Peter van Ulsen

Structure of the translocator domain of a bacterial autotransporter

Autotransporters are virulence‐related proteins of Gram‐negative bacteria that are secreted via an outer‐membrane‐based C‐terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N‐terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro‐folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12‐stranded β‐barrel with a hydrophilic pore of 10 × 12.5 Å that is filled by an N‐terminal α‐helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger‐domain transport through the hydrophilic channel within the β‐barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger‐domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer‐membrane proteins, on which autotransporters are dependent.

A Neisserial autotransporter NalP modulating the processing of other autotransporters

Autotransporters constitute a relatively simple secretion system in Gram‐negative bacteria, depending for their translocation across the outer membrane only on a C‐terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid‐moiety as a result of additional N‐terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass‐spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild‐type strain, whereas only the lower molecular‐weight forms predominated in the culture supernatant of the nalP mutant. The serine‐protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.

Polar Localization of the Autotransporter Family of Large Bacterial Virulence Proteins

Autotransporters are an extensive family of large secreted virulence-associated proteins of gram-negative bacteria. Secretion of such large proteins poses unique challenges to bacteria. We demonstrate that autotransporters from a wide variety of rod-shaped pathogens, including IcsA and SepA of Shigella flexneri, AIDA-I of diffusely adherent Escherichia coli, and BrkA of Bordetella pertussis, are localized to the bacterial pole. The restriction of autotransporters to the pole is dependent on the presence of a complete lipopolysaccharide (LPS), consistent with known effects of LPS composition on membrane fluidity. Newly synthesized and secreted BrkA is polar even in the presence of truncated LPS, and all autotransporters examined are polar in the cytoplasm prior to secretion. Together, these findings are consistent with autotransporter secretion occurring at the poles of rod-shaped gram-negative organisms. Moreover, NalP, an autotransporter of spherically shaped Neisseria meningitidis contains the molecular information to localize to the pole of Escherichia coli. In N. meningitidis, NalP is secreted at distinct sites around the cell. These data are consistent with a model in which the secretion of large autotransporters occurs via specific conserved pathways located at the poles of rod-shaped bacteria, with profound implications for the underlying physiology of the bacterial cell and the nature of bacterial pathogen-host interactions.

Type V secretion: From biogenesis to biotechnology☆

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

NalP-Mediated Proteolytic Release of Lactoferrin-Binding Protein B from the Meningococcal Cell Surface

ABSTRACT 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.

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal β-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The mechanism of passenger translocation and folding is still unclear but involves a conserved region at the C terminus of the passenger domain, the so-called autochaperone domain. This domain functions in the stepwise translocation process and in the folding of the passenger domain after translocation. In the autotransporter hemoglobin protease (Hbp), the autochaperone domain consists of the last rung of the β-helix and a capping domain. To examine the role of this region, we have mutated several conserved aromatic residues that are oriented toward the core of the β-helix. We found that non-conservative mutations affected secretion with Trp1015 in the cap region as the most critical residue. Substitution at this position yielded a DegP-sensitive intermediate that is located at the periplasmic side of the OM. Further analysis revealed that Trp1015 is most likely required for initiation of processive folding of the β-helix at the cell surface, which drives sequential translocation of the Hbp passenger across the OM.

Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach

Isobaric labeling reagents such as Tandem Mass Tags (TMT®) enable the genome‐wide quantification of protein expression levels under different conditions using a gel‐free MS/MS‐based approach. Here, we applied a TMTduplex approach with two isobaric tags to study the response of the human pathogen Neisseria meningitidis to deprivation of iron, a condition met in the human body. In total, 609 proteins were identified in samples of three independent growth experiments, in which we compared cultures grown in the presence and absence of iron. Expression of 35 proteins was found to be induced or repressed under iron‐limiting conditions, including 11 proteins whose ORFs were not previously identified in DNA array studies as being regulated by iron availability at the transcriptional level. These 11 proteins include proteins likely involved in iron metabolism.

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

ABSTRACT Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

Estimating the size of the active translocation pore of an autotransporter.

Autotransporters (ATs) are large virulence factors secreted by Gram-negative bacteria. The passenger domain, carrying the virulence functions, is transported across the bacterial outer membrane in a step that is facilitated by a C-terminal β-domain. This domain folds into a β-barrel with a central aqueous pore of ∼1 nm inner diameter according to crystal structures. However, these static dimensions are not compatible with the observed secretion of passengers that may contain natural short-spaced disulfide bonds or artificially fused folded elements. Here, we have systematically analyzed the dimensions of the active AT passenger translocator by inserting peptides of different length and structural complexity in the passenger of the AT hemoglobin protease. The peptides were introduced in a short loop protruding from the main structure and flanked by two single cysteines. Our results show that the attained secondary structure may be more critical for secretion than the length of peptide inserted. Furthermore, the data suggest that, during passenger translocation, at least four extended polypeptides or an extended polypeptide and an α-helix are accommodated in the translocator, indicating that the diameter of the active translocation pore is up to 1.7 nm. If the β-domain functions as the translocator, it must be forced into an expanded conformation during passenger translocation.

YidC is required for the assembly of the MscL homopentameric pore

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.