Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

Abstract

Intimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REI v , ubiquitin, calmodulin, ␤-lactamase inhibitor protein, or ␤-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.The dual membrane envelopes of gram-negative bacteria provide two barriers of unlike nature that pose formidable problems concerning the transport of molecules into and out of these organisms. Nutrients and essential cofactors must be actively transported into the cells, and end products of metabolism, toxic molecules, and proteins need to be extruded. While gram-positive bacteria, eukaryotes, and archaea exhibit just three known secretory systems for protein transport across the cytoplasmic and endoplasmic reticulum membranes (7, 37, 45), gram-negative bacteria have evolved multiple systems for protein transport across the whole-cell envelope; the proteins may remain attached to the surface or be released into the extracellular milieu (8,25,40,50). They serve as, for example, substrate-degrading enzymes, adhesion anchors, or pathogenicity factors that interfere with host metabolism or immune defense.Most machineries for translocating proteins across gramnegative bacterial membranes are composed of numerous proteins that form heterooligomeric structures, which mediate the simultaneous export of a passenger protein across both membranes (25). Two exceptions are known: the type V secretion pathway (19) and the autodisplay of intimins and invasins (34). In these cases, all of the necessary elements for translocation across the outer membrane are located within their own polypeptide sequences. Members of the family of type V secreted virulence factors comprise three functional domains in a single autoexport protein: an N-terminal targeting sequence, a C-terminal translocation domain, and the passenger domain in between. The C-terminal domain is supposed to for...

Probing the Borrelia burgdorferi Surface Lipoprotein Secretion Pathway Using a Conditionally Folding Protein Domain

“…In one study, replacement of an internal passenger subdomain of the E. coli type V "autotransporter" secretion protein Hbp with CaM blocked secretion, leading to envelope stress and degradation of the fusion protein by the periplasmic protease DegP (27). A similar CaM-dependent secretory block was seen with E. coli intimin, an intimin/invasin-type protein (1). Based on the absence of a secretion block phenotype with the OspA wt -CaM fusion proteins, one might therefore argue that the association of the mutant OspA-CaM fusion proteins with the periplasmic OM leaflet is "off-pathway."…”

“…The Ca 2ϩ -free apo-form is largely unfolded, whereas the Ca 2ϩ -loaded protein forms a stable dumbbell structure (30). Hence, the folding state of CaM can be controlled by experimentally manipulating the Ca 2ϩ level (1,24,27,59). To test whether the addition of a tightly folded Ca 2ϩ -CaM domain could block the secretion of a Borrelia surface lipoprotein, we generated OspA-CaM fusion proteins (Fig.…”

The inverse autotransporter family: Intimin, invasin and related proteins

“…Indirect evidence for inverse autotransport was provided by studies replacing the passenger domain of intimin with heterologous passengers (Adams et al, 2005;Salema et al, 2013;Wentzel et al, 2001). The intimin translocation domain efficiently exports foreign domains, unless these domains had a propensity for stable folding or contained disulfide bridges (Adams et al, 2005). This is reminiscent of classical autotransporters, where secretion of heterologous payloads has been exploited for biotechnological applications in a system termed 'autodisplay' (Jose, 2006;Jose and Meyer, 2007).…”

“…2). Indirect evidence for inverse autotransport was provided by studies replacing the passenger domain of intimin with heterologous passengers (Adams et al, 2005;Salema et al, 2013;Wentzel et al, 2001). The intimin translocation domain efficiently exports foreign domains, unless these domains had a propensity for stable folding or contained disulfide bridges (Adams et al, 2005).…”

Correlating Levels of Type III Secretion and Secreted Proteins with Fecal Shedding of Escherichia coli O157:H7 in Cattle

“…Both intimin and Tir are the products of ORFs present in LEE5, but only Tir requires the T3SS for its translocation and subsequent insertion into the host epithelial cell membranes by adopting a hairpin loop conformation. Intimin, on the other hand, belongs to an autotransporter family of proteins, which uses its unique structural features to translocate to bacterial outer cell membranes (2).…”