Abstract
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.
Highlights
There is a general paucity of cysteine residues within the passenger domains of autotransporter proteins
plasmid-encoded toxin (Pet) possesses D2A in place of D3, and the Pet amino acid sequence directly adjacent to D2A (QPDWET) is almost completely conserved with Hbp; it has been shown that these residues provide a platform for the projection of a stable external loop capable of independent movement [48]
Stalled Intermediates Interact with BamA and BamD during outer membrane (OM) Translocation—We recently demonstrated that BamA and BamD are the only components of the barrel assembly machinery (BAM) complex essential for AT biogenesis [8]
Summary
There is a general paucity of cysteine residues within the passenger domains of autotransporter proteins. The translocation of sizable folded domains comprising disulfidebonded segments of heterologous passenger domains was reported (30 –32), a greater number of studies have demonstrated that periplasmic formation of either long disulfidebonded loops or tightly folded/rigid structures is incompatible with OM translocation/secretion of AT passenger domains [20, 28, 29, 33,34,35,36] These dichotomous sets of studies have given rise to two mechanistic models of AT biogenesis, one proposing that passenger domain translocation occurs through a folded AT -barrel pore, and the other proposing BamA as an accessory factor mediating passenger translocation before the -domain adopts a final folded conformation. We demonstrate that when present, native Cys pairs within AT passenger domains are intrinsically closely spaced to avoid jamming of the translocator pore by large disulfide-bonded regions
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