Gram-negative bacteria use different protein secretion systems, ranging from type I through type IX, to invade the host and cause infections. Proteins of the type V secretion system, called autotransporters, can autonomously transport a part of their own polypeptide chain to the bacterial cell surface through an outer membrane-embedded β-barrel domain. The transported part of the protein is called the passenger. Thus, autotransporters can be considered self-contained secretion systems, with several described subclasses. Intimin, an adhesin of enteropathogenic Escherichia coli, is a prototypical member of the Type Ve secretion system or inverse autotransporter pathway. Intimin has been proposed to export its passenger though the β-barrel domain via a hairpin intermediate. During studies on Intimin autotransport, a double HA tag was inserted into the N-terminus of the passenger resulting in a stalled secretion intermediate caught in the hairpin conformation. In this project, to study the molecular details of hairpin formation and passenger secretion, I have made mutations in three regions of the β-barrel of Intimin. The mutations were made both in wild-type Intimin and the stalled variant to see the effect on passenger secretion and hairpin formation, respectively. All mutant proteins, except the mutation where a β-strand on the extracellular side of the β-barrel was deleted, were produced, correctly folded and inserted into the membrane. Using the stalled variant, the formation of the hairpin was studied by exposure of the HA tag at the cell surface. Using the mutant proteins in the secretion-competent background, the exposure of the C-terminus of Intimin was studied. After introducing mutations in the three regions of the barrel, a region with a β-strand on the extracellular side of the β-barrel was observed to be important for hairpin formation and successful passenger secretion. This β-strand is a part of a β-sheet formed with the β-strands located on extracellular loop 4 and 5 of the β-barrel. The β-strand is present at the very C-terminus of the linker. The β-barrel forms a pore in the outer membrane of the bacteria through which the passenger is translocated. In this study, I propose two alternative models: one, where the sequence forming the β-strand directs the linker into the OM pore formed by the -barrel, stabilizes the hairpin and promotes passenger secretion and the other, where the hairpin is formed by unknown means and the β-strand interacts with the extracellular loops locking the hairpin in its configuration thereby promoting passenger translocation.