Thioester-containing Protein Research Articles

In silico analysis of proteins and microRNAs related to human African trypanosomiasis in tsetse fly

Human African trypanosomiasis (HAT), also known as sleeping sickness, causes millions of deaths worldwide. HAT is primarily transmitted by the vector tsetse fly (Glossina morsitans). Early diagnosis remains a key objective for treating this disease. MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that play key roles in vector-borne diseases. To date, the roles of proteins and miRNAs in HAT disease have not been thoroughly elucidated. In this study, we have re-annotated the function of protein-coding genes and identified several miRNAs based on a series of bioinformatics tools. A batch of 81.1 % of tsetse fly proteins could be determined homology in mosquito genome, suggesting their probable similar mechanisms in vector-borne diseases. A set of 11 novel salivary proteins and 14 midgut proteins were observed in the tsetse fly, which could be applied to the development of vaccine candidates for the control of HAT disease. In addition, 35 novel miRNAs were identified, among which 10 miRNAs were found to be unique in tsetse fly. Pathway analysis of these 10 miRNAs indicated that targets of miR-15a-5p were significantly enriched in the HAT-related neurotrophin signaling pathway. Besides, topological analysis of the miRNA-gene network indicated that miR-619-5p and miR-2490-3p targeted several genes that respond to trypanosome infection, including thioester-containing protein Tep1 and heat shock protein Hsp60a. In conclusion, our work helps to elucidate the function of miRNAs in tsetse fly and establishes a foundation for further investigations into the molecular regulatory mechanisms of HAT disease.

Thioester-containing Proteins in the Drosophila melanogaster Immune Response against the Pathogen Photorhabdus.

The fruit fly Drosophila melanogaster forms a magnificent model for interpreting conserved host innate immune signaling and functional processes in response to microbial assaults. In the broad research field of host-microbe interactions, model hosts are used in conjunction with a variety of pathogenic microorganisms to disentangle host immune system activities and microbial pathogenicity strategies. The pathogen Photorhabdus is considered an established model for analyzing bacterial virulence and symbiosis due to its unique life cycle that extends between two invertebrate hosts: an insect and a parasitic nematode. In recent years, particular focus has been given to the mechanistic participation of the D. melanogaster thioester-containing proteins (TEPs) in the overall immune capacity of the fly upon response against the pathogen Photorhabdus alone or in combination with its specific nematode vector Heterorhabditis bacteriophora. The original role of certain TEPs in the insect innate immune machinery was linked to the antibacterial and antiparasite reaction of the mosquito malaria vector Anopheles gambiae; however, revamped interest in the immune competence of these molecules has recently emerged from the D. melanogaster-Photorhabdus infection system. Here, we review the latest findings on this topic with the expectation that such information will refine our understanding of the evolutionary immune role of TEPs in host immune surveillance.

A New Assessment of Thioester-Containing Proteins Diversity of the Freshwater Snail Biomphalaria glabrata.

Thioester-containing proteins (TEPs) superfamily is known to play important innate immune functions in a wide range of animal phyla. TEPs are involved in recognition, and in the direct or mediated killing of several invading organisms or pathogens. While several TEPs have been identified in many invertebrates, only one TEP (named BgTEP) has been previously characterized in the freshwater snail, Biomphalaria glabrata. As the presence of a single member of that family is particularly intriguing, transcriptomic data and the recently published genome were used to explore the presence of other BgTEP related genes in B. glabrata. Ten other TEP members have been reported and classified into different subfamilies: Three complement-like factors (BgC3-1 to BgC3-3), one α-2-macroblobulin (BgA2M), two macroglobulin complement-related proteins (BgMCR1, BgMCR2), one CD109 (BgCD109), and three insect TEP (BgTEP2 to BgTEP4) in addition to the previously characterized BgTEP that we renamed BgTEP1. This is the first report on such a level of TEP diversity and of the presence of macroglobulin complement-related proteins (MCR) in mollusks. Gene structure analysis revealed alternative splicing in the highly variable region of three members (BgA2M, BgCD109, and BgTEP2) with a particularly unexpected diversity for BgTEP2. Finally, different gene expression profiles tend to indicate specific functions for such novel family members.

Disruption of mosGILT in Anopheles gambiae impairs ovarian development and Plasmodium infection.

Plasmodium infection in Anopheles is influenced by mosquito-derived factors. We previously showed that a protein in saliva from infected Anopheles, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT), inhibits the ability of sporozoites to traverse cells and readily establish infection of the vertebrate host. To determine whether mosGILT influences Plasmodium within the mosquito, we generated Anopheles gambiae mosquitoes carrying mosaic mutations in the mosGILT gene using CRISPR/CRISPR associated protein 9 (Cas9). Here, we show that female mosaic mosGILT mutant mosquitoes display defects in ovarian development and refractoriness to Plasmodium. Following infection by either Plasmodium berghei or Plasmodium falciparum, mutant mosquitoes have significantly reduced oocyst numbers as a result of increased thioester-containing protein 1 (TEP1)-dependent parasite killing. Expression of vitellogenin (Vg), the major yolk protein that can reduce the parasite-killing efficiency of TEP1, is severely impaired in mutant mosquitoes. MosGILT is a mosquito factor that is essential for ovarian development and indirectly protects both human and rodent Plasmodium species from mosquito immunity.

Alternative splicing, spatiotemporal expression of TEP family genes in Yesso scallop (Patinopecten yessoensis) and their disparity in responses to ocean acidification

The complement system constitutes a highly sophisticated and powerful body defense machinery acting in the innate immunity of both vertebrates and invertebrates. As central components of the complement system, significant effects of thioester-containing protein (TEP) family members on immunity have been reported in most vertebrates and in some invertebrates, but the spatiotemporal expression and regulatory patterns of TEP family genes under environmental stress have been less widely investigated in scallops. In this study, expression profiling of TEP family members in the Yesso scallop Patinopecten yessoensis (designated PyTEPs) was performed at all developmental stages, in different healthy adult tissues, and in mantles during exposure to different levels of acidification (pH = 6.5 and 7.5) for different time points (3, 6, 12 and 24 h); this profiling was accomplished through in silico analysis of transcriptome and genome databases. Spatiotemporal expression patterns revealed that PyTEPs had specific functional differentiation in all stages of growth and development of the scallop. Expression analysis confirmed the inducible expression patterns of PyTEPs during exposure to acidification. Gene duplication and alternative splicing events simultaneously occurred in PyTEP1. Seven different cDNA variants of PyTEP1 (designated PyTEP1-A–PyTEP1-G) were identified in the scallop mantle transcriptome during acidic stress. These variants were produced by the alternative splicing of seven differentially transcribed exons (exons 18–24), which encode the highly variable central region. The responses to immune stress may have arisen through the gene duplication and alternative splicing of PyTEP1. The sequence diversity of PyTEP1 isoforms and their different expression profiles in response to ocean acidification (OA) suggested a mechanism used by scallops to differentiate and regulate PyTEP1 gene expression. Collectively, these results demonstrate the gene duplication and alternative splicing of TEP family genes and provide valuable resources for elucidating their versatile roles in bivalve innate immune responses to OA challenge.

A Gut Symbiotic Bacterium Serratia marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses.

The malaria development in the mosquito midgut is a complex process that results in considerable parasite losses. The mosquito gut microbiota influences the outcome of pathogen infection in mosquitoes, but the underlying mechanisms through which gut symbiotic bacteria affect vector competence remain elusive. Here, we identified two Serratia strains (Y1 and J1) isolated from field-caught female Anopheles sinensis from China and assessed their effect on Plasmodium development in An. stephensi. Colonization of An. stephensi midgut by Serratia Y1 significantly renders the mosquito resistant to Plasmodium berghei infection, while Serratia J1 has no impact on parasite development. Parasite inhibition by Serratia Y1 is induced by the activation of the mosquito immune system. Genome-wide transcriptomic analysis by RNA-seq shows a similar pattern of midgut gene expression in response to Serratia Y1 and J1 in sugar-fed mosquitoes. However, 24 h after blood ingestion, Serratia Y1 modulates more midgut genes than Serratia J1 including the c-type lectins (CTLs), CLIP serine proteases and other immune effectors. Furthermore, silencing of several Serratia Y1-induced anti-Plasmodium factors like the thioester-containing protein 1 (TEP1), fibrinogen immunolectin 9 (FBN9) or leucine-rich repeat protein LRRD7 can rescue parasite oocyst development in the presence of Serratia Y1, suggesting that these factors modulate the Serratia Y1-mediated anti-Plasmodium effect. This study enhances our understanding of how gut bacteria influence mosquito-Plasmodium interactions.

Anopheles gambiae TEP1 forms a complex with the coiled-coil domain of LRIM1/APL1C following a conformational change in the thioester domain.

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms exhibit significant variation in stability from hours to days at room temperature. In particular, the refractory allele TEP1*R1 is significantly more stable than the susceptible allele TEP1*S1. This raises the question of whether the stability of cleaved TEP1 is linked to allelic variation and varying immunophenotypes. We have analyzed the stability of the cleaved form of additional TEP1 alleles and constructs. We show that stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains of TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1cut/LRIM1/APL1C complex is related to the conformational change that induces the precipitation of cleaved TEP1.

Evidence of trained immunity in teleost fish: Conserved features in carp macrophages

Thioester-containing proteins (TEPs), characterized by a unique intrachain β-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in CfTEP, a previously identified thioester-containing protein in scallop Chlamys farreri, were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of CfTEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The CfTEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the CfTEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of CfTEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or β-glucan. Two bands (100 and 55 kDa) were detected using anti-CfTEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942–1472), and a single band (46 kDa) was detected by using anti-CfTEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452–496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of CfTEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum. All these results suggested that CfTEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops.

Identification and functional analysis of a thioester-containing protein from Portunus trituberculatus reveals its involvement in the prophenoloxidase system, phagocytosis and AMP synthesis

Thioester-containing proteins (TEPs) are an ancient superfamily of secreted effector proteins that perform essential roles in the innate immune response. Herein, a TEP gene, designated as PtTEP, was identified from the swimming crab Portunus trituberculatus. The open reading frame (ORF) of PtTEP was 4434 bp in length, encoded a polypeptide with 1478 amino acids containing the conserved sequence features of insect TEP. PtTEP was highly expressed in most immune-related tissues, such as intestine, gill and hepatopancreas, and the expression of PtTEP was significantly up-regulated in hemocytes after bacterial and fungal challenges. Compared to that in fertilized eggs, the transcripts of PtTEP were decreased obviously in the cleavage stage and followed by a significant up-regulation in the blastula, gastrula and heart-beating stages. Combined with the higher mRNAs in crab ovary, it is indicated that PtTEP be a maternal immune factor. RNA interference-mediated suppression of PtTEP could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1–3 and PtSPH) but inhibit the expression of PtLSZ and phagocytosis-related genes (PtMyosin and PtRab5). These results were further supported by the PO and lysozyme activities in hemolymph of the PtTEP-silenced crabs. We also observed that silencing of PtTEP reduced the expression of antimicrobial peptide genes (PtALF1–3, PtCrustin1 and PtCrustin3) and the genes involved in activation of the Toll and NF-κB pathways and additionally increased the mortality of Vibrio alginolyticus infected crabs. Taken together, our study suggests that PtTEP might function in crab innate immune defense via regulating the proPO-activating system, phagocytosis and AMP synthesis.

CD109: a multifunctional GPI-anchored protein with key roles in tumor progression and physiological homeostasis.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein and a member of the α2 -macroglobulin/C3,C4,C5 family of thioester-containing proteins first identified as being expressed on blood cells, including activated T cells and platelets, and a subset of CD34 + bone marrow cells containing megakaryocyte progenitors. Although CD109 carries the biallelic platelet-specific alloantigen Gov, the physiological functions or roles of CD109 in human disease remain largely unknown. It was recently demonstrated that CD109 is expressed in many malignant tumors, including various squamous cell carcinomas and adenocarcinomas, and plays a role as a multifunctional coreceptor. CD109 reportedly associates with transforming growth factor (TGF)-β receptors and negatively regulates TGF-β signaling in keratinocytes. Additionally, CD109 is potentially related to signal transducer and activator of transcription-3 signaling and aberrant cell proliferation. In this review, we describe recent evidence of CD109-specific significance in malignant tumors shown in mouse models and human tissues. Furthermore, we discuss the physiological functions of CD109 in vitro and in vivo, including results of phenotype analyses of CD109-deficient mice exhibiting epidermal hyperplasia and osteopenia.

Complement-like proteins TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the mosquito Anopheles gambiae

The mosquito immune and circulatory systems are functionally integrated. During an infection, hemocytes aggregate around the ostia (valves) of the dorsal vessel – areas of the heart called the periostial regions – where they phagocytose live and melanized pathogens. Although periostial hemocyte aggregation is an immune response that occurs following infection with bacteria and malaria parasites, the molecular basis of this process remains poorly understood. Here, we show that the thioester-containing proteins, TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the African malaria mosquito, Anopheles gambiae. RNAi-based knockdown of TEP1, TEP3 and TEP4 resulted in fewer periostial hemocytes following Escherichia coli infection, without affecting the adjacent population of non-periostial, sessile hemocytes. Moreover, knockdown of TEP1, TEP3 and TEP4 expression resulted in reduced bacterial accumulation and melanin deposition at the periostial regions. Finally, this study confirmed the role that TEP1 plays in reducing infection intensity in the hemocoel. Overall, this research shows that the complement-like proteins, TEP1, TEP3 and TEP4, are positive regulators of the functional integration between the immune and circulatory systems of mosquitoes.

Anopheles stephensi Dual Oxidase Silencing Activates the Thioester-Containing Protein 1 Pathway to Suppress Plasmodium Development

We characterized the dual oxidase (Duox) gene in the major Indian malaria vector Anopheles stephensi, which regulates the generation of reactive oxygen species. The AsDuox gene encodes for a 1,475-amino-acid transmembrane protein that contains an N-terminal noncytoplasmic heme peroxidase domain, a calcium-binding domain, seven transmembrane domains, and a C-terminal cytoplasmic NADPH domain. Phylogenetic analyses revealed that A. stephensi Duox protein is highly conserved and shares 97–100% amino acid identity with other anopheline Duoxes. AsDuox is expressed in all the developmental stages of A. stephensi and the pupal stages revealed relatively higher expressions. The Duox gene is induced in Plasmodium-infected mosquito midguts, and RNA interference-mediated silencing of this gene suppressed parasite development through activation of the thioester-containing protein 1 pathway. We propose that this highly conserved anopheline Duox, being a Plasmodium agonist, is an excellent target to control malaria parasite development inside the insect host.

BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata.

Insect thioester-containing protein (iTEP) is the most recently defined group among the thioester-containing protein (TEP) superfamily. TEPs are key components of the immune system, and iTEPs from flies and mosquitoes were shown to be major immune weapons. Initially characterized from insects, TEP genes homologous to iTEP were further described from several other invertebrates including arthropods, cniderians, and mollusks albeit with few functional characterizations. In the freshwater snail Biomphalaria glabrata, a vector of the schistosomiasis disease, the presence of a TEP protein (BgTEP) was previously described in a well-defined immune complex involving snail lectins (fibrinogen-related proteins) and schistosome parasite mucins (SmPoMuc). To investigate the potential role of BgTEP in the immune response of the snail, we first characterized its genomic organization and its predicted protein structure. A phylogenetic analysis clustered BgTEP in a well-conserved subgroup of mollusk TEP. We then investigated the BgTEP expression profile in different snail tissues and followed immune challenges using different kinds of intruders during infection kinetics. Results revealed that BgTEP is particularly expressed in hemocytes, the immune-specialized cells in invertebrates, and is secreted into the hemolymph. Transcriptomic results further evidenced an intruder-dependent differential expression pattern of BgTEP, while interactome experiments showed that BgTEP is capable of binding to the surface of different microbes and parasite either in its full length form or in processed forms. An immunolocalization approach during snail infection by the Schistosoma mansoni parasite revealed that BgTEP is solely expressed by a subtype of hemocytes, the blast-like cells. This hemocyte subtype is present in the hemocytic capsule surrounding the parasite, suggesting a potential role in the parasite clearance by encapsulation. Through this work, we report the first characterization of a snail TEP. Our study also reveals that BgTEP may display an unexpected functional dual role. In addition to its previously characterized anti-protease activity, we demonstrate that BgTEP can bind to the intruder surface membrane, which supports a likely opsonin role.

Identification and characterization of TEP family genes in Yesso scallop (Patinopecten yessoensis) and their diverse expression patterns in response to bacterial infection

Thioester-containing protein (TEP) family members are characterized by their unique intrachain β-cysteinyl-γ-glutamyl thioesters, and they play important roles in innate immune responses. Although significant effects of TEP members on immunity have been reported in most vertebrates, as well as certain invertebrates, the complete TEP family has not been systematically characterized in scallops. In this study, five TEP family genes (PyC3, PyA2M, PyTEP1, PyTEP2 and PyCD109) were identified from Yesso scallop (Patinopecten yessoensis) through whole-genome scanning, including one pair of tandem duplications located on the same scaffold. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the five genes (PyTEPs). The vast distribution of PyTEPs in TEP subfamilies confirmed that the Yesso scallop contains relatively comprehensive types of TEP members in evolution. The expression profiles of PyTEPs were determined in hemocytes after bacterial infection with gram-positive (Micrococcus luteus) and gram-negative (Vibrio anguillarum) using quantitative real-time PCR (qRT-PCR). Expression analysis revealed that the PyTEP genes exhibited disparate expression patterns in response to the infection by gram bacteria. A majority of PyTEP genes were overexpressed after bacterial stimulation at most time points, especially the notable elevation displayed by duplicated genes after V. anguillarum challenge. Interestingly, at different infection times, PyTEP1 and PyTEP2 shared analogous expression patterns, as did PyC3 and PyCD109. Taken together, these results help to characterize gene duplication and the evolutionary origin of PyTEPs and supplied valuable resources for elucidating their versatile roles in bivalve innate immune responses to bacterial pathogen challenges.

Thioester-Containing Proteins 2 and 4 Affect the Metabolic Activity and Inflammation Response in Drosophila.

Drosophila melanogaster is an outstanding model for studying host antipathogen defense. Although substantial progress has been made in understanding how metabolism and immunity are interrelated in flies, little information has been obtained on the molecular players that regulate metabolism and inflammation in Drosophila during pathogenic infection. Recently, we reported that the inactivation of thioester-containing protein 2 (Tep2) and Tep4 promotes survival and decreases the bacterial burden in flies upon infection with the virulent pathogens Photorhabdus luminescens and Photorhabdus asymbiotica Here, we investigated physiological and pathological defects in tep mutant flies in response to Photorhabdus challenge. We find that tep2 and tep4 loss-of-function mutant flies contain increased levels of carbohydrates and triglycerides in the presence or absence of Photorhabdus infection. We also report that Photorhabdus infection leads to higher levels of nitric oxide and reduced transcript levels of the apical caspase-encoding gene Dronc in tep2 and tep4 mutants. We show that Tep2 and Tep4 are upregulated mainly in the fat body rather than the gut in Photorhabdus-infected wild-type flies and that tep mutants contain decreased numbers of Photorhabdus bacteria in both tissue types. We propose that the inactivation of Tep2 or Tep4 in adult Drosophila flies results in lower levels of inflammation and increased energy reserves in response to Photorhabdus, which could confer a survival-protective effect during the initial hours of infection.

Detection of antibacterial protein in Bacillus sphaericus-treated Culex pipiens (Diptera: Culicidae)

Antimicrobial peptides (AMPs) constitute a major arm of defense in mosquitoes against microbes. The purpose of this study was to determine which of the peptides are produced in the mosquito hemolymph after bacterial treatment. Culex pipiens (Diptera: Culicidae) larvae, collected from drainage canal in Suez Governorate, Egypt, were treated with LC80 of Bacillus sphaericus strain 2362 (0.035 ppm). The hemolymph of bacteria-treated Cx. pipiens mosquitoes and non-treated mosquitoes as control were extracted and undergo electrophoresis, using a Bio-Rad Mini-protean II cell. In Cx. pipiens mosquitoes emerging from bacteria-treated larvae, a band of protein was detected at 165 kDa. This protein was more likely to be Thioester-containing protein 1 (TEP1). Understanding mosquito resistance mechanism to bacterial control is crucial to build up programs to overcome such resistance.

Thioester-containing proteins in the tsetse fly (Glossina) and their response to trypanosome infection.

Thioester‐containing proteins (TEPs) are conserved proteins with a role in innate immune immunity. In the current study, we characterized the TEP family in the genome of six tsetse fly species (Glossina spp.). Tsetse flies are the biological vectors of several African trypanosomes, which cause sleeping sickness in humans or nagana in livestock. The analysis of the tsetse TEP sequences revealed information about their structure, evolutionary relationships and expression profiles under both normal and trypanosome infection conditions. Phylogenetic analysis of the family showed that tsetse flies harbour a genomic expansion of specific TEPs that are not found in other dipterans. We found a general expression of all TEP genes in the alimentary tract, mouthparts and salivary glands. Glossina morsitans and Glossina palpalis TEP genes display a tissue‐specific expression pattern with some that are markedly up‐regulated when the fly is infected with the trypanosome parasite. A different TEP response was observed to infection with Trypanosoma brucei compared to Trypanosoma congolense, indicating that the tsetse TEP response is trypanosome‐specific. These findings are suggestive for the involvement of the TEP family in tsetse innate immunity, with a possible role in the control of the trypanosome parasite.

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.

Rapid seasonal evolution in innate immunity of wild Drosophila melanogaster.

Understanding the rate of evolutionary change and the genetic architecture that facilitates rapid adaptation is a current challenge in evolutionary biology. Comparative studies show that genes with immune function are among the most rapidly evolving genes across a range of taxa. Here, we use immune defence in natural populations of Drosophila melanogaster to understand the rate of evolution in natural populations and the genetics underlying rapid change. We probed the immune system using the natural pathogens Enterococcus faecalis and Providencia rettgeri to measure post-infection survival and bacterial load of wild D. melanogaster populations collected across seasonal time along a latitudinal transect along eastern North America (Massachusetts, Pennsylvania and Virginia). There are pronounced and repeatable changes in the immune response over the approximately 10 generations between spring and autumn collections, with a significant but less distinct difference observed among geographical locations. Genes with known immune function are not enriched among alleles that cycle with seasonal time, but the immune function of a subset of seasonally cycling alleles in immune genes was tested using reconstructed outbred populations. We find that flies containing seasonal alleles in Thioester-containing protein 3 (Tep3) have different functional responses to infection and that epistatic interactions among seasonal Tep3 and Drosomycin-like 6 (Dro6) alleles underlie the immune phenotypes observed in natural populations. This rapid, cyclic response to seasonal environmental pressure broadens our understanding of the complex ecological and genetic interactions determining the evolution of immune defence in natural populations.

Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps

BackgroundMembers of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPqΔ, lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4.ResultsSurvival analyses with TEPqΔ flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPqΔ flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation.ConclusionsCollectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.