Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins.

<p>Abstract</p> <p>Background</p> <p>The discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, <it>Anopheles gambiae</it>, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR) containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM) proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains.</p> <p>Results</p> <p>The identification and characterisation of genes related to <it>LRIM1 </it>and <it>APL1C </it>revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of <it>LRIM1 </it>and <it>APL1C </it>identified more than 20 <it>LRIM</it>-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: <it>An. gambiae</it>, <it>Aedes aegypti</it>, and <it>Culex quinquefasciatus</it>. Comparative sequence analyses revealed that this family of mosquito <it>LRIM</it>-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains.</p> <p>Conclusions</p> <p>The evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation of LRIM protein complexes or mediate interactions with other immune proteins. The conserved LRIM cysteine residue patterns are likely to be important for structural fold stability and the formation of protein complexes. These sequence-structure-function relations of mosquito LRIMs will serve to guide the experimental elucidation of their molecular roles in mosquito immunity.</p

Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

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Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei

Mosquito immune responses and compatibility between Plasmodium parasites and anopheline mosquitoes

<p>Abstract</p> <p>Background</p> <p>Functional screens based on dsRNA-mediated gene silencing identified several <it>Anopheles gambiae </it>genes that limit <it>Plasmodium berghei </it>infection. However, some of the genes identified in these screens have no effect on the human malaria parasite <it>Plasmodium falciparum</it>; raising the question of whether different mosquito effector genes mediate anti-parasitic responses to different <it>Plasmodium </it>species.</p> <p>Results</p> <p>Four new <it>An. gambiae </it>(G3) genes were identified that, when silenced, have a different effect on <it>P. berghei </it>(Anka 2.34) and <it>P. falciparum </it>(3D7) infections. Orthologs of these genes, as well as <it>LRIM1 </it>and <it>CTL4</it>, were also silenced in <it>An. stephensi </it>(Nijmegen Sda500) females infected with <it>P. yoelii </it>(17XNL). For five of the six genes tested, silencing had the same effect on infection in the <it>P. falciparum-An. gambiae </it>and <it>P. yoelii-An. stephensi </it>parasite-vector combinations. Although silencing <it>LRIM1 </it>or <it>CTL4 </it>has no effect in <it>An. stephensi </it>females infected with <it>P. yoelii</it>, when <it>An. gambiae </it>is infected with the same parasite, silencing these genes has a dramatic effect. In <it>An. gambiae </it>(G3), TEP1, LRIM1 or LRIM2 silencing reverts lysis and melanization of <it>P. yoelii</it>, while <it>CTL4 </it>silencing enhances melanization.</p> <p>Conclusion</p> <p>There is a broad spectrum of compatibility, the extent to which the mosquito immune system limits infection, between different <it>Plasmodium </it>strains and particular mosquito strains that is mediated by TEP1/LRIM1 activation. The interactions between highly compatible animal models of malaria, such as <it>P. yoelii </it>(17XNL)-<it>An. stephensi </it>(Nijmegen Sda500), is more similar to that of <it>P. falciparum </it>(3D7)-<it>An. gambiae </it>(G3).</p

The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1(cut) that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals

Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei

Background: The compaction of DNA in chromatin in eukaryotes allowed the expansion of genome size and coincided with significant evolutionary diversification. However, chromatin generally represses DNA function, and mechanisms coevolved to regulate chromatin structure and its impact on DNA. This included the selection of specific nucleosome positions to modulate accessibility to the DNA molecule. Trypanosoma brucei, a member of the Excavates supergroup, falls in an ancient evolutionary branch of eukaryotes and provides valuable insight into the organization of chromatin in early genomes. Results: We have mapped nucleosome positions in T. brucei and identified important differences compared to other eukaryotes: The RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined −1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent variant surface glycoprotein (VSG) gene cassettes showed extensive arrays of well-positioned nucleosomes, which may repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within silent VSG gene cassettes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions. Conclusions: Our results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility