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Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.

The mechanosensitive channel with large conductance (MscL) of Escherichia coli is formed by a homopentameric assembly of MscL proteins. Here, we describe MscL biogenesis as determined using in vivo approaches. Evidence is presented that MscL is targeted to the inner membrane via the signal recognition particle (SRP) pathway, and is inserted into the lipid bilayer independently of the Sec machinery. This is consistent with published data. Surprisingly, and in conflict with earlier data, YidC is not critical for membrane insertion of MscL. In the absence of YidC, assembly of the homopentameric MscL complex was strongly reduced, suggesting a late role for YidC in the biogenesis of MscL. The data are consistent with the view that YidC functions as a membrane-based chaperone 'module' to facilitate assembly of a subset of protein complexes in the inner membrane of E. coli. © 2009 FEBS.

Neisseria meningitidis and Neisseria gonorrheae are Gram-negative bacteria that are only human pathogens. Most of the time N. meningitidis is commensal and colonizes the upper respiratory tract. Occasionally, N. meningitidis crosses the mucosal barrier and causes sepsis and meningitis with a high rate of mortality in children and young adults. Secretion of virulence factors and surface exposed proteins are associated with the pathogenicity of the bacteria. Secreted virulence factors in N. meningitidis are following two pathways, type I and type V. Type I secretion pathway consists in a multimeric complex composed of a trimeric OM-embedded channel protein, an IM-embedded ATP-binding cassettes (ABC) transporter and a periplasmic membrane fusion protein. The type V secretion system regroups three distinct classes of protein being the type Va, the classical autotransporters, the type Vb, being the two partner secretion system and the type Vc being the trimeric autotransporters. The largest group of secreted virulence factors present in Neisseria are the autotransporters. Autotransporters are modular proteins that consist in three domains: an N-terminal signal sequence for the transport across the inner membrane, a C-terminal translocator domain (TD) for the transport across the outer membrane and, in between, a secreted passenger domain. The name autotransporter was coined because translocation across the outer membrane was thought to depend only of the presence of the translocator domain. Genome analysis showed that N. meningitidis possesses eight different autotransporters. The autotransporter IgA protease was first described and extensively studied in N. gonorrheae but its function is not completely understood. In the chapter 2, we addressed the fate of the IgA protease that remains behind in the outer membrane of N. meningitidis. Dependent on the strain and whether or not the autotransporter nalP was expressed, three different variants of the TD of IgA protease were found, being the -barrel core domain or this domain extended with either one or two extracellular domains, known as the linker domain and the -peptide. These domains are abundant and really stable and might make them good vaccine candidates. In the chapter 3, we characterized the TD of Hbp, an Escherichia coli autotransporter, by comparing its biophysical properties to the neisserial autotransporter NalP and IgA protease. We could show that all TDs possess similar pore activity and stability. The autotransporter NalP is a protease and is known to process several autotransporters at the cell surface, i.e. IgA protease, App and AusI. In the chapter 4, we demonstrated that NalP is also able to release specifically the surface exposed lipoprotein Lactoferrin binding protein B, LbpB, reducing the complement mediated killing of the bacteria when incubated with a LbpB-specific bactericidal antiserum. Finally in chapter 5, NalP is the only neisserial autotransporter that is a lipoprotein. We show by using several variants lacking the lipid anchor that the biogenesis of NalP was not influenced but that the processing of the different targets was affected. This suggests that the lipid anchor is needed to dock the active site of NalP at the proper location being close to the cell surface.