right or a single one of these peptides, as indicated. All transgenes were present in 2 copies in a Bom∆^55C background. Each curve represents the pooled results of 3 independent experiments involving ≥25 flies per genotype. Survival curves were compared using the Gehan-Breslow-Wilcoxon test. Significance is shown relative to the no transgene control (Bom∆^55C) and adjusted for multiple comparisons (*** p < 0.0001; ns, not significant, p > 0.05).

Fig. 3

Short-form Bom peptides mediate survival after infection with C. glabrata. The graph plots the survival of adults at indicated intervals after infection with C. glabrata. Bom transgenes were expressed under control of either their endogenous promoter or pBOM3. All transgenes were present in 2 copies in a Bom∆^55C background. Data were captured and analyzed as in Figure 2.

Fig. 4

Fungicidal activity of hemolymph is Bom-dependent. Hemolymph activity was assayed for 4 sets of flies: w¹¹¹⁸ (wild-type), Bom∆^55C, and Bom∆^55C carrying either a 55C-Right (four-gene) or Bom065 (single-gene) construct. All flies had been pricked 1 day earlier with heat-killed M. luteus to induce Toll-mediated gene expression. Fungicidal activity was assayed by incubating cell-free hemolymph with C. glabrata for 1 h at room temperature (RT) or for 24 h at 4°C before spreading the mixture onto a yeast (YPD) plate. Percent killing was calculated by comparing the colony count for each sample to that for yeast mixed with a w¹¹¹⁸ uninduced hemolymph control. Experiments were performed in triplicate; error bars represent standard error of the mean. One-way ANOVA followed by Tukey's test was performed for each incubation condition. Significance is shown relative to the w¹¹¹⁸ uninduced control (⚫⚫⚫ and ◆◆◆ = p < 0.0001; ns, not significant, p > 0.05).

Fig. 5

Analysis of hemolymph from adult Drosophila by MALDI-TOF MS. Hemolymph (50 nL) was collected from a single fly from each genotype and analyzed by MALDI-TOF MS using the linear (a, b, d, f) or reflectron mode (c, e, g) (see Materials and Methods). Representative MALDI spectra from ≥5 such samples are shown for hemolymph from a w¹¹¹⁸ fly either uninduced (control) (a), or induced 24 h earlier by Toll pathway stimulation with heat-killed M. luteus (b, c), and induced flies of the following genotypes: Bom∆^55C (d, e) and Bom∆^55C; {pBOM3-Bom068} (f, g). AU, arbitrary units. The y axis was scaled to the tallest peak within the window examined. Numbering and naming of induced peptides corresponds to published conventions [9]. As observed previously [9], a prominent peak appears in control spectra that becomes insignificant in the context of Toll induction. Drs, drosomycin; Mtk, metchnikowin.