Briefing Document: Native Structure of Mosquito Salivary Protein Uncovers Domains Relevant to Pathogen Transmission

Dates: Received - 04 October 2022 | Accepted - 07 February 2023 | Published - 17 February 2023
Source: Liu, S., Xia, X., Calvo, E., & Zhou, Z. H. (2023). Native structure of mosquito salivary protein uncovers domains relevant to pathogen transmission. Nature Communications, 14(1), 1-20. https://doi.org/10.1038/s41467-023-36577-y

Executive Summary:

This study, published in Nature Communications, presents the first native structure of the Salivary Gland Surface protein 1 (SGS1) from the Aedes aegypti mosquito, a key vector for numerous human pathogens. Utilizing cryo-electron microscopy (cryo-EM), the researchers determined the structure of this high-molecular weight protein, revealing a complex architecture comprised of a Tc toxin-like Rhs/YD shell, four receptor domains, and a set of partially shielded C-terminal daisy-chained helices predicted to form transmembrane helices. The structural insights shed light on the potential mechanisms by which SGS1 facilitates pathogen invasion into salivary glands and manipulates the host's immune response during blood feeding, a process responsible for approximately one million deaths annually.

Main Themes and Important Ideas/Facts:

• SGS1: A Crucial Protein in Pathogen Transmission:
• Female mosquitoes inject saliva containing various proteins into vertebrate hosts during blood feeding, facilitating pathogen transmission. This process is responsible for significant human morbidity and mortality.
• SGS1 is identified as one of the most abundant and conserved proteins secreted by female salivary glands of key disease-vectoring mosquitoes like Aedes aegypti, Culex pipiens, and Anopheles gambiae.
• Previous research indicated that SGS1 is essential for the invasion of Aedes aegypti salivary glands by Plasmodium gallinaceum sporozoites and positively affects Zika virus transmission.
• Orthologs of SGS1 in other mosquito species are thought to enhance the pathogenicity of arboviruses and Plasmodium parasites by modulating the host's immune response.
• The protein is exclusively expressed in the salivary glands of adult female mosquitoes, suggesting a specific role in blood-feeding and pathogen transmission.
• Cryo-EM Reveals the Native Structure of SGS1:
• The study successfully determined the native structure of full-length, uncleaved SGS1 (3364 amino acids) using cryoID and cryo-EM at an overall resolution of 3.3 Å.
• The structure is "cocoon shaped, with dimensions of 210Å × 115 Å × 86 Å" and is organized into six domains: two β-propeller domains, a rearrangement hotspot/tyrosine-aspartate (Rhs/YD)-repeats domain, a carbohydrate-binding module (CBM), a lectin carbohydrate-recognition domain (lectin-CRD), and a wedge domain.
• The C-terminal region, predicted to form transmembrane (TM) helices, is surprisingly "almost fully buried within the chamber inside the cocoon shell and forms daisy-chained helices."
• The Rhs/YD Shell: A Protective and Potentially Activating Environment:
• The Rhs/YD-repeats domain forms a "hollow shell (i.e., Rhs/YD shell)" structurally similar to that found in bacterial Tc toxins and eukaryotic teneurins.
• This shell encapsulates the C-terminal moiety, suggesting a mechanism for maintaining these potentially membrane-interacting helices in a soluble environment.
• Sequence analysis reveals a conserved aspartyl auto-protease (AP) site within the Rhs core of SGS1, similar to Tc toxins. Mass spectrometry data indicates that "an SGS1 peptide covering this auto-cleavage site was present in the salivary gland extract but was not detected in the saliva of Aedes aegypti," suggesting that auto-cleavage occurs upon secretion into saliva.
• The authors propose that a conformational change in SGS1 is likely required to activate this catalytic activity during secretion.
• Partially Folded Transmembrane Helices within the Chamber:
• The ~230 amino acid long C-terminal moiety, predicted to form six TM helices, is found to be "almost fully embedded inside the SGS1 chamber."
• Cryo-EM reveals that these predicted TM helices are only "partially folded and entirely sheltered in the chamber," interacting extensively with the interior of the Rhs/YD-repeats and the β-propeller 2 domain via hydrophobic contacts.
• Despite not being fully folded in the observed structure, the high conservation of these sequences among different mosquito species suggests an "essential function."
• The authors hypothesize that these metastable, extended structures might facilitate their detachment from the inner shell and potential insertion into host cell membranes upon injection.
• Receptor Domains on the Shell Surface:
• The exterior of the SGS1 Rhs/YD shell is decorated with four potential receptor domains: β-propeller 1, β-propeller 2, a carbohydrate-binding module (CBM), and a lectin carbohydrate-recognition domain (lectin-CRD).
• These domains are proposed to "mediate protein-protein interactions and/or facilitate carbohydrate binding" during pathogen transmission.
• The presence of these receptor domains is conserved among SGS proteins of disease-transmitting mosquito species.
• Proposed Model for Cleavage and Secretion:
• The study proposes a model where the C-terminal Tox-SGS domain is initially cleaved by furin protease in the salivary gland. This is supported by the absence of density for Tox-SGS in the cryo-EM map and mass spectrometry results indicating no peptides after residue 3035 were recovered from salivary glands.
• The remaining SGS1 fragment, potentially with the help of its receptor domains, may facilitate sporozoite/arbovirus invasion of the salivary gland.
• Subsequently, the SGS1 fragment is secreted into saliva, where the aspartyl auto-protease has already catalyzed its cleavage.
• The putative TM helices remain embedded within the Rhs/YD shell in saliva, protected from the soluble environment.
• Upon injection into the host during blood feeding, SGS1 may interact with host cells via its receptor domains and potentially release its TM helices, which could modulate host immune responses to benefit pathogen transmission.

Key Quotes:

• "Among the most abundant and conserved proteins secreted by female salivary glands is a high-molecular weight protein called salivary gland surface protein 1 (SGS1) that facilitates pathogen trans-mission, but its mechanism remains elusive."
• "Here, we determine the native structure of SGS1 by the cryoID approach, showing that the 3364 amino-acid protein has a Tc toxin-like Rhs/YD shell, four receptor domains, and a set of C-terminal daisy-chained helices."
• "These helices are partially shielded inside the Rhs/YD shell and poised to transform into predicted transmembrane helices. This transformation, and the numerous receptor domains on the surface of SGS1, are likely key in facilitating sporozoite/arbovirus invasion into the sali-vary glands and manipulating the host’s immune response."
• "The SGS1 structure is cocoon shaped, with dimensions of 210Å × 115 Å × 86 Å. It is organized into 6 domains: two β-propeller domains, a rearrangement hotspot/tyrosine-aspartate (Rhs/ YD)-repeats domain, a carbohydrate-binding module (CBM), a lectin carbohydrate-recognition domain (lectin-CRD), and a wedge domain."
• "Sequence alignment indicate...