Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics

Abstract

Background: Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution.

Results: We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression.

Conclusions: Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Figure 1

Flowchart of analyses performed on the set of L. hesperus venom gland specific transcripts (VSTs). Colored boxes indicate subsets of sequences resulting from specific analyses. Boxes below the dashed line indicate analyses with the combined proteomic and transcriptomic datasets.

Figure 2

Phylogenetic tree of latrotoxin protein sequences. Previously published sequences labeled with NCBI accession numbers and newly assembled transcript sequences from L. hesperus with a predicted open reading frame of at least 500 amino acids from this study (in bold). The midpoint-rooted tree is a 50% majority-rule consensus of 3002 trees sampled in Bayesian analysis. Values at nodes show posterior probabilities ≥ 0.95, followed after the slash by ML bootstrap values when >= 70%. Shaded boxes indicate clades of known latrotoxin subtypes associated with specific phyletic targets with representative targets shown to the right; illustrations by Emily Damstra and used here with her permission. An asterisk symbol (*) after the name of the sequence indicates exclusive expression in the venom gland (zero eCPM in other tissues) otherwise the minimum fold difference in expression between the venom gland and the other two tissues is indicated. Underlined sequences vary in placement between the Bayesian and ML trees, as described in the text.

Figure 3

Representation of domain structure for selected previously published latrotoxins and latrotoxin sequences from this study. Predictions from InterProScan are shown for ankyrin repeats (blue ovals) and the latrotoxin C-terminal domain (red rectangles). 1 = venom_comp_106397_c0_seq1, 2 = L. tredecimguttatus δ-latroinsectotoxin, 3 = Contig2826, 4 = L. tredecimguttatus α –latroinsectotoxin, 5 = venom_Contig10081, 6 = venom_comp110241_c0_seq1, 7 = L. hesperus α –latrotoxin. The red bar at the N-terminus of sequence 7 indicates 9 amino acids not present in the published sequence that are predicted from the orthologous transcript in this study.

Figure 4

Amino acid sequences from L. hesperus transcripts containing predicted inhibitory cystine knot (ICK) motifs. Sequences with BLAST homology to known ICK toxin sequences (A) or lacking a BLAST hit but possessing a predicted ICK scaffold (B). The cysteine spacing is numbered by the sequence in the mature toxin. The predicted signal peptide is shaded gray, and the KNOTER1D predicted disulfide connectivity is indicated by colored bars and cysteine residues. Cysteines not predicted to participate in disulfide bonds are underlined. 1=venom_comp104578_c0_seq1, 2=venom_comp104578_c0_seq3, 3=venom_comp104578_c0_seq6, 4=Contig7465, 5=venom_comp72844_c0_seq1, 6=Contig3061, 7=Contig5795, 8=Contig7277, 9=venom_comp98528_c0_seq1, 10=venom_comp75139_c0_seq1, 11= Contig20358.

Figure 5

Bayesian tree of predicted protein sequences from BLAST-identified ICK toxins of L. hesperus and other spiders. Prefixed identifiers are included for sequences retrieved from the UniProt database. The tree is a midpoint-rooted 50% majority-rule consensus of 3002 trees sampled in Bayesian analysis. Values at nodes are posterior probabilities where they are ≥ 0.95, followed after the slash by ML bootstrap values when >= 70%. Sequences from L. hesperus from this study are in bold and the distinct L. hesperus clade is shaded in red. Red text delineates sequences for which information is available from prior functional studies (see text for details). Tissue expression levels (eCPM) for sequences derived from this study are shown in chart form (Ceph.=cephalothorax, Ven.=venom gland). The two underlined sequences are flipped in position in the ML tree.

Figure 6

Bayesian tree of CRISP proteins. Midpoint rooted 50% majority-rule consensus of 15002 trees. Values at nodes are posterior probabilities where ≥ 0.95, followed by a slash and bootstrap values where ≥ 70% (see also Additional file 5). L. hesperus sequences are bold, followed by three tissue expression levels (eCPM) (C = cephalothorax/S = silk gland/V = venom gland). UniProt accession numbers precede species name for other sequences. L. hesperus venom gland specific CRISPs are shaded red. Sequences from venomous species in red text, followed by a red dot if venom gland expression is confirmed. Sequences from hematophagous species in blue text, followed by a blue dot if salivary gland expression is confirmed. Sequences from non-venomous/non-hematophagous species in black. Ixodes ricinius = castor bean tick, I. scapularis = deer tick, Bombyx mori = domesticated silkmoth, Danaus plexippus = monarch butterfly, Drosophila = fruitfly, Musca domestica = housefly, Culex quinquefasciatus = southern house mosquito, Dipetalogaster maximus = kissing bug, Rhodnius prolixus = assassin bug, Camponotus floridanus = Florida carpenter ant, Crassostrea gigas = Pacific oyster, Solenopsis invicta = red imported fire ant, Polistes annularis = red paper wasp, Vespula pensylvanica = western yellow jacket, Vespula germanica = European wasp, Rhynchium brunneum = potter wasp, Microctonus hyperodae = braconid wasp, Daphnia pulex = water flea, Coptotermes formosanus = Formosan subterranean termite, Psorophora albipes = mosquito, Pediculus humanus = body louse, Caligulus rogercresseyi = sea louse, Isometroides vescus = spider hunting scorpion, Hottentotta judaicus = scorpion, Urodacus manicatus = black rock scorpion, Opisthocanthus cayaporum = South American scorpion, Amblyomma maculatum = Gulf coast tick, Rhipicephalus pulchellus = questing tick, Trittame loki = brush foot trapdoor spider, Grammostola rosea = Chilean rose tarantula, Lycosa singoriensis = spotted wolf spider, Tityus serrulatus = Brazilian yellow scorpion, Lychas buchari = Buchar’s scorpion.

Figure 7

Results of BLAST-based clustering analysis of L. hesperus predicted proteins from VSTs. Clustering of sequences was performed across a range of sequence overlap and identity values.

Figure 8

Summary of diversity and expression of L. hesperus venom gland specific transcripts. (A) The proportion of total distinct venom gland specific transcripts assigned to several known toxin types or enzymes by BLASTx significant similarity, and assigned to all other categories labeled as “other”, or lacking a significant BLAST hit. The numbers of sequences in the smaller categories were summed for clarity. (B) Overall expression as % of total FPKM in the venom gland specific set using these same categories.