Figure 5 - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
Analysis of skin perfused blood during a mosquito bite or SGE inoculation from sialokinin-gene edited Ae. aegypti (A) Diagram of workflow of laser speckle experiments during a mosquito bite. (B) Speckle tissue perfusion images of mouse lower back during WT or KO mosquito bites (sialoK i5 and sialoK Δ8 ). (C) Graphs showing the blood perfusion units in each group during probing, feeding, and 2 min post-feeding. High blood perfusion is visualized in red and low blood perfusion is in blue. The results were normalized to the blood perfusion units at the bite site before probing and are shown as the mean of measurements of 10 mosquitoes per group (WT, sialoK i5 , and sialoK Δ8 ) ± SEMs. The results from sialoK i5 and sialoK Δ8 were combined for representation and analysis. (D) Diagram of workflow of laser speckle experiments during injection of SGE. (E) Speckle tissue perfusion images of mouse lower back during injection of SGE from WT or KO mosquitoes. (F) Graphs showing the blood perfusion units in each group before and 1 min and 2 min after SGE injection. The results were normalized to the blood perfusion units at the
Source publication
Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosqui...
Contexts in source publication
Context 1
... determined the effect of sialokinin on host vasculature during a mosquito bite using laser speckle contrast imaging to analyze blood flow at the bite site ( Figure 5A). We observed an increase in blood perfusion at the bite site during probing, feeding, and 2 min post-feeding of WT mosquitoes (Figures 5B and 5C), which was visualized in our experiments as an accumulation of red signal. ...
Context 2
... observed an increase in blood perfusion at the bite site during probing, feeding, and 2 min post-feeding of WT mosquitoes (Figures 5B and 5C), which was visualized in our experiments as an accumulation of red signal. The bite of sialokinin-KO mosquitoes produced lower blood perfusion during probing and feeding ( Figure 5C). We performed similar analyses by the intradermal inoculation of SGEs collected from sialokinin-KO Ae. aegypti. ...
Context 3
... performed similar analyses by the intradermal inoculation of SGEs collected from sialokinin-KO Ae. aegypti. Our results showed that SGEs from WT mosquitoes induced greater vasodilation 1 and 2 min post-injection than SGEs from both sialokinin-KO mosquitoes ( Figures 5D-5F). These results demonstrate that sialokinin is the main salivary vasodilator in Ae. aegypti. ...
Citations
... First, we exploited 100 ng of purified salivary proteins inoculated into a mouse footpad to assess the role of these proteins in triggering neutrophil influx. Indeed, the dosage for studying mosquito saliva proteins is varied in different studies (Jin et al, 2018;Martin-Martin et al, 2022;Uraki et al, 2019). Schmid et al reported a total protein amount of approximately 370-600 ng in the salivary gland extraction per individual mosquito (Schmid et al, 2016). ...
Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein ( Aa NRP), facilitates the transmission of Zika and dengue viruses. Aa NRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, Aa NRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces Aa NRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.
... Gene editing techniques are valuable tools for studying gene function and have been used to characterize the importance of arthropod salivary gland genes and their coded proteins (22,23). In this work, we evaluated the importance of the two salivary D7 long-forms in mosquito blood feeding and pathogen transmission using gene editedknockout (KO) mosquitoes. ...
... These experiments confirmed that the effects of D7L1 and D7L2 in vertebrates are essential for keeping mosquito probing time short. Similar results were observed with Ae. aegypti KO mosquitoes that lacked the salivary vasodilator sialokinin (23). In Anopheles gambiae, D7 long form L2 (AGAP008279) weakly binds leukotrienes B4 and D4 and had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI (20). ...
... Aedes aegypti (Liverpool strain, LVP) mosquitoes were reared in standard insectary conditions (27°C, 80% humidity with a 12-h light/dark cycle) at either the Department of Entomology and Fralin Life Science Institute, Virginia Tech, or the Laboratory of Malaria and Vector Research, NIAID, NIH. Salivary gland dissections and saliva collection were performed as previously described (23). ...
Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti . The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo . We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection.
IMPORTANCE
During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti , the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.
... To assess the effect of hepatic Lrp1 deficiency on RVFV infection, we used a well-characterized footpad infection model of RVF, which is commonly used to mimic mosquito bite (21)(22)(23)(24). Mixed sex mice of both genotypes (4 to 12 weeks old; n = 25 and 36 for Lrp1 f/f Alb-Cre and Lrp1 f/f controls, respectively) were inoculated by footpad with 20 plaque-forming units (PFUs) of RVFV ZH501, which corresponds to at least 20 times the 50% lethal dose (LD 50 ) (15). ...
Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.
... Unlike microbes in other tissues, the salivary gland microbiome may affect mosquito-borne diseases by modulating the host immune system. Salivary factors can adjust the immune responses of vertebrate hosts (Mellink and Vos, 1977;Demeure et al., 2005;Depinay et al., 2006;Gavor et al., 2022;Martin-Martin et al., 2022), and some arboviruses exploit these mechanisms to evade immune destruction and find host immune cells as their initial replication sites (Schneider et al., 2010;Styer et al., 2011;Briant et al., 2014;Conway et al., 2014;Schmid et al., 2016;Sun et al., 2020). Therefore, it is possible that mosquito commensal microbes participate in the recruitment of host innate immune cells and the initiation of infection. ...
Mosquito-borne diseases present a significant threat to human health, with the possibility of outbreaks of new mosquito-borne diseases always looming. Unfortunately, current measures to combat these diseases such as vaccines and drugs are often either unavailable or ineffective. However, recent studies on microbiomes may reveal promising strategies to fight these diseases. In this review, we examine recent advances in our understanding of the effects of both the mosquito and vertebrate microbiomes on mosquito-borne diseases. We argue that the mosquito microbiome can have direct and indirect impacts on the transmission of these diseases, with mosquito symbiotic microorganisms, particularly Wolbachia bacteria, showing potential for controlling mosquito-borne diseases. Moreover, the skin microbiome of vertebrates plays a significant role in mosquito preferences, while the gut microbiome has an impact on the progression of mosquito-borne diseases in humans. As researchers continue to explore the role of microbiomes in mosquito-borne diseases, we highlight some promising future directions for this field. Ultimately, a better understanding of the interplay between mosquitoes, their hosts, pathogens, and the microbiomes of mosquitoes and hosts may hold the key to preventing and controlling mosquito-borne diseases.
... Physiological function MSPs have antivasoconstriction, anticoagulation, antiplatelet aggregation, antimicrobial, and prodigestive functions (James et al., 1989;James & Rossignol, 1991;Ribeiro & Francischetti, 2003;Luplertlop et al., 2011;Chagas et al., 2014;Li et al., 2020;Conway, 2021;Martin-Martin et al., 2022) Direct interaction with mosquito-borne viruses (MBVs) ...
Mosquito-borne viruses (MBVs) are a large class of viruses transmitted mainly through mosquito bites, including dengue virus, Zika virus, Japanese encephalitis virus, West Nile virus, and chikungunya virus, which pose a major threat to the health of people around the world. With global warming and extended human activities, the incidence of many MBVs has increased significantly. Mosquito saliva contains a variety of bioactive protein components. These not only enable blood feeding but also play a crucial role in regulating local infection at the bite site and the remote dissemination of MBVs as well as in remodeling the innate and adaptive immune responses of host vertebrates. Here, we review the physiological functions of mosquito salivary proteins (MSPs) in detail, the influence and the underlying mechanism of MSPs on the transmission of MBVs, and the current progress and issues that urgently need to be addressed in the research and development of MSP-based MBV transmission blocking vaccines.
... The vasodilator sialokinin, related to the tachykinin protein family, was found in Ae. aegypti salivary glands [31,32]. Sialokinin is crucial during blood feeding by inducing nitric oxide release and stimulating the permeability of endothelial cells [33]. Although Anopheline mosquitoes do not produce vasodilatory substances, the salivary peroxidase/catechol oxidase isolated from An. albimanus exerts a vasodilatory activity by rescinding hemostatically active biogenic amines that are released during blood feeding [34,35]. ...
Arthropod-borne viruses present important public health challenges worldwide. Viruses such as DENV, ZIKV, and WNV are of current concern due to an increasing incidence and an expanding geographic range, generating explosive outbreaks even in non-endemic areas. The clinical signs associated with infection from these arboviruses are often inapparent, mild, or nonspecific, but occasionally develop into serious complications marked by rapid onset, tremors, paralysis, hemorrhagic fever, neurological alterations, or death. They are predominately transmitted to humans through mosquito bite, during which saliva is inoculated into the skin to facilitate blood feeding. A new approach to prevent arboviral diseases has been proposed by the observation that arthropod saliva facilitates transmission of pathogens. Viruses released within mosquito saliva may more easily initiate host invasion by taking advantage of the host’s innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, especially because of the lack of licensed vaccines against most of these viruses. This review aims to provide an overview of the effects on the host immune response by the mosquito salivary proteins and how these phenomena alter the infection outcome for different arboviruses, recent attempts to generate mosquito salivary-based vaccines against flavivirus including DENV, ZIKV, and WNV, and the potential benefits and pitfalls that this strategy involves.
... Mass spectrometry was performed as previously described 37,38 . Salivary gland extracts and saliva samples were subjected to mass spectrometry at Research and Technology Branch (NIAID, NIH). ...
Female mosquitoes inject saliva into vertebrate hosts during blood feeding. This process transmits mosquito-borne human pathogens that collectively cause ~1,000,000 deaths/year. 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 transmission, 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 salivary glands and manipulating the host’s immune response. Female mosquitoes inject saliva into vertebrate hosts during blood feeding, transmitting mosquito-borne pathogens. Here, cryo-EM of mosquito salivary gland extract uncovers the native SGS1 structure and domains relevant to pathogen transmission.
... When injected into C3H/HeJ mice, sialokinin decreased production of T H 1 cytokines and increased production of T H 2 cytokines by mouse splenocytes [36]. Recent studies have also shown that sialokinin enhances arboviral infection by modulating endothelial barrier function and altering immune cell recruitment to the bite site [33,37]. However, these studies were performed in wild-type C57BL/6 and BALB/c mice and gave limited insight on human immune responses. ...
... Two sialokinin knockout Ae. aegypti (Liverpool) mosquito lines were created by using the CRISPR/Cas9 system and donated by the Adelman and Calvo laboratories [37]. The sialokinin d5 knockout line has a 5 base pair insertion, while the knockout line sialokinin d8 has an 8 base pair deletion. ...
... Both knockout lines were homozygous and contain frameshift mutations that resulted in premature stop codons. Sialokinin knockouts were verified via mass spectrometry and immunofluorescence for the presence of sialokinin in the saliva and salivary glands, respectively [37]. Our laboratory received sialokinin knockout mosquito lines as eggs and were reared in the same manner as wild-type mosquitoes. ...
Mosquito saliva is a mix of numerous proteins that are injected into the skin while the mosquito searches for a blood meal. While mosquito saliva is known to be immunogenic, the salivary components driving these immune responses, as well as the types of immune responses that occur, are not well characterized. We investigated the effects of one potential immunomodulatory mosquito saliva protein, sialokinin, on the human immune response. We used flow cytometry to compare human immune cell populations between humanized mice bitten by sialokinin knockout mosquitoes or injected with sialokinin, and compared them to those bitten by wild-type mosquitoes, unbitten, or saline-injected control mice. Humanized mice received 4 mosquito bites or a single injection, were euthanized after 7 days, and skin, spleen, bone marrow, and blood were harvested for immune cell profiling. Our results show that bites from sialokinin knockout mosquitoes induced monocyte and macrophage populations in the skin, blood, bone marrow, and spleens, and primarily affected CD11c- cell populations. Other increased immune cells included plasmacytoid dendritic cells in the blood, natural killer cells in the skin and blood, and CD4+ T cells in all samples analyzed. Conversely, we observed that mice bitten with sialokinin knockout mosquitoes had decreased NKT cell populations in the skin, and fewer B cells in the blood, spleen, and bone marrow. Taken together, we demonstrated that sialokinin knockout saliva induces elements of a TH1 cellular immune response, suggesting that the sialokinin peptide is inducing a TH2 cellular immune response during wild-type mosquito biting. These findings are an important step towards understanding how mosquito saliva modulates the human immune system and which components of saliva may be critical for arboviral infection. By identifying immunomodulatory salivary proteins, such as sialokinin, we can develop vaccines against mosquito saliva components and direct efforts towards blocking arboviral infections.
Dengue virus (DENV) is a continuing global threat that puts half of the world’s population at risk for infection. This mosquito-transmitted virus is endemic in over 100 countries. When a mosquito takes a bloodmeal, virus is deposited into the epidermal and dermal layers of human skin, infecting a variety of permissive cells, including keratinocytes, Langerhans cells, macrophages, dermal dendritic cells, fibroblasts, and mast cells. In response to infection, the skin deploys an array of defense mechanisms to inhibit viral replication and prevent dissemination. Antimicrobial peptides, pattern recognition receptors, and cytokines induce a signaling cascade to increase transcription and translation of pro-inflammatory and antiviral genes. Paradoxically, this inflammatory environment recruits skin-resident mononuclear cells that become infected and migrate out of the skin, spreading virus throughout the host. The details of the viral–host interactions in the cutaneous microenvironment remain unclear, partly due to the limited body of research focusing on DENV in human skin. This review will summarize the functional role of human skin, the cutaneous innate immune response to DENV, the contribution of the arthropod vector, and the models used to study DENV interactions in the cutaneous environment.
Factors contained within mosquito saliva enhance transmission of mosquito-borne viruses to the mammalian host. In this issue, Wang et al (2024) identify AaNRP as a crucial pro-viral factor in Aedes aegypti mosquito saliva that promotes recruitment of Zika and dengue virus-susceptible myeloid cells.
