Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

N-terminal characterization of the Bordetella pertussis filamentous haemagglutinin

The major adhesin of Bordetella pertussis , filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium. Mature FHA derives from a large precursor, FhaB, that undergoes several post-translational maturations. In this work, we demonstrate by site-directed mutagenesis that the N-terminal signal peptide of FHA is composed of 71 amino acids, including a 22-residue-long 'N-terminal extension' sequence. This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis. The entire N-terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence. After this maturation, the N-terminal glutamine residue is modified to a pyroglutamate residue. This modification is not crucial for heparin binding, haemagglutination or secretion. Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives. In addition, it is dependent in B. pertussis on the presence of all three cysteines contained in the signal peptide of FhaB. These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery.     

Effect of signal peptide changes on the extracellular processing of streptokinase from Escherichia coli : requirement for secondary structure at the cleavage junction

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfils the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction. Received: 22 September 1997 / Accepted: 17 December 1997     

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin

In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.     

Secretion of active truncated CD4 into Escherichia coli periplasm

Summary A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus. Offprint requests to: C.-S. C. Tomich     

Regulated,sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin

Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C‐terminal ~1,200 amino acid ‘prodomain’ is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C‐terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein.     

Effect of signal peptide changes on the extracellular processing of streptokinase from Escherichia coli : requirement for secondary structure at the cleavage junction

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfils the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction.     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.     

Topology and maturation of filamentous haemagglutinin suggest a new model for two-partner secretion

Two-partner secretion (TPS) is the most widely distributed secretion pathway known. These systems export large exoproteins through highly conserved channel-forming beta-barrel proteins. Filamentous haemagglutinin (FHA), expressed by Bordetella species, is the prototypical TPS family member. Here we show that the C-terminus of mature FHA, as opposed to the N-terminus as previously proposed, is exposed on the cell surface and is required for mediating adherence to cultured epithelial cells. We show that the C-terminus of the FHA pro-protein (FhaB) is required for FHA function in vitro and in vivo and we show that cleavage of FhaB to form FHA is not the mechanism by which FHA is released from the cell. Based on these data, we propose a new model for TPS. This model provides an explanation for the energetics of export of globular protein domains across membranes in the absence of ATP and it suggests a new mechanism for the control of protein folding.     

Regulation of Toll‐like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β), is critical for the pathogenesis of Escherichia coli K1 meningitis. Since Hsp90 chaperones Toll‐like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2^?/? mice are resistant to E. coli K1 meningitis, while TLR4^?/? mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA? E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC‐α while the other from TLR2 through MyD88, ERK1/2 and NF‐κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC.     

The VANA glycopeptide resistance protein is related to d-alanyl-d-alanine ligase cell wall biosynthesis enzymes

Summary Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

Natural‐host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C‐terminal domain,but not the RGD motif,during infection

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human‐adapted (e.g. Bordetella pertussis) strains produce a surface‐exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg‐Gly‐Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural‐host animal models should apply to B. pertussis FHA as well. We also show that the C‐terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage‐like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.     

The prodomain of the Bordetella two‐partner secretion pathway protein FhaB remains intracellular yet affects the conformation of the mature C‐terminal domain

Two‐partner secretion (TPS) systems use β‐barrel proteins of the Omp85‐TpsB superfamily to transport large exoproteins across the outer membranes of Gram‐negative bacteria. The Bordetella FHA/FhaC proteins are prototypical of TPS systems in which the exoprotein contains a large C‐terminal prodomain that is removed during translocation. Although it is known that the FhaB prodomain is required for FHA function in vivo, its role in FHA maturation has remained mysterious. We show here that the FhaB prodomain is required for the extracellularly located mature C‐terminal domain (MCD) of FHA to achieve its proper conformation. We show that the C‐terminus of the prodomain is retained intracellularly and that sequences within the N‐terminus of the prodomain are required for this intracellular localization. We also identify sequences at the C‐terminus of the MCD that are required for release of mature FHA from the cell surface. Our data support a model in which the intracellularly located prodomain affects the final conformation of the extracellularly located MCD. We hypothesize that maturation triggers cleavage and degradation of the prodomain.     

Fusion expression and immunogenicity of <Emphasis Type="Italic">Bordetella pertussis</Emphasis> PTS1-FHA protein: implications for the vaccine development

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.     

重组蛋白融合信号肽在大肠杆菌周质空间表达的研究进展

重组蛋白在大肠杆菌中表达时,往往面临着形成包涵体的问题,而重组蛋白若是分泌至周质空间则基本解决了这一问题,周质空间的周质蛋白不仅能帮助重组蛋白正确折叠还有利于二硫键的生成。信号肽是一段由15-30个氨基酸组成,被融合在重组蛋白N端的短肽,按照结构、功能的不同可以划分为N区、H区和C区,具有引导重组蛋白转运至细胞周质空间的作用。本文综述了信号肽的结构组成、作用机理和基本分泌途径,讨论了信号肽的高效转运和筛选方法,总结了在大肠杆菌中重组蛋白融合信号肽实现周质表达的新进展,并对未来高效信号肽选择方面的研究进行了探讨。     

Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coliα-haemolysin translocator

The 1706-residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy-proximai glycine and aspartate-rich repeats. CyaA does not have a cleavable amino-terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the α-haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N–terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/ToiC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp-1587 to lle-1631. Interestingly, a conserved acidic' motif (Glu/Asp)-(X)₁₁-Asp-(X)_3/5-(Glu/Asp)-(X)₁₄-Asp was found in the C-terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer