Positional Cloning and RNA Interference Assay Demonstrated Plasmodium-Melanizing Function of a Serine Protease Within the Plasmodium Encapsulation 1 Locus (Pen1) of the Malaria Mosquito Anopheles Gambiae

Doctoral Dissertation

Abstract

Due to its public health impact as the most important vector of malaria in Africa, the Anopheles gambiae mosquito and its Plasmodium parasites have been the subject of many studies both in the laboratory and out in the fields. For successful transmission of disease, the parasites must undergo a period of development in the mosquito from gametocytes to sporozoites. During this period, susceptibility or refractoriness is determined by the ability of some mosquitoes to encapsulate ookinetes in a melanin/protein matrix following their penetration of the midgut epithelium. Melanin is deposited onto the surface of the parasites, causing the dead parasites to appear brownish black. Melanin synthesis in insects involves a biochemical pathway named after the first and most important enzyme, phenoloxidase (PO). This enzyme is synthesized in an inactive form and its activity depends on proteolytic activation by a serine protease usually called prophenoloxidase-activating enzyme (PPAE). This serine protease has at least one extra domain called a Clip domain (Jiang and Kanost, 2000). Melanin-associated refractoriness to the primate parasite P. cynomolgi B and the rodent parasite P. berghei is influenced by at least three Quantitative trait loci (QTLs). A genetic crossing experiment determined that the major QTL lies on chromosome arm 2R between division 8C-8D and this locus was named Plasmodium Encapsulation 1 (Pen1) to reflect its major role in melanotic encapsulation of parasites (Zheng et al., 1997). This thesis describes the positional cloning of Pen1 from the construction of a physical map to the genomic, bioinformatic, molecular and functional analyses supporting the conclusion that Pen1 encodes a Clip domain serine protease with PPAE function. Specifically, we show that CLIPB17 is found within the Pen1 region and that it encodes a serine protease with three Clip domains and a serine protease domain with sequence similarity to all known PPAEs. In a selected strain with a fully refractory phenotype (L35 strain), CLIPB17 gene expression is up-regulated at the time of parasite melanization. In the counter-selected strain 4Arr, no such induction is evident. The regulatory region of CLIPB17 contains multiple strain-specific nucleotide polymorphisms clustered within three putative NF-kB sites that could explain the L35-specific gene induction. We also show that RNAi knockdown abolishes CLIPB17 gene induction in L35 and significantly reduces refractoriness to both Plasmodium berghei and P. cynomolgi B. Melanotic encapsulation represents the fastest and most important endogenous refractory mechanism against pathogens too large or too numerous for other immune mechanisms to cope with (Vey, 1993). Interest in this immune mechanism as a novel malaria control strategy prompted its genetic selection in a laboratory strain. Our determination of Pen1 molecular identity and function significantly enhances the future prospect to explore this mechanism for controlling one of the most devastating diseases of man.

Attributes

Attribute NameValues
URN
  • etd-12162005-135656

Author Lucas Q. Ton
Advisor Frank H. Collins
Contributor Michael Ferdig, Committee Member
Contributor David Severson, Committee Member
Contributor Frank H. Collins, Committee Chair
Contributor Joseph OTousa, Committee Member
Degree Level Doctoral Dissertation
Degree Discipline Biological Sciences
Degree Name PhD
Defense Date
  • 2005-12-07

Submission Date 2005-12-16
Country
  • United States of America

Subject
  • mosquito

  • Encapsulation

  • Plasmodium

  • Pen1

  • Anopheles gambiae

  • Serine Protease

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility and Access Public
Content License
  • All rights reserved

Departments and Units

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