Autotransporters are a superfamily of proteins that use the type V secretion pathway for their delivery to the surface of Gram-negative bacteria. At first glance, autotransporters look to contain all the functional elements required to promote their own secretion: an amino-terminal signal peptide to mediate translocation across the inner membrane, a central passenger domain that is the secreted functional moiety, and a channel-forming carboxyl terminus that facilitates passenger domain translocation across the outer membrane. However, recent discoveries of common structural themes, translocation intermediates and accessory interactions have challenged the perceived simplicity of autotransporter secretion. Here, we discuss how these studies have led to an improved understanding of the mechanisms responsible for autotransporter biogenesis.

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a ␤-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-␤ or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in ϳ80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct proteinprotein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker "autotransporter."Secretion of proteins to the surface of gram-negative bacteria requires passage through the inner membrane (IM), the periplasm, and the outer membrane (OM). This formidable series of obstacles can be overcome only by complex biological processes. The autotransporter (AT) system, probably the most common gram-negative secretion mechanism (13), is characterized by formation of an OM ␤-barrel comprised of the C terminus of the periplasmic species. The precise events required for AT translocation across the OM, however, are controversial. The original model for OM translocation comprised targeting to the periplasm via the Sec apparatus, followed by formation of an OM ␤-barrel, which mediates passage of an unfolded or partially folded N-terminal passenger domain to the extracellular milieu (30). Three models of AT translocation have gained some acceptance (3, 16). According to the hairpin model, translocation of the passenger domain is initiated with the C-terminal end of the passenger forming a hairpin structure inside the AT ␤-barrel, followed by movement of the rest of the passenger through the barrel's pore in a C-to-N direction. Under the Omp85 model, the pore-forming Omp85 (YaeT in Escherichia coli) OM protein (OMP) facilitates insertion of the AT translocator domain into the OM, whereupon the AT passenger domain translocates th...

Background Although a majority of patients with PSC have colitis [PSC-IBD; primary sclerosing cholangitis-inflammatory bowel disease], this is phenotypically different from ulcerative colitis [UC]. We sought to define further the pathophysiological differences between PSC-IBD and UC, by applying a comparative and integrative approach to colonic gene expression, gut microbiota and immune infiltration data. Methods Colonic biopsies were collected from patients with PSC-IBD [n = 10], UC [n = 10], and healthy controls [HC; n = 10]. Shotgun RNA-sequencing for differentially expressed colonic mucosal genes [DEGs], 16S rRNA analysis for microbial profiling, and immunophenotyping were performed followed by multi-omic integration. Results The colonic transcriptome differed significantly between groups [p = 0.01]. Colonic transcriptomes from HC were different from both UC [1343 DEGs] and PSC-IBD [4312 DEGs]. Of these genes, only 939 had shared differential gene expression in both UC and PSC-IBD compared with HC. Imputed pathways were predominantly associated with upregulation of immune response and microbial defense in both disease cohorts compared with HC. There were 1692 DEGs between PSC-IBD and UC. Bile acid signalling pathways were upregulated in PSC-IBD compared with UC [p = 0.02]. Microbiota profiles were different between the three groups [p = 0.01]; with inferred function in PSC-IBD also being consistent with dysregulation of bile acid metabolism. Th17 cells and IL17-producing CD4 cells were increased in both PSC-IBD and UC when compared with HC [p < 0.05]. Multi-omic integration revealed networks involved in bile acid homeostasis and cancer regulation in PSC-IBD. Conclusions Colonic transcriptomic and microbiota analysis in PSC-IBD point toward dysregulation of colonic bile acid homeostasis compared with UC. This highlights important mechanisms and suggests the possibility of novel approaches in treating PSC-IBD.

Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or "binding boxes" within the PAP sequence. In the important human pathogen Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a

Background: There is a general paucity of cysteine residues within the passenger domains of autotransporter proteins. Results: Distantly spaced cysteines forming disulfide-bonded loops or those enclosing structural elements are secretion-incompetent. Conclusion: Only closely spaced cysteine pairs are compatible with the autotransporter pathway. Significance: Secretion of folded peptides by the autotransporter pathway is limited; hence autotransporters lack large disulfide-bonded loops to remain secretion-competent.Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein.The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.Gram-negative bacteria possess seven secretion pathways (numbered I-VI and the chaperone-usher pathway) that facilitate navigation of secreted proteins through the inner membrane, periplasm, and outer membrane (OM).2 These pathways generally use specialized machineries that span the width of the cell envelope and that differ in complexity, structural features, and mechanism of protein translocation. At first glance, the simplicity of the type Va secretion pathway appeared to be the exception; all the functional elements required for secretion appeared to be contained within a single protein with the N-terminal signal peptide mediating inner membrane translocation, the central passenger domain being the secreted functional moiety, and the C terminus forming a ␤-barrel structure in the OM, the latter element being essential for passenger domain translocation to the bacterial cell surface. Accordingly, the superfamily of proteins that exploit this pathway for their delivery to the surface of Gram-negative bacteria was termed autotransporters (ATs) (1). However, recent studies demonstrating that passenger domain secretion requires the ...