Esteban Veiga

Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains

An investigation was made into the oligomerization, the ability to form pores and the secretion‐related properties of the 45 kDa C‐terminal domain of the IgA protease (C‐IgAP) from Neisseria gonorrhoeae. This protease is the best studied example of the autotransporters (ATs), a large family of exoproteins from Gram‐negative bacteria that includes numerous virulence factors from human pathogens. These proteins contain an N‐terminal passenger domain that em bodies the secreted polypeptide, while the C‐domain inserts into the outer membrane (OM) and trans locates the linked N‐module into the extracellular medium. Here we report that purified C‐IgAP forms an oligomeric complex of ∼500 kDa with a ring‐like structure containing a central cavity of ∼2 nm diameter that is the conduit for the export of the N‐domains. These data overcome the previous model for ATs, which postulated the passage of the N‐module through the hydrophilic channel of the β‐barrel of each monomeric C‐domain. Our results advocate a secretion mechanism not unlike other bacterial export systems, such as the secretins or fimbrial ushers, which rely on multimeric complexes assembled in the OM.

Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain.

The mechanism of protein secretion mediated by the β‐domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram‐negative bacteria called autotransporters, has been examined using a single‐chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the β‐module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N‐terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S–S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N‐passenger domain can be translocated through the β‐domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re‐evaluation of the prevailing model for secretion of the N‐domain of autotransporters.

Candida albicans internalization by host cells is mediated by a clathrin‐dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E‐ or N‐cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes. InlA mediates entry of L. monocytogenes into host cells through binding to E‐cadherin. A role in internalization, for a non‐classical stimulation of the clathrin‐dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans. Using live‐cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans, we observed that host E‐cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N‐cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin‐dependent manner. These data indicate that C. albicans, like L. monocytogenes, hijacks the clathrin‐dependent endocytic machinery to invade host cells.

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

During antigen‐specific T‐cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin‐related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin‐rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T‐cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1‐specific inhibitor mdivi‐1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T‐cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T‐cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1‐dependent mitochondrial positioning and activity controls T‐cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

Structural tolerance of bacterial autotransporters for folded passenger protein domains

In this report we investigate the capacity of bacterial autotransporters (AT) to translocate folded protein domains across the outer membrane (OM). Polypeptides belonging to the AT family contain a C‐terminal domain that supports the secretion of the N‐domain (the passenger) across the OM of Gram‐negative bacteria. Despite some controversial data, it has been widely accepted that N‐passenger domains of AT must be unfolded and devoid of disulphide bonds for efficient translocation. To address whether or not AT are able to translocate folded protein domains across the OM, we employed several types of recombinant antibodies as heterologous N‐passengers of the transporter C‐domain of IgA protease (C‐IgAP) of Neisseria gonorroheae. The N‐domains used were single chain Fv fragments (scFv) and variable mono‐domains derived from camel antibodies (VHH) selected on the basis of their distinct and defined folding properties (i.e. enhanced solubility, stability and presence or not of disulphide bonds). Expression of these hybrids in Escherichia coli shows that stable scFv and VHH domains are efficiently (>99%) translocated towards the bacterial surface regardless of the presence or not of disulphide bonds on their structure. Antigen‐binding assays demonstrate that surface‐exposed scFv and VHH domains are correctly folded and thus able to bind their cognate antigens. Expression of scFv‐ or VHH‐C‐IgAP hybrids in E. coli dsbA or fkpA mutant cells reveals that these periplasmic protein chaperones fold these N‐domains before their translocation across the OM. Furthermore, large N‐passengers composed of strings of VHH domains were secreted in a folded state by AT with no loss of efficacy (>99%) despite having multiple disulphide bonds. Thus AT can efficiently translocate toward the cell surface folded N‐passengers composed of one, two or three immunoglobulin (Ig) domains, each with a folded diameter between ∼2 nm and having disulphide bonds. This tolerance for folded protein domains of ∼2 nm fits with the diameter of the central hydrophilic channel proposed for the ring‐like oligomeric complex assembled by C‐IgAP in the OM.

Endosomal clathrin drives actin accumulation at the immunological synapse.

Antigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell–cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell–cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

Miro-1 links mitochondria and microtubule dynein motors to control lymphocyte migration and polarity

ABSTRACT The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.

F-actin-binding protein drebrin regulates CXCR4 recruitment to the immune synapse.

The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

Autotransporters as Scaffolds for Novel Bacterial Adhesins: Surface Properties of Escherichia coli Cells Displaying Jun/Fos Dimerization Domains

Hybrid proteins containing the beta-autotransporter domain of the immunoglobulin A (IgA) protease of Neisseria gonorrhoea (IgA beta) and the partner leucine zippers of the eukaryotic transcriptional factors Fos and Jun were expressed in Escherichia coli. Such fusion proteins targeted the leucine zipper modules to the cell surface. Cells displaying the Jun beta sequence flocculated shortly after induction of the hybrid protein. E. coli cells expressing separately Fos beta and Junbeta chimeras formed stable bacterial consortia. These associations were physically held by tight intercell ties caused by the protein-protein interactions of matching dimerization domains. The role of autotransporters in the emergence of new adhesins is discussed.

Neutralization of Enteric Coronaviruses with Escherichia coli Cells Expressing Single-Chain Fv-Autotransporter Fusions

ABSTRACT We report here that fusions of single-chain antibodies (scFvs) to the autotransporter β domain of the IgA protease of Neisseria gonorrhoeae are instrumental in locating virus-neutralizing activity on the cell surface of Escherichia coli. E. coli cells displaying scFvs against the transmissible gastroenteritis coronavirus on their surface blocked in vivo the access of the infectious agent to cultured epithelial cells. This result raises prospects for antiviral strategies aimed at hindering the entry into target cells by bacteria that naturally colonize the same intestinal niches.