Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56.

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Fibronectin-Functionalized Fibrous Meshes as a Substrate to Support Cultures of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.

Thymic crosstalk restrains the pool of cortical thymic epithelial cells with progenitor properties.

Cortical (cTEC) and medullary (mTEC) thymic epithelial cells establish key microenvironments for T-cell differentiation and arise from thymic epithelial cell progenitors (TEP). However, the nature of TEPs and the mechanism controlling their stemness in the postnatal thymus remain poorly defined. Using TEC clonogenic assays as a surrogate to survey TEP activity, we found that a fraction of cTECs generates specialized clonal-derived colonies, which contain cells with sustained colony-forming capacity (ClonoTECs). These ClonoTECs are EpCAM+MHCII-Foxn1lo cells that lack traits of mature cTECs or mTECs but co-express stem-cell markers, including CD24 and Sca-1. Supportive of their progenitor identity, ClonoTECs reintegrate within native thymic microenvironments and generate cTECs or mTECs in vivo. Strikingly, the frequency of cTECs with the potential to generate ClonoTECs wanes between the postnatal and young adult immunocompetent thymus, but it is sustained in alymphoid Rag2-/-Il2rg-/- counterparts. Conversely, transplantation of wild-type bone marrow hematopoietic progenitors into Rag2-/-Il2rg-/- mice and consequent restoration of thymocyte-mediated TEC differentiation diminishes the frequency of colony-forming units within cTECs. Our findings provide evidence that the cortical epithelium contains a reservoir of epithelial progenitors whose abundance is dynamically controlled by continual interactions with developing thymocytes across lifespan.

Intracellular trafficking of AIP56, an NF-κB-cleaving toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.

Two thioredoxin-superfamily members from sea bass (Dicentrarchus labrax, L.): characterization of PDI (PDIA1) and ERp57 (PDIA3).

PDI (PDIA1) and ERp57 (PDIA3), members of the PDI family and of the thioredoxin (Trx) superfamily, are multifunctional proteins with wide physiological roles and have been implicated in several pathologies. Importantly, they are both involved in the MHC class I antigen presentation pathway. This paper reports the isolation and characterization of full cDNA and genomic clones from sea bass (Dicentrarchus labrax, L.) PDI (Dila-PDI) and ERp57 (Dila-ERp57). The genes are ~12.4 and ~7.1 kb long, originating 2155 and 2173 bp transcripts and encoding 497 and 484 amino acids mature proteins, for Dila-PDI and -ERp57, respectively. The PDI gene consists of eleven exons and ERp57 of thirteen. As described in other species, both molecules are composed of four Trx-like domains (abb'a') followed by a C-terminal tail, retaining two CGHC active sites and an ER-signalling sequence, suggestive of a conserved function. Additionally, three-dimensional homology models further support Dila-PDI and Dila-ERp57 as orthologs of mammalian PDI and ERp57, respectively. Finally, high similarity is observed to their vertebrate counterparts (>69% identity), especially among the few ones from closely related teleosts (>79% identity). Hence, these results provide relevant primary data and will enable further studies to clarify the roles of PDI and ERp57 in European sea bass immunity.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) calreticulin.

Mammalian calreticulin (CRT) is a key molecular chaperone and regulator of Ca(2+) homeostasis in endoplasmic reticulum (ER), also being implicated in a variety of physiological/pathological processes outside the ER. Importantly, it is involved in assembly of MHC class I molecules. In this work, sea bass (Dicentrarchus labrax) CRT (Dila-CRT) gene and cDNA have been isolated and characterized. The mature protein retains two conserved motifs, three structural/functional domains (N, P and C), three type 1 and 2 motifs repeated in tandem, a conserved pair of cysteines and ER-retention motif. It is a single-copy gene composed of 9 exons. Dila-CRT three-dimensional homology models are consistent with the structural features described for mammalian molecules. Together, these results are supportive of a highly conserved structure of CRT through evolution. Moreover, the present data provides information that will allow further studies on sea bass CRT involvement in immunity and in particular class I antigen presentation.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) MHC class I heavy chain and ß2-microglobulin.

In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) ß2-microglobulin (Dila-ß2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-ß2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-ß2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of ß2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other ß2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) Tapasin.

Mammalian tapasin (TPN) is a key member of the major histocompatibility complex (MHC) class I antigen presentation pathway, being part of the multi-protein complex called the peptide loading complex (PLC). Several studies describe its important roles in stabilizing empty MHC class I complexes, facilitating peptide loading and editing the repertoire of bound peptides, with impact on CD8(+) T cell immune responses. In this work, the gene and cDNA of the sea bass (Dicentrarchus labrax) glycoprotein TPN have been isolated and characterized. The coding sequence has a 1329 bp ORF encoding a 442-residue precursor protein with a predicted 24-amino acid leader peptide, generating a 418-amino acid mature form that retains a conserved N-glycosylation site, three conserved mammalian tapasin motifs, two Ig superfamily domains, a transmembrane domain and an ER-retention di-lysine motif at the C-terminus, suggestive of a function similar to mammalian tapasins. Similar to the human counterpart, the sea bass TPN gene comprises 8 exons, some of which correspond to separate functional domains of the protein. A three-dimensional homology model of sea bass tapasin was calculated and is consistent with the structural features described for the human molecule. Together, these results support the concept that the basic structure of TPN has been maintained through evolution. Moreover, the present data provides information that will allow further studies on cell-mediated immunity and class I antigen presentation pathway in particular, in this important fish species.

Transporters associated with antigen processing (TAP) in sea bass (Dicentrarchus labrax, L.): molecular cloning and characterization of TAP1 and TAP2.

The transporters associated with antigen processing (TAP), play an important role in the MHC class I antigen presentation pathway. In this work, sea bass (Dicentrarchus labrax) TAP1 and TAP2 genes and transcripts were isolated and characterized. Only the TAP2 gene is structurally similar to its human orthologue. As other TAP molecules, sea bass TAP1 and TAP2 are formed by one N-terminal accessory domain, one core membrane-spanning domain and one canonical C-terminal nucleotide-binding domain. Homology modelling of the sea bass TAP dimer predicts that its quaternary structure is in accordance with that of other ABC transporters. Phylogenetic analysis segregates sea bass TAP1 and TAP2 into each subfamily cluster of transporters, placing them in the fish class and suggesting that the basic structure of these transport-associated proteins is evolutionarily conserved. Furthermore, the present data provides information that will enable more studies on the class I antigen presentation pathway in this important fish species.

Molecular characterization, 3D modelling and expression analysis of sea bass (Dicentrarchus labrax L.) interleukin-10.

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1beta and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1beta.

Molecular cloning and characterization of sea bass (Dicentrarchus labrax L.) CD8alpha.

In this work, the gene and cDNA of the sea bass CD8alpha have been isolated and characterized. The coding sequence has an ORF of 666 bp. It retains the Ig motif that interacts with MHC and the two cysteines responsible for an intra-chain disulfide bridge. The hinge region contains the two essential cysteines involved in dimerization. The transmembrane region is well conserved in all analysed sequences. Similar to other teleosts, the cytoplasmic region lacks the consensus p56(lck) motif common in higher vertebrates. Analysis of the expression pattern using RT-PCR shows the highest expression in the thymus. Like in the human gene, the sea bass CD8alpha genomic structure is organized into six exons, which roughly correspond to separate functional domains of the protein. Southern blotting shows that CD8alpha exists as a single copy gene. Together, these results support the concept that the basic structure of CD8alpha has been maintained through evolution.