The C-Type Lectin Domain Gene Family in Aedes aegypti and Their Role in Arbovirus Infection

Abstract

Several medically important flaviviruses that are transmitted by mosquitoes have been shown to bind to the C-type lectin fold that is present in either vertebrate or invertebrate proteins. While in some cases this interaction is part of a neutralizing anti-viral immune response, many reports have implicated this as critical for successful virus entry. Despite the establishment of mosquito C-type lectin domain containing proteins (CTLDcps) as known host factors in assisting the infectious process for flaviviruses, little is known about the structural characteristics of these proteins and their relationships to each other. In this report, we describe the manual annotation and structural characterization of 52 Aedes aegypti CTLDcps. Using existing RNAseq data, we establish that these genes can be subdivided into two classes: those highly conserved with expression primarily in development (embryo/early larvae) and those with no clear orthologs with expression primarily in late larvae/pupae or adults. The latter group contained all CTLDcps that are regulated by the Toll/Imd immune pathways, all known microbiome-regulating CTLDcps, and almost all CTLDcps that are implicated as flavivirus host factors in A. aegypti. Finally, we attempt to synthesize results from multiple conflicting gene expression profiling experiments in terms of how flavivirus infection changes steady-state levels of mRNA encoding CTLDcps.

Keywords: Aedes aegypti; C-type lectin; arbovirus; dengue; host factor; immunity; mosquito.

Figure 1

A. aegypti C-type lectin domain containing proteins (CTLDcps). (A) Primary structure and categorization of CTLDcps. CTLD-S (orange box), CTLD-E (blue box), CTLD-SP (yellow box), and CTLD-X (red box) are indicated. All of the models were drawn to scale using IBS v1.0.3. Predicted signal peptides are indicated at the N-terminus of each sequence (pink), along with predicted transmembrane spanning domains (TM; gold), CTLD (dark blue), and elastase-serine protease (ESP). For CTLD-X members, domains are as indicated and the reader is directed to supplemental Table S1 for a full list of domains including accession numbers for each; (B) Alignment of the N-terminus of CTLD-E gene AAEL018265 and its orthologs from other mosquitoes. Identical (dark blue) and similar (light blue) residues are indicated. Initiation methionine (M1) and alternative methionine (M2) are indicated; (C) Signal Peptide/transmembrane domain prediction output from Phobius [14] for CTLD-E gene AAEL018265 and its orthologs based on the M1 or M2 methionines; and, (D) Predicted amino acid lengths of CTLD genes in A. aegypti and three other mosquitoes, by category. For A. aegypti, CTLD-S (orange), CTLD-E (blue), CTLD-SP (yellow), and CTLD-X (red) groups are indicated. Horizontal line indicates the median length.

Figure 2

Developmental expression of A. aegypti mRNA transcripts encoding CTLDcps. Color indicates the log10 FPKM expression value. No scaling is applied to rows; clustering was performed using Morpheus (https://software.broadinstitute.org/morpheus) with one minus pearson correlation and average linkage. CTLD-S (■), -SP (■), -E (■), and -X (■) groups are indicated. NBF, non-bloodfed; BF, bloodfed; L, larvae; P, pupae.

Figure 3

Neighbor-joining tree based on alignment of 183 CTLDs extracted from A. aegypti (●), A. albopictus (●), C. quinquefasciatus (▲), and A. gambiae (♦) CTLD containing genes. Ambiguous positions were removed for each sequence pair; there were a total of 235 positions in the final dataset; branch support of more than 50% (1000 replicates) is indicated. Conserved clades containing CTLD-X (red) and CTLD-E (blue) members are indicated. Other highlighted clusters contain CTLD-SP (yellow) and CTLD-S proteins with both A. gambiae and A. aegypti orthologs with branch support over 50% (orange). Individual CTLDcps previously implicated as host (green) or resistance (red) factors for dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile encephalitis virus (WNV), P. falciparum (Pfal) bacteria (ProB/AntiB), or the prophenol oxidase response (PPO) are indicated, as are genes specifically expressed in the salivary glands (SG) and testes (Ts).

Figure 4

Expansion of CTLD-S and CTLD-SP genes in A. aegypti. Syntenic regions of the A. aegypti and A. gambiae genomes containing related clusters of CTLDcps. Genes encoding CTLD-S (orange), CTLD-SP (yellow), and CTLD-E (blue) are indicated (not to scale). Dotted lines indicated A. aegypti CTLDs that clustered together in Figure 3. Three letter codes indicate the residues present in the putative sugar-binding region of each CTLD.

Figure 5

Differentially-regulated CTLDcp-encoding transcripts after virus challenge or immune activation. Heat map of log2 values corresponding to the change in gene expression after virus infection [20,21,23,24,25,37], dsRNA treatment [21,36], or bloodmeal-induced activation of Rel1/Rel2 [22]. Only those genes described as being significantly different in the initial publications are included. Transcripts that are associated with CTLDcps identified as virus host factors (DENV, JEV, WNV) [1,2,3] or probiotic (ProB)/antibiotic factors (AntiB) [6] are indicated.