A potential role for phenylalanine hydroxylase in mosquito immune responses

Abstract

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense 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.