Chitin biosynthesis and the mosquito peritrophic matrix

描述(由申请人提供)：蚊媒疾病的死灰复燃使更好地描述影响媒介能力的机制的研究变得越来越重要。在与血餐一起摄入后，所有蚊媒传播的病原体必须在中肠环境中存活，并穿过营养周围基质(PM)和中肠上皮，到达发育和/或传播所需的特定组织；因此，PM是一种屏障，如果病原体要成功传播，必须绕过它。PM是一种蛋白多糖基质，由几丁质、蛋白质和糖蛋白组成，由中肠产生，用于将血块与中肠上皮分开。这项研究的重点是为了增加我们对埃及伊蚊PM形成所需的中肠几丁质生物合成的了解。在之前的支持期间，启动了对三种关键酶的研究，以证实它们参与了几丁质的合成，并开始评估控制其表达的因素。目前已知的谷氨酰胺合成酶(GS)、氨基葡萄糖：果糖-6-磷酸氨基转移酶(GFAT)和几丁质合成酶(CS)在中肠几丁质合成中起主要作用。详细研究血粉诱导中肠这些基因表达上调的因素的一个主要障碍是提取过程中蛋白酶诱导的核蛋白降解。在支持的最初阶段，实验方案被开发来缓解这个问题；因此，凝胶迁移率改变分析(EMSA)现在已经确定了几个重要的顺式作用元件和假定的反式作用因子，它们结合这些元件并选择性地控制中肠组织中GS的表达。然而，几丁质是如何从中肠细胞的细胞质运输到中肠腔的细胞外空间的，在那里形成PM仍然是未知的。同样，还有三种酶，氨基葡萄糖-6-磷酸N-乙酰基转移酶(GNAT)、磷酸乙酰氨基葡萄糖变位酶(AGM)和尿苷二磷酸-N-乙酰氨基葡萄糖焦磷酸酶(UAP)也参与了这一途径，但从未在昆虫中克隆或鉴定过。在这一竞争性的继续应用中，将对蚊子Ae使用各种显微镜、生化和分子技术。埃及，通过(1)克隆和表征编码GNAT、AGM和UAP的基因并确定血液喂养后中肠的时空转录和翻译，(2)确定GS、GFAT和CS基因中负责血餐后中肠特异性表达上调的关键调控元件，(3)利用转导病毒系统进行基因沉默研究，以获得这些酶在甲壳素生物合成中所起作用的直接证据，(4)获得结构-功能信息以评估甲壳素是如何被运输出中肠细胞的，并从机械上确定GFAT在反馈抑制甲壳素合成中的调节功能。

DESCRIPTION (provided by the applicant): The resurgence of mosquito-borne diseases places a growing importance on research that will better delineate mechanisms influencing vector competence. Following ingestion with a blood meal, all mosquito-borne pathogens must survive the midgut environment and traverse the peritrophic matrix (PM) and midgut epithelium to reach specific tissues required for development and/or transmission; therefore the PM serves as a barrier that must be circumvented if a pathogen is to be successfully transmitted. The PM is a proteoglycan matrix composed of chitin, proteins and glycoproteins that is produced by the midgut and serves to separate the blood bolus from the midgut epithelium. The focus of this research proposal is aimed at increasing our understanding of midgut chitin biosynthesis required for PM formation in the mosquito, Aedes aegypti. During the previous period of support, studies were initiated on three key enzymes to verify their involvement in chitin synthesis and to begin an assessment of factors controlling their expression. It now is known that glutamine synthetase (GS), glucosamine: fructose-6-phosphate amidotransferase (GFAT) and chitin synthase (CS) play major roles in chitin synthesis in the midgut. A major hurdle to detailed studies of factors responsible for the blood meal-induced upregulation of expression of these genes in midguts has been the protease-induced degradation of nuclear proteins during extraction. During the initial period of support, experimental protocols were developed to alleviate this problem; consequently, electrophoretic mobility shift assays (EMSA) now have identified several important cis-acting elements and putative trans-acting factors that bind those elements and selectively control GS expression in midgut tissues. However, It still is not known how chitin is trafficked from the cytoplasm of midgut cells to the extracellular space of the midgut lumen where PM formation occurs. Likewise, three additional enzymes, glucosamine-6-phosphate N-acetyltransferase (GNAT), phosphoacetyl glucosamine mutase (AGM), and uridine diphosphate-N-acetylglucosamine pyrophophrylase (UAP) are also involved in this pathway but have never been cloned or characterized in any insect. In this competing continuation application various microscopical, biochemical and molecular techniques will be used with the mosquito, Ae. aegypti, to extend studies on the chitin biosynthetic pathway by (1) cloning and characterizing the genes encoding GNAT, AGM, and UAP and determining temporal and spatial transcription and translation in midguts following blood feeding, (2) identifying key regulatory elements in the GS, GFAT and CS genes that are responsible for midgut-specific up regulation of expression following a blood meal, (3) performing gene silencing studies, using a transducing virus system, to obtain direct evidence of the roles these enzymes play in chitin biosynthesis, and (4) obtaining structure-function information to evaluate how chitin is trafficked out of midgut cells and also mechanistically determining the regulatory function of GFAT in feedback inhibition of chitin synthesis.