Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V

A general secretion signal for the mycobacterial type VII secretion pathway

Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes

Evaluation of pharmacological aids on physical performance after a transmeridian flight

Effets de la caféine et de la mélatonine sur les problèmes liés au décalage horaire (baisse des performances, fatigue...) chez des réservistes de l'U.S. Air Force ayant effectués un vol sur sept fuseaux horaires en direction de l'est

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein Esp

Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8(+) T cells

Contains fulltext : 215684.pdf (Publisher’s version ) (Open Access)Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c(+) DCs to OTI CD8(+) T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a(+) professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8(+) T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8(+) T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8(+) T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8(+) T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8(+) T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8(+) T cells is provided

An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines.

Background: The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule. Results: As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface. Conclusions: The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines