Mechanistic link between barrel assembly and the initiation of autotransporter secretion

Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2013; 110(10):E938-47. DOI: 10.1073/pnas.1219076110
Source: PubMed


Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal β barrel ("β") domain that anchors the protein to the outer membrane. The β domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the β domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the β domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the β domain into the outer membrane, but do not delay the binding of the β domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the β domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of β barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.

    • "The observation was later attributed to additional accessory factors and interspecies incompatibilities in the outer membrane protein machinery (Robert et al. 2006). Since there is clear evidence for the involvement of the Omp85/BamA machinery in the outer membrane insertion of autotransporters and surface translocation of its passengers, the term " auto " transporter appears to be slightly misleading (Gawarzewski et al. 2013a;Ieva et al. 2011;Pavlova et al. 2013). For clarity reasons, the term autodisplay is still used for surface display of recombinant proteins using autotransporters (Jose et al. 2012;Jose and Meyer 2007), in particular, the AIDA-I autotransporter (Maurer et al. 1997). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.
    No preview · Article · Aug 2014 · Applied Microbiology and Biotechnology
  • Source
    • "Given this, it is important to consider the genomic makeup of these accessory systems in conjunction with the gene structure and protein domain organization of ATs for determining the mechanisms of type 5 secretion in each organism. A recent model for the secretion pathway of a classical AT, EspP of E. coli, illustrates the contribution of the BAM complex and several periplasmic chaperones to type 5a secretion (Pavlova et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The genus Rickettsia (Alphaproteobacteria; Rickettsiales; Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, though others (e.g., josamycin, ciprofloxacin, chloramphenicol and azithromycin) are also effective. Much of the recent research geared towards understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial “life on the inside”.This article is protected by copyright. All rights reserved.
    Full-text · Article · Aug 2014 · FEMS microbiology reviews
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Pathogenic Gram-negative bacteria have evolved several secretion mechanisms to translocate adhesins, enzymes, toxins and other virulence factors across the inner and outer membrane. Currently, eight different secretion systems, type I - type VIII (T1SS - T8SS) plus the chaperone-usher (CU) pathway have been identified, which act in one-step or two-step mechanisms to traverse both membrane barriers. The type V secretion system (T5SS) is dependent first on the Sec translocon within the inner membrane. The periplasmic intermediates are then secreted through aqueous pores formed by β-barrels in the outer membrane. Until now, transport across the outer membrane has not been understood on a molecular level. With respect to special characteristics revealed by crystal structure analysis, bioinformatic and biochemical data, five subgroups of T5SS were defined. Here, we compare the transport moieties of members of four subgroups based on X-ray crystal structures. For the fifth subgroup, which was identified only recently no structures have so far been reported. We also discuss different models for the translocation process across the outer membrane with respect to recent findings.
    No preview · Article · Aug 2013 · Biological Chemistry
Show more