APL1C protein is a pathogen binding factor for the midgut ookinete stage of Plasmodium 

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Basis of microbial immune sensing and evolution

One of the challenges in immunology is to understand how the host organism detects  the presence of infectious agents and mounts efficient killing mechanisms against  these intruders, without destroying self-tissues. Sensing intruder as “non-self” or as  lacking “self-markers” constitutes the starting point of the host response as a defence  reaction in all living organisms. For microbial sensing, host-derived factors need to come in contact with the pathogen and generate the signals that will be transduced into a downstream immune response leading to the clearance of the microbes. Even such simple organisms as prokaryotes have defence mechanisms based on restriction enzymes and clustered regularly interspaced palindromic repeats (CRISPRs) that target invading pathogenic phages for degradation48. The foundational defence strategies in eukaryotic organisms are generally classified as the innate immune system, a host defence system in which plants and animals share similar inherited molecular modules including for pathogen sensing. The principle of innate immune recognition is based on the detection of constitutive and conserved products from structural microbial surface proteins or from their metabolism. Different pathogens share unique structures, for example lipopolysaccharide (LPS) or 456 peptidoglycan, which are bacterial surface molecules, characterized as Pathogen Associated Molecular Patterns (PAMPs) and which are not synthetized by eukaryotic cells. The structure of these components is conserved, as they are often essential components for microbial survival and fitness. PAMPs are sensed by host germline-encoded proteins serving as pathogen recognition receptors (PRRs). Activation of PRRs through PAMP recognition transduces an immune signal inside the cells that leads to diverse killing mechanisms of the invaded pathogen (opsonization, activation of complement, phagocytosis, activation of pro-inflammatory signalling pathways, induction of apoptosis etc.).

LRR domain proteins in immunity

LRR motifs have been identified in proteins ranging from viruses to eukaryotes. Despite distinct evolutionary origin, many plant and animal immune receptors or factors including cell-adhesion molecules, hormone receptors, tyrosine kinase receptors or extracellular matrix-binding glycoproteins contain LRR domain. LRR proteins serve as significant mediators in both innate and, replacing immunoglobulin (Ig) domain, in alternative adaptive immune systems where they play a dual function. Firstly, serving in pathogen sensing, through direct or indirect recognition of the pathogens, and secondly, as signal mediator, through the elicitors of downstream immune signalling they activate. LRR domains vary in their lengths and pattern of conserved residues, mediating ligand binding interactions54. In the next parts, I give an overview of distinct conserved immune protein families from different organisms, all carrying the LRR domain required for achieving their protective function.

Toll-like receptors (TLR)

Identification of Toll receptors in Drosophila opened new perspectives to understand the innate immune system, which was understudied in higher organisms in favour to the adaptive immune response. In deuterostomes, Toll-like receptors (TLRs), which are transmembrane proteins, were identified as the major PRRs that underwent purifying and diversifying selection, ranging from 222 TLR genes in sea urchin Strongylocentrotus purpuratus to 12 TLR in mouse66, in order to adapt to a variety of coevolving pathogens. Among mammals, human encodes at least 10 TLRs. In contrast to Drosophila, where the Toll-dependent pathway is induced by Gram-positive and fungal pathogens, TLR in mammals are activated upon structurally variant PAMPs from different organisms and that constitute a large repertoire of microbial structures (Table 1).

LRR-coding receptors in adaptive-like immunity of jawless vertebrates

Jawless vertebrates (agnathans) and jawed vertebrates (gnathostomes) diverged approximately 550 million years ago. While jawed vertebrates are predominant, the jawless clade has only two extant representatives: hagfishes and lampreys. As mentioned above, the arsenal of adaptive immune recognition in gnathostomes is based on Ig domain B and T lymphocyte receptors (BCRs and TCRs) and major  histocompatibility complex (MHC) molecules. While none of these proteins are present  in agnathans, hagfishes and lampreys were shown to mount adaptive-like immune  responses to repetitive antigenic challenges through the expression of unique antigen receptors named variable lymphocyte receptors (VLRs). These VLRs generate antigen-recognition diversity through a gene conversion-like mechanism involving a  variable LRR segment domain91. Three VLR genes have been identified, VLRA, VLRB and VLRC and they share a similar protein organization with a N-ending LRR-capping  motif (LRRNT) followed by 18-residue N-terminal LRR module (LRR1), diverse region composed of multiple, variable LRR modules (LRRV), 24-residue 684 called end LRRV (LRRVe), a connecting peptide (CP) and a C-terminal LRR-capping motif (LRRCT, Figure 4)92.

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Table of contents :

SUMMARY
ACKNOWLEDGMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ANNEX CONTENT
ABBREVIATIONS
I. GENERAL INTRODUCTION
1.1 Malaria – overview
1.1.1 Female Anopheles mosquitoes – vector of malaria
1.1.2 History of malaria, mosquito-vector discovery and vector control
1.1.3 Why is Africa the most malaria endemic continent – what makes a competent mosquito vector? .
1.1.4 The complex lifecycle of Plasmodium parasite in vertebrate and mosquito
1.1.5 Two bottlenecks of Plasmodium development in mosquito vector
1.2 Microbial sensing by the immune system
1.2.1 Basis of microbial immune sensing and evolution
1.2.2 LRR domain proteins in immunity
1.2.2.1 LRR proteins in the innate immune system
1.2.2.1.1 Drosophila Toll receptors
1.2.2.1.2 Toll-like receptors (TLR)
1.2.2.1.3 Nucleotide-binding leucine-rich repeat receptors
1.2.2.1.4 Plant LRRs
1.2.2.2 LRR-coding receptors in adaptive-like immunity of jawless vertebrates
II. CURRENT KNOWLEDGE, GAPS AND OBJECTIVES
2.1 Natural resistance to Plasmodium highlighted a family of LRR genes termed APL1 (Anopheles Plasmodium-responsive leucine-rich repeat)
2.2 APL1 functional activity and the mosquito complement system
2.3 Scope of the thesis
III. RESULTS
3.1 APL1C protein is a pathogen binding factor for the midgut ookinete stage of Plasmodium
3.1.1 Blood feeding induces an extracellular layer of APL1C surrounding the midgut
3.1.2 APL1C presence on the basal side of the midgut epithelium is microbiome independent
3.1.3 APL1C binds to Plasmodium ookinetes exiting the basal side of the midgut epithelium
3.1.4 LRR proteins bind autonomously to the ookinete surface (coll.: C. Lavazec, Institut Cochin, Paris) .
3.1.5 Phagocytic hemocytes are required for wildtype APL1C levels in the hemolymph
3.1.6 Nitration pathway activity is required for full APL1C abundance in hemolymph
3.2 APL1C protein is a pathogen binding factor for the hemocoel sporozoite stage of Plasmodium
3.2.1 Sporozoites affect APL1C protein abundance
3.2.2 APL1C activity in the hemocoel limits sporozoite invasion of salivary glands
3.2.3 APL1C binds to free Plasmodium sporozoites in the mosquito hemolymph LE OF
.4 APL1C protective activity against sporozoites does not require the complement 109 proteins TEP1 or TEP3
3.3 Implication of the APL1 LRR family in immune signal transduction
3.3.1 APL1A and APL1C gene silencing in A. coluzzii
3.3.2 Kinetics of APL1A and APL1C silencing
3.3.3 RNAseq and bioinformatic analysis
3.3.4 qPCR validation of the RNAseq gene candidates.
3.3.5 APL1C controls expression of immune-like genes in the mosquito
IV. DISCUSSION
4.1 Dual functionality for APL1C as guard and PRR-like factor
4.2 The HdMvs, a source of secreted APL1C, mediated by the nitration pathway?
4.3 APL1C presence in hemolymph at the basal side of the midgut epithelium is not PAMP dependent
4.4 The sophisticated sporogony control by APL1C
4.5 Whole transcriptome analysis reveals differential signalling function of APL1 genes
V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES
VI. MATERIALS AND METHODS
REFERENCES
ANNEX
ABSTRACT

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