The unusual extended signal peptide region is not required for secretion and function of an Escherichia coli autotransporter

Leyton, Denisse L.; Luna, Maria das Graças de; Sevastsyanovich, Yanina R.; Jensen, Karina Tveen; Browning, Douglas F.; Scott-Tucker, Anthony; Henderson, Ian R.

doi:10.1111/j.1574-6968.2010.02081.x

Cited by 17 publications

(15 citation statements)

References 18 publications

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“…The 1,461-aa protein is a typical TAA, possessing all of the expected features of this family. Analysis of the amino acid sequence revealed a putative signal sequence possessing a conserved N-terminal extended signal peptide region (ESPR), which is common to many autotransporter proteins (4,17,19,49), and is predicted to be cleaved after amino acid 50. The analysis also revealed a 1,333-aa passenger domain and a Cterminal translocation unit indicated by the Pfam:YadA (PF03895) domain.…”

Section: Resultsmentioning

confidence: 99%

SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

et al. 2011

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Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.

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Section: Resultsmentioning

confidence: 99%

SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

et al. 2011

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“…pBADPet has been described previously 46 . To construct pBADPetG 1061 A, pBADPetG 1076 A, pBADPetG 1151 A and pBADPetY 1103 A, megaprimer PCR was performed as described previously 47,48 with some variation.…”

Section: Methodsmentioning

confidence: 99%

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

et al. 2014

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Bacterial autotransporters comprise a 12-stranded membrane-embedded b-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring b-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

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“…pBADPet has been described previously (39). A 363-bp SpeI-XbaI fragment comprising sequence coding for Pet without D2A was synthesized de novo and cloned into pUC57 (GenScript).…”

Section: Methodsmentioning

confidence: 99%

“…Analysis of Pet Biogenesis-Growth, expression, and precipitation of Pet from TOP10 and TOP10 dsbA::kan transformed with pBADPet derivatives were performed as described previously (39). Where necessary, 10 mM ␤-mercaptoethanol (␤-ME; Sigma-Aldrich) was added to the culture medium to reduce disulfide bonds formed in the periplasm (20).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, Pet from TOP10 cells transformed with pBADPet derivatives was expressed, and clarified supernatants were concentrated 100-fold as described previously (39). Supernatants were buffer-exchanged into thiol modification buffer (50 mM Tris, pH 7.5), and protein concentrations were calculated using Bradford protein assay reagent according to the manufacturer's instructions (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

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Size and Conformation Limits to Secretion of Disulfide-bonded Loops in Autotransporter Proteins

Leyton

Sevastsyanovich

Browning

et al. 2011

Journal of Biological Chemistry

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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 ...

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The unusual extended signal peptide region is not required for secretion and function of an Escherichia coli autotransporter

Cited by 17 publications

References 18 publications

SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

Size and Conformation Limits to Secretion of Disulfide-bonded Loops in Autotransporter Proteins

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