Thioester-containing Protein Research Articles

Thioester-containing proteins of the tick Ixodes ricinus: Gene expression, response to microbial challenge and their role in phagocytosis of the yeast Candida albicans

The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens.

The structure and function of thioester-containing proteins in arthropods.

Thioester-containing proteins (TEPs) form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. Two families of TEPs, complement factors and α2-macroglobulins, have been known and studied in vertebrates for many years, but only in the last decade have crystal structures become available. In the same period, the presence of two additional classes of TEPs has been revealed in arthropods. In this review, we discuss the common structural features TEPs and how this knowledge can be applied to the many arthropod TEPs of unknown function. TEPs perform a wide variety of functions that are driven by different quaternary structures and protein-protein interactions between a common set of folded domains. A common theme is regulated conformational change triggered by proteolysis. Structure-function analysis of the diverse arthropod TEPs may identify not just new mechanisms in innate immunity but also interfaces between immunity, development and cell death.

Complement-related proteins control the flavivirus infection of Aedes aegypti by inducing antimicrobial peptides.

The complement system functions during the early phase of infection and directly mediates pathogen elimination. The recent identification of complement-like factors in arthropods indicates that this system shares common ancestry in vertebrates and invertebrates as an immune defense mechanism. Thioester (TE)-containing proteins (TEPs), which show high similarity to mammalian complement C3, are thought to play a key role in innate immunity in arthropods. Herein, we report that a viral recognition cascade composed of two complement-related proteins limits the flaviviral infection of Aedes aegypti. An A. aegypti macroglobulin complement-related factor (AaMCR), belonging to the insect TEP family, is a crucial effector in opposing the flaviviral infection of A. aegypti. However, AaMCR does not directly interact with DENV, and its antiviral effect requires an A. aegypti homologue of scavenger receptor-C (AaSR-C), which interacts with DENV and AaMCR simultaneously in vitro and in vivo. Furthermore, recognition of DENV by the AaSR-C/AaMCR axis regulates the expression of antimicrobial peptides (AMPs), which exerts potent anti-DENV activity. Our results both demonstrate the existence of a viral recognition pathway that controls the flaviviral infection by inducing AMPs and offer insights into a previously unappreciated antiviral function of the complement-like system in arthropods.

Gene discovery and differential expression analysis of humoral immune response elements in female Culicoides sonorensis (Diptera: Ceratopogonidae).

BackgroundFemale Culicoides sonorensis midges (Diptera: Ceratopogonidae) are vectors of pathogens that impact livestock and wildlife in the United States. Little is known about their biology on a molecular-genetic level, including components of their immune system. Because the insect immune response is involved with important processes such as gut microbial homeostasis and vector competence, our aims were to identify components of the midge innate immune system and examine their expression profiles in response to diet across time.MethodsIn our previous work, we de novo sequenced and analyzed the transcriptional landscape of female midges under several feeding states including teneral (unfed) and early and late time points after blood and sucrose. Here, those transcriptomes were further analyzed to identify insect innate immune orthologs, particularly humoral immune response elements. Additionally, we examined immune gene expression profiles in response to diet over time, on both a transcriptome-wide, whole-midge level and more specifically via qRTPCR analysis of antimicrobial peptide (AMP) expression in the alimentary canal.ResultsWe identified functional units comprising the immune deficiency (Imd), Toll and JAK/STAT pathways, including humoral factors, transmembrane receptors, signaling components, transcription factors/regulators and effectors such as AMPs. Feeding altered the expression of receptors, regulators, AMPs, prophenoloxidase and thioester-containing proteins, where blood had a greater effect than sucrose on the expression profiles of most innate immune components. qRTPCR of AMP genes showed that all five were significantly upregulated in the alimentary canal after blood feeding, possibly in response to proliferating populations of gut bacteria.ConclusionsIdentification and functional insight of humoral/innate immune components in female C. sonorensis updates our knowledge of the molecular biology of this important vector. Because diet alone influenced the expression of immune pathway components, including their effectors, subsequent study of the role of innate immunity in biological processes such as gut homeostasis and life history are being pursued. Furthermore, since the humoral response is a key contributor in gut immunity, manipulating immune gene expression will help in uncovering genetic components of vector competence, including midgut barriers to infection. The results of such studies will serve as a platform for designing novel transmission-blocking strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-388) contains supplementary material, which is available to authorized users.

Ability of TEP1 in intestinal flora to modulate natural resistance of Anopheles dirus

Blocking transmission of malaria is a reliable way to control and eliminate infection. However, in-depth knowledge of the interaction between Plasmodium and mosquito is needed. Studies suggest that innate immunity is the main mechanism inhibiting development of malaria parasites in the mosquito. Recent studies have found that use of antibiotics that inhibit the mosquito gut flora can reduce the immune response of Anopheles gambiae, thereby contributing to the development of malaria parasites. In our study, we used the non susceptible model of Anopheles dirus–Plasmodium yoelii to explore the effect of Anopheles intestinal flora on the natural resistance of A. dirus to P. yoelii. We found that in mosquitoes infected with Plasmodium, the intestinal flora can regulate expression of thioester-containing protein (TEP1) via an RNAi gene-silencing approach. Our results suggest that in the absence of TEP1, the natural microbiota cannot suppress the development of P. yoelii in A. dirus. This suggests that AdTEP1 plays an important role in the resistance of A. dirus to P. yoelii. The intestinal flora may modulate the development of P. yoelii in A. dirus by regulating TEP1 expression.

AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

Retraction

genesisVolume 51, Issue 5 p. 381-381 RetractionFree Access Retraction First published: 22 May 2013 https://doi.org/10.1002/dvg.22394AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The following article from Genesis, “The gene encoding the thioester-containing protein, CPAMD8, is essential for construction of adult tissues in the chordate, Ciona intestinalis” by Terumi Matsuoka, Satoko Awazu, Michio Ogasawara, Kazuo Inaba, Nori Satoh and Yasunori Sasakura, published online on 26 February 2013 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor in Chief Dr. Sally A. Moody, and Wiley Periodicals. The retraction has been agreed due to disagreements over authorship. Reference The gene encoding the thioester-containing protein, CPAMD8, is essential for construction of adult tissues in the chordate, Ciona intestinalis Terumi Matsuoka, Satoko Awazu, Michio Ogasawara, Kazuo Inaba, Nori Satoh and Yasunori Sasakura DOI: 10.1002/dvg.22383 Volume51, Issue5Special Issue: Chick GenomicsMay 2013Pages 381-381 ReferencesRelatedInformation

Retracted: The gene encoding the thioester-containing protein, CPAMD8, is essential for construction of adult tissues in the chordate, Ciona intestinalis

Retracted: The gene encoding the thioester-containing protein, CPAMD8, is essential for construction of adult tissues in the chordate, <i>Ciona intestinalis</i>

Interaction of the tick immune system with transmitted pathogens

Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes is likely mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia), rickettsiae (Anaplasma), and protozoans (Babesia). Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens.

Molecular Basis for Genetic Resistance of Anopheles gambiae to Plasmodium: Structural Analysis of TEP1 Susceptible and Resistant Alleles

Thioester-containing protein 1 (TEP1) is a central component in the innate immune response of Anopheles gambiae to Plasmodium infection. Two classes of TEP1 alleles, TEP1*S and TEP1*R, are found in both laboratory strains and wild isolates, related by a greater or lesser susceptibility, respectively to both P. berghei and P. falciparum infection. We report the crystal structure of the full-length TEP1*S1 allele which, while similar to the previously determined structure of full-length TEP1*R1, displays flexibility in the N-terminal fragment comprising domains MG1-MG6. Amino acid differences between TEP1*R1 and TEP1*S1 are localized to the TED-MG8 domain interface that protects the thioester bond from hydrolysis and structural changes are apparent at this interface. As a consequence cleaved TEP1*S1 (TEP1*S1cut) is significantly more susceptible to hydrolysis of its intramolecular thioester bond than TEP1*R1cut. TEP1*S1cut is stabilized in solution by the heterodimeric LRIM1/APL1C complex, which preserves the thioester bond within TEP1*S1cut. These results suggest a mechanism by which selective pressure on the TEP1 gene results in functional variation that may influence the vector competence of A. gambiae towards Plasmodium infection.

Evolution of the thioester-containing protein genes in the arthropoda

Evolution of the thioester-containing protein genes in the arthropoda

Pattern recognition receptors acting in innate immune system of shrimp against pathogen infections

Invertebrates, including shrimp, have developed very complicated innate immune system against pathogens. Much work has been performed on the innate immunity of shrimp, including immune recognition, signal transduction, effector molecules and antiviral responses due to its great economic value. Pattern recognition is the first step of innate immunity. Pattern recognition receptors (PRRs) sense the presence of infection and activate immune responses. The studies on shrimp PRRs revealed the recognition mechanism of shrimp at a certain degree. To date, 11 types of pattern recognition receptors (PRRs) have been identified in shrimp, namely, β-1,3-glucanase-related proteins, β-1,3-glucan-binding proteins, C-type lectins, scavenger receptors, galectins, fibrinogen-related proteins, thioester-containing protein, Down syndrome cell adhesion molecule, serine protease homologs, trans-activation response RNA-binding protein and Toll like receptors. A number of PRRs have been functionally studied and have been found to have different binding specificities and immune functions. The present review aims to summarize the current knowledge on the PRRs of shrimp.

Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes.

Plasmodium falciparum lines differ in their ability to infect mosquitoes. The Anopheles gambiae L3-5 refractory (R) line melanizes most Plasmodium species, including the Brazilian P. falciparum 7G8 line, but it is highly susceptible to some African P. falciparum strains such as 3D7, NF54, and GB4. We investigated whether these lines differ in their ability to evade the mosquito immune system. Silencing key components of the mosquito complement-like system [thioester-containing protein 1 (TEP1), leucine-rich repeat protein 1, and Anopheles Plasmodium-responsive leucine-rich repeat protein 1] prevented melanization of 7G8 parasites, reverting the refractory phenotype. In contrast, it had no effect on the intensity of infection with NF54, suggesting that this line is able to evade TEP1-mediated lysis. When R females were coinfected with a line that is melanized (7G8) and a line that survives (3D7), the coinfection resulted in mixed infections with both live and encapsulated parasites on individual midguts. This finding shows that survival of individual parasites is parasite-specific and not systemic in nature, because parasites can evade TEP1-mediated lysis even when other parasites are melanized in the same midgut. When females from an extensive genetic cross between R and susceptible A. gambiae (G3) mosquitoes were infected with P. berghei, encapsulation was strongly correlated with the TEP1-R1 allele. However, P. falciparum 7G8 parasites were no longer encapsulated by females from this cross, indicating that the TEP1-R1 allele is not sufficient to melanize this line. Evasion of the A. gambiae immune system by P. falciparum may be the result of parasite adaptation to sympatric mosquito vectors and may be an important factor driving malaria transmission.

Abstract 4250: Deficiency of CD109, a negative regulator of TGF-β signaling, leads epidermal hyperplasia and appendage abnormalities in mice

Abstract CD109, a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, is a member of the β2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is highly expressed in various tumor cell lines and in tumor tissues including squamous cell carcinomas (SCCs) of the lung, oral cavity, esophagus and uterine cervix, malignant melanoma of the skin, and urothelial carcinoma of the urinary bladder. CD109 is, therefore, thought to be a cancer-related gene. On the other hand, CD109 is a component of TGF-β receptor system and negatively regulates TGF-β signaling. However, its physiological function in vivo remains largely unknown. To investigate the physiological function of CD109 in vivo, we generated CD109-deficient (CD109-/-) mice in which exon 1 and 2 of CD109 genomic locus were replaced with the lacZ gene. CD109 protein is expressed in the skin, tongue, and testis of adult CD109+/+ mice, but not in CD109-/- mice. Macroscopically, CD109-/- mice showed hair growth impairment from postnatal day 7 (P7) and persisted until P28. Microscopically, hyperplasia of the epidermis and sebaceous glands became apparent at P7 in CD109-/- mice and sustained until P70, and the hair follicles of CD109-/- mice exhibited ectasia with kinked hair shafts at P14. The epidermis of CD109-/- mice showed apparent thickening of the basal / suprabasal layers, which are positive for CK14, compared with CD109+/+ mice, whereas no significant differences in Ki-67 or cleaved caspase-3-positive ratios were detected between the two groups. These results suggest that CD109 regulates the differentiation of keratinocytes in vivo, and impairment of hair growth and ectatic hair follicles may be secondary changes caused by narrowing of the infundibular portion of the hair follicles due to epithelial hyperplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4250. doi:1538-7445.AM2012-4250

Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity.

Plasmodium ookinetes traverse midgut epithelial cells before they encounter the complement system in the mosquito hemolymph. We identified a heme peroxidase (HPX2) and NADPH oxidase 5 (NOX5) as critical mediators of midgut epithelial nitration and antiplasmodial immunity that enhance nitric oxide toxicity in Anopheles gambiae. We show that the two immune mechanisms that target ookinetes-epithelial nitration and thioester-containing protein 1 (TEP1)-mediated lysis-work sequentially, and we propose that epithelial nitration works as an opsonization-like system that promotes activation of the mosquito complement cascade.

An insect TEP in a crustacean is specific for cuticular tissues and involved in intestinal defense

In an attempt to identify genes encoding thioester-containing proteins in the freshwater crayfish, Pacifastacus leniusculus, three different cDNAs were found. A phylogenetic analysis of these proteins indicates that they can be classified into two subfamilies: two alpha-2-macroglobulins ( Pl-A2M1, Pl-A2M2) showing a close similarity to shrimp A2M, and one insect TEP-like protein ( Pl-TEP). This is the first report of an insect TEP-like protein in a crustacean. Crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP cDNAs encode proteins with 1480, 1586 or 1507 amino acids, respectively . Pl-A2M1, Pl-A2M2 and Pl-TEP have the basic domain structure and functionally important residues for each molecule, and their mRNA was detected in different parts of the body, suggesting that they may have different functions. Pl-A2M1 was mainly expressed in hemocytes and Pl-A2M2 was highly expressed in heart and nerve, while Pl-TEP was exclusively expressed in cuticular tissues such as gill and intestine. RNA interference of Pl-TEP in vivo resulted in that these animals were slightly less resistant when fed with the bacterium, Pseudomonas libanensis/gessardii. Furthermore, when TEP activity was blocked using methylamine followed by bacterial feeding, the animals were killed to a higher extent compared to a control group. Taken together, this indicates that Pl-TEP and/or Pl-A2M1, Pl-A2M2 may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues.

An In Vivo Transfection Approach Elucidates a Role for Aedes aegypti Thioester-Containing Proteins in Flaviviral Infection

Mosquitoes transmit pathogens that cause infectious diseases of global importance. Techniques to easily introduce genes into mosquitoes, however, limit investigations of the interaction between microbes and their arthropod vectors. We now show that a cationic liposome significantly enhances delivery and expression of plasmid DNA in Aedes aegypti and Anopheles gambiae mosquitoes. We then introduced the genes for Ae. aegypti thioester-containing proteins (AeTEPs), which are involved in the control of flaviviral infection, into mosquitoes using this technique. In vivo transfection of AeTEP-1 into Ae. aegypti significantly reduced dengue virus infection, suggesting that the approach can further our understanding of pathogen-mosquito interactions.

Chemical labelling of active serum thioester proteins for quantification

The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.

Evolution of the thioester-containing proteins (TEPs) of the arthropoda, revealed by molecular cloning of TEP genes from a spider, Hasarius adansoni

The thioester-containing protein (TEP) family of genes, found in most Eumetazoan genomes, is classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily. Many A2M subfamily members, including insect TEP (iTEP), have been reported from the Arthropoda, whereas the C3 subfamily members have been reported only from two horseshoe crab species thus far. To elucidate the evolution of these genes among the Arthropoda, TEP genes were isolated from a spider, Hasarius adansoni (Chelicerata), by reverse transcription polymerase chain reaction (RT–PCR) amplification using universal degenerate primers specific for the thioester region. Four different TEP genes were identified. Phylogenetic analysis using the entire amino acid sequences of these and various other TEP sequences from the Eumetazoa indicated that two of the spider genes are type C3 ( HaadC3-1 and HaadC3-2), one is type A2M ( HaadA2M) and the other is closely related to iTEP ( HaadiTEP). These results suggest that the common ancestor of the Arthropoda possessed at least three TEP genes, C3, A2M and iTEP and that they were lost differentially in the Crustacean and Hexapodan lineages.