Bacteria have developed several mechanisms for iron uptake during colonization of mammalian hosts, where the availability of free iron is limiting for growth. Neisseria meningitidis expresses under iron-limiting conditions a receptor complex consisting of the lactoferrin-binding proteins A (LbpA) and LbpB to acquire iron from lactoferrin, which is abundantly present on the mucosal surfaces of the human nasopharynx. LbpA is an integral outer membrane-embedded iron transporter, whereas LbpB is a cell surface-exposed lipoprotein. In this study, we demonstrate that LbpB is also released into the culture medium. We identified NalP, an autotransporter known to be involved in the processing of other autotransporters, as the protease responsible for LbpB release. This release of LbpB reduced the complement-mediated killing of the bacteria when incubated with an LbpB-specific bactericidal antiserum. Since antibodies directed against LbpB are found in convalescent-patient sera, the release of an immunogenic protein as LbpB may represent a novel means for N. meningitidis to escape the human immune response.The Gram-negative bacterial species Neisseria meningitidis and Neisseria gonorrhoeae are the only Neisseriaceae that are pathogenic to humans, who are also the only known reservoir of these bacteria. Normally, N. meningitidis behaves as a commensal and colonizes the upper respiratory tract without any obvious clinical symptoms. However, in rare cases, it crosses the mucosal barriers and causes sepsis and meningitis with a high mortality and morbidity. This occurs most frequently in children and young adults. In the human body, the concentration of free soluble iron is too low to support bacterial growth. Iron in the human body is bound intracellularly to heme, hemoglobin, or ferritin and in serum and on mucosal surfaces to transferrin and lactoferrin, respectively (13). Bacteria have developed several different mechanisms of iron utilization, one of which involves the synthesis and secretion of siderophores (25). N. meningitidis and N. gonorrhoeae do not produce siderophores (3, 38). However, when grown under iron limitation, they express surface-exposed receptors for human iron-binding compounds, including transferrin (10, 19), lactoferrin (6, 28, 30), hemoglobin (34), and haptoglobin (20).The lactoferrin receptor is thought to be an important virulence factor of N. meningitidis. The main site of entry into the human body is the nasopharynx, where lactoferrin is abundant and could provide a major source of iron (24). Using an affinity isolation procedure, a single lactoferrin-binding protein was originally identified (32). The gene encoding this receptor, designated LbpA (lactoferrin-binding protein A), was subsequently characterized (6,28,30). LbpA showed a high degree of similarity to the TbpA (transferrin-binding protein A)-component of the transferrin receptor, which further consists of a lipoprotein designated TbpB (19). Upstream of lbpA, an open reading frame was identified, the deduced amino acid sequence of whic...
As with all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a modular protein consisting of an N-terminal signal sequence, a passenger domain and a Cterminal translocator domain (TD) that assists in the secretion of the passenger domain across the outer membrane. The passenger of IgA protease consists of three separate domains: the protease domain, the c-peptide and the a-peptide that contains nuclear localization signals (NLSs). The protease domain is released into the extracellular milieu either via autocatalytic processing or via cleavage by another autotransporter, NalP, expression of which is phase-variable. NalP-mediated cleavage results in the release of a passenger that includes the a-and c-peptides. Here, we studied the fate of the a-peptide when NalP was not expressed and observed strain-dependent differences. In meningococcal strains where the a-peptide contained a single NLS, the a-peptide remained covalently attached to the TD and was detected at the cell surface. In other strains, the a-peptide contained four NLSs and was separated from the TD by an IgA protease autoproteolytic cleavage site. In many of those cases, the a-peptide was found non-covalently associated with the cells as a separate polypeptide. The cell surface association of the a-peptides may be relevant physiologically. We report a novel function for the a-peptide, i.e. the binding of heparin -an immune-modulatory molecule that in the host is found in the extracellular matrix and connected to cell surfaces.
Autotransporters of Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain and the secreted passenger domain in between. The autotransporter NalP of Neisseria meningitidis includes a protease domain that facilitates the release of several immunogenic proteins from the cell surface into the extracellular milieu. Rather exceptionally among autotransporters, NalP is a lipoprotein. We investigated the role of lipidation in the biogenesis and function of the protein. To this end, the N-terminal cysteine, which is lipidated in the wild-type protein, was substituted by alanine. Like the wild-type protein, the mutant protein was secreted into the medium, demonstrating that lipidation is not required for biogenesis of the protein. However, the non-lipidated NalP variant had a drastically reduced capacity to cleave its substrate proteins from the cell surface, suggesting that the lipid moiety is important for function. Kinetic experiments demonstrated that the autocatalytic processing of the non-lipidated protein at the cell surface was much faster than that of the wild-type protein. Thus, the lipid moiety delays the release of NalP from the cell surface, thereby allowing it to release other surface-exposed proteins into the milieu.
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.
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