A potential role for phenylalanine hydroxylase in mosquito immune responses
Introduction
The innate immune response of insects involve both humoral and cellular components and is highly effective in protecting these organisms from specific pathogens and parasites (Beernsten et al., 2000). Although the cellular response of insects has received little attention from the scientific community, the humoral response, especially of Drosophila melanogaster, has been studied in great detail (Hoffman et al., 1999). In addition to these humoral responses, Drosophila and other insects are able to utilize the cellular arm of their immune system to destroy invading pathogens by phagocytosis and/or encapsulation. When insects are exposed to multicellular pathogens too large to be phagocytosed, the most common response involves encapsulation. This has been referred to as ‘failed phagocytosis’, whereby numerous hemocytes encase the pathogen, often with multiple layers of cells. Ultrastructural and in vitro observations of mosquito melanotic defense responses suggest that hemocytes play an essential role in recognition and subsequent destruction of the pathogen (Chen and Laurence, 1985, Chikilian et al., 1994, Christensen and Forton, 1986). Encapsulation is often accompanied by the deposition of melanin and protein-polyphenol complexes at the surface of the pathogen in a reaction termed melanotic encapsulation (Liu et al., 1997, Rizki and Rizki, 1990).
Melanin biosynthesis is a multienzymatic pathway that plays numerous roles in insects. The majority of the research relating to insect melanogenesis involves phenoloxidase (PO) and PO activating factors. PO catalyzes the hydroxylation of tyrosine to dopa and the oxidation of o-diphenols to o-quinones. It is now known that dopa decarboxylase and dopachrome conversion enzyme also play critical roles in the production of melanin in mosquitoes (Ferdig et al., 1996, Johnson et al., 2001, Zhao et al., 1995). Because the rate of tyrosine hydroxylation by PO is about one-order magnitude lower than that of dopa- or 5, 6-dihydroxyindole oxidation by the same enzyme, the hydroxylation of tyrosine to dopa is considered as a rate-limiting step for melanin biosynthesis. Many investigations have focused on the metabolic fate of tyrosine; however, little attention has been given to tyrosine biosynthesis and the role it might play in melanotic immune responses. Although significant quantities of tyrosine undoubtedly are obtained by female mosquitoes during blood feeding, the ability to produce melanin for various physiological events is essential in non-blood feeding developmental stages (i.e. cuticular tanning), and in adult females during periods when they do not blood feed (i.e. wound healing and immune responses). Tyrosine, acquired during the larval feeding stages, is found as an inert storage form in the hemolymph in several insects, including Drosophila (Lunan and Mitchell, 1969, Mitchell and Lunan, 1964), Musca domestica (Bodnaryk, 1974), and lepidopteran species (Lu et al., 1982). However, studies by Munkirs et al. (1990) found no inert tyrosine storage form in Aedes aegypti. In mammals, the hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH) is the exclusive means of phenylalanine catabolism to produce tyrosine and this is likely the case in mosquitoes. Previous studies in insects demonstrated the hydroxylation of phenylalanine to tyrosine by PAH (Belzecka et al., 1964, Fukuda, 1956, Geltosky and Mitchell, 1980, Morales et al., 1990, Silva et al., 1992). Furthermore, these reports on Drosophila and Bombyx mori have explored the role of PAH in cuticle formation and the production of silk proteins, respectively, and not in innate immunity.
In both mammals and insects, the metabolism of phenylalanine is complex. The hydroxylation of phenylalanine to tyrosine by PAH is not only the rate-limiting step in the catabolism of phenylalanine, but also in the biosynthesis of other amino acids and proteins. This PAH-catalyzed reaction requires the reduced cofactor tetrahydrobiopterin (BH4), enzyme-bound iron, and oxygen (Lazarus et al., 1981). A separate enzymatic system, involving dihydropteridine reductase, regenerates the obligatory pteridine cofactor back to its reduced form (Hufton et al., 1995). Mammalian PAH is tightly regulated in vivo by phosphorylation (Abita et al., 1976, Donlon and Kaufman, 1978, Hufton et al., 1995), its substrate (Shiman and Gray, 1980), and at the transcriptional level (Dahl and Mercer, 1986, Konecki et al., 1992). It is reasonable to predict that these same regulatory mechanisms influence insect PAH.
We hypothesize that PAH and endogenous tyrosine play important roles in mosquito melanotic immune responses by providing ample substrate necessary for an effective response against parasites. Herein we: (1) report the isolation of a PAH cDNA (AaPah1) from the yellow fever mosquito, A. aegypti; (2) verify up-regulation of gene expression in the hemocytes during the melanotic encapsulation of filarial worms; and (3) localize PAH in the hemolymph to the oenocytoid hemocytes. This is the first study to report the potential involvement of PAH in the innate immune response of any organism.
Section snippets
Chemicals and reagents
NBT and BCIP were purchased from Fisher Scientific Co. All other chemicals were purchased from Sigma Chemical Co. or as otherwise noted.
Mosquito rearing and maintenance
The Liverpool strain A. aegypti used in this study was obtained originally from the University of London in 1977 and reared according to the methods described by Christensen and Sutherland (1984).
Isolation of the AaPah1 cDNA clone
A partial mosquito PAH was generated using RT-PCR. Degenerate primers were designed from the conserved regions of PAH sequence from D. melanogaster, rat, human, and an
AaPAH1 cDNA nucleotide and deduced amino acid sequence
Sequencing the A. aegypti PAH cDNA (AaPah1; GenBank accession number AY099427) revealed a 1610 bp full-length clone with a 1341 bp open reading frame. The AaPah1 translation product is 447 amino acids with a predicted molecular mass of 51 kDa. The cDNA gene product shows the greatest sequence similarities with the A. gambiae and D. melanogaster PAH. AaPah1 is 93.5, 86 and 70 % similar to the A. gambiae PAH (NCB accession number EAA10731), Drosophila PAH (NCB accession number CAA66797), and Mus
Discussion
Mosquitoes possess a defense system that is capable of restricting or preventing infection by various pathogens and parasites. Melanotic encapsulation plays a critical role in mosquito immunity against protozoan and metazoan parasites, whereby successful parasite sequestering and killing requires the participation of both hemocytes and melanin. Melanin substrates are synthesized through a metabolic pathway commencing with tyrosine, a substrate that is generated endogenously by PAH from
Acknowledgements
We thank Federick Oduol, Jiannong Xu and Ken Vernick for the A. gambiae PAH cDNA clone and Linda Christensen, Jeremy Fuchs and Shelley Schmidt for rearing the mosquitoes. This work was supported by NIH grant AI19769 to BMC, NIH grant AI37789 to JL, and NSC grants 89-2320-B-010-008-Y and 89-2320-B-010-037 to C.C. Chen.
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2017, Advances in Insect PhysiologyMolecular cloning and characterization of a phenylalanine hydroxylase from the common cutworm Spodoptera litura
2016, Journal of Asia-Pacific EntomologyCitation Excerpt :In mammals, PAH dysfunction induces error in various physiological events, including amino acid metabolism, phenylketonuria (PKU), vitiligo, and milder forms of perphenylalaninemia (Flydal and Martinez, 2013; Schallreuter et al., 2005). Furthermore, compared with mammal PAH, insect derived PAH is involved in melanin synthesis for immune responses, integument coloration, cuticle formation, and synthesis of fibrous proteins (Chen et al., 2013; Fukuda, 1956; Geltosky and Mitchell, 1980; Infanger et al., 2004; Johnson et al., 2003; Morales et al., 1990). In D. melanogaster, DmPah is mainly expressed in the fat body during the embryogenesis and larval stages and plays a functional role in cuticle formation (Geltosky and Mitchell, 1980; Morales et al., 1990).
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2014, Current Opinion in Insect ScienceScallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α
2011, Fish and Shellfish ImmunologyCitation Excerpt :PAH is responsible for catalyzing the reaction from phenylalanine to tyrosine by using molecular oxygen and BH4. The previous studies have demonstrated that PAH is involved in immune defence response by supplying the starting material tyrosine for the synthesis of catecholamines and melanin [16–19]. In the present study, a PAH gene was identified from the scallop C. farreri.