Serine proteases as mediators of mosquito immune responses

Gorman, Maureen J.; Paskewitz, Susan M.

doi:10.1016/s0965-1748(00)00145-4

Cited by 176 publications

(110 citation statements)

References 29 publications

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“…Several innate immune pathways in mosquitoes have been elucidated for defense against bacterial and macroparasite infections (18,(37)(38)(39)(40); however, no antiviral mechanisms or pathways have been described. In vertebrate species, there are innate immune mechanisms that recognize and mount responses to dsRNA, including the interferon and protein kinase R pathways, but neither has been detected in the mosquito.…”

Section: Discussionmentioning

confidence: 99%

RNA interference acts as a natural antiviral response to O'nyong-nyong virus ( Alphavirus ; Togaviridae) infection of Anopheles gambiae

Keene

Foy

Sánchez-Vargas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

315

272

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RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control ␤-galactosidase dsRNA (ds␤gal; note that ''ds'' is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNVeGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with ds␤gal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than ds␤gal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.innate immunity ͉ mosquito ͉ vector competence

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Section: Discussionmentioning

confidence: 99%

RNA interference acts as a natural antiviral response to O'nyong-nyong virus ( Alphavirus ; Togaviridae) infection of Anopheles gambiae

Keene

Foy

Sánchez-Vargas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

315

272

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“…While these series of reactions have been widely recognized as an integral part of the defense system, only circumstantial experimental evidence is available so far for its role in microbe killing. For example, melanotic encapsulation takes place in some mosquitoes that are refractory to parasites (Gorman and Paskewitz, 2001). One electron transfer from dopa to oxygen could have happened to produce dopa semiquinone and superoxide anion during melanization (Nappi et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

Zhao

Wang

et al. 2007

Insect Biochemistry and Molecular Biology

168

109

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Although quinone production and melanin formation are widely recognized as an integral part of the insect defense system, experimental evidence is lacking that the proteolytic activation of prophenoloxidase participates in the direct killing of invading microbes-active phenoloxidase generates quinones that polymerize to form melanin. Here, we report the antimicrobial effect of reactive intermediates produced in phenoloxidase-catalyzed reactions. After being treated with Manduca sexta phenoloxidase and dopamine, Escherichia coli and Bacillus subtilis ceased to grow, whereas the growth of Pichia pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive compounds were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium) and 52-99% of the Gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus) were killed. In the presence of phenoloxidase, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD) did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of DHI was detected using P. pastoris, Saccharomyces cerevisiae, Candida albicans, and Beauveria bassiana. These results established that prophenoloxidase activation is an integral component of the insect defense system involving a multitude of enzymes (e.g. proteinases, oxidases, and dopachrome conversion enzyme (DCE)), which immobilizes and kills invading microorganisms.

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“…gambiae is a dipteran hematophage and its saliva is rich in anti-inflammatory and anti-hemostatic enzymes which interfere with blood coagulation and inhibit the pain response, allowing the mosquito to have a blood-meal with a minimal chance of detection [4,37]. Proteases in the midgut, orthologs of trypsin and chymotrypsin found in higher organisms, are involved in rapid digestion [38], whereas others are involved in immune response [39]. The salivary gland and midgut regions of the mosquito have attracted a great deal of attention, as this is where the Plasmodium develops and matures in its vector.…”

Section: Functional Assignments For the Salivary Gland Proteinsmentioning

confidence: 99%

A proteomic analysis of salivary glands of femaleAnopheles gambiae mosquito

Kalume¹,

Okulate²,

Zhong³

et al. 2005

Proteomics

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Understanding the development of the malaria parasite within the mosquito vector at the molecular level should provide novel targets for interrupting parasitic life cycle and subsequent transmission. Availability of the complete genomic sequence of the major African malaria vector, Anopheles gambiae, allows discovery of such targets through experimental as well as computational methods. In the female mosquito, the salivary gland tissue plays an important role in the maturation of the infective form of the malaria parasite. Therefore, we carried out a proteomic analysis of salivary glands from female An. gambiae mosquitoes. Salivary gland extracts were digested with trypsin using two complementary approaches and analyzed by LC-MS/MS. This led to identification of 69 unique proteins, 57 of which were novel. We carried out a functional annotation of all proteins identified in this study through a detailed bioinformatics analysis. Even though a number of cDNA and Edman degradation-based approaches to catalog transcripts and proteins from salivary glands of mosquitoes have been published previously, this is the first report describing the application of MS for characterization of the salivary gland proteome. Our approach should prove valuable for characterizing proteomes of parasites and vectors with sequenced genomes as well as those whose genomes are yet to be fully sequenced.

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Serine proteases as mediators of mosquito immune responses

Cited by 176 publications

References 29 publications

RNA interference acts as a natural antiviral response to O'nyong-nyong virus ( Alphavirus ; Togaviridae) infection of Anopheles gambiae

RNA interference acts as a natural antiviral response to O'nyong-nyong virus ( Alphavirus ; Togaviridae) infection of Anopheles gambiae

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

A proteomic analysis of salivary glands of femaleAnopheles gambiae mosquito

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