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.     

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.     

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.     

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro

Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface‐displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT‐FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration‐dependent manner and the N‐terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C‐terminal segment of FHA with ～650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C‐terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host‐directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.     

FhaC takes a bow to FHA in the two‐partner do‐si‐do

FhaC is an outer membrane transporter from Bordetella pertussis belonging to the t wo‐ p artner s ecretion (TPS) pathway with its primary role being the secretion of the virulence factor f ilamentous h aem a gglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β‐augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N‐terminal α‐helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. Guérin et al. (2014) report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.     

Processing and targeting of cathepsin L (TbCatL) to the lysosome in Trypanosoma brucei

Cathepsin L (TbCatL) is an essential lysosomal thiol protease in African trypanosomes. TbCatL is synthesized as two precursor forms (P/X) that are activated to mature form (M) with the removal of the prodomain upon arrival in the lysosome. We examine TbCatL trafficking in a novel system: truncated TbCatL reporter without the C‐terminal domain (CTD; TbCatL?) ectopically expressed in an RNA interference (RNAi) cell line targeting the CTD/3′ untranslated region (UTR) of endogenous mRNA. TbCatL? is synthesized as P′/X′/M′ species, localizes to the lysosome, and rescues the lethal TbCatL RNAi phenotype. Inactive TbCatLΔ:C150A is only processed to M′ in the presence of endogenous TbCatL indicating trans‐auto‐catalytic activation. X′ is formed with active endoplasmic reticulum (ER)‐retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER by cis‐auto‐cleavage within the prodomain of newly synthesized P′. Modelling the TbCatL prodomain on the human CatL structure suggests three solvent accessible features that could contain post‐Golgi targeting signals: the N‐terminus, the helix 1/turn 1 junction, and a separate turn (T3). We demonstrate that the critical motif for lysosomal targeting is an asparagine‐proline dipeptide in T3 that is strictly conserved in all Kinetoplastida. These findings show novel insights on the maturation of TbCatL, which is a critical virulence factor in mammalian infection.     

Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secre tion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8–9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M_r as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. perfussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.     

The metalloprotease of Listeria monocytogenes is activated by intramolecular autocatalysis

The metalloprotease (Mpl) of Listeria monocytogenes is a thermolysin-like protease that mediates the maturation of a broad-range phospholipase C, whose function contributes to the ability of this food-borne bacterial pathogen to survive intracellularly. Mpl is made as a proprotein that undergoes maturation by proteolytic cleavage of a large N-terminal prodomain. In this study, we identified the N terminus of mature Mpl and generated Mpl catalytic mutants to investigate the mechanism of Mpl maturation. We observed that Mpl activity was a prerequisite for maturation, suggesting a mechanism of autocatalysis. Furthermore, using a strain of L. monocytogenes expressing both the wild-type form and a catalytic mutant form of Mpl simultaneously, we determined that in vivo maturation of Mpl occurs exclusively by an intramolecular autocatalysis mechanism.     

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post‐translational modification has a C‐terminal GPI‐anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed ω→ω + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C‐terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3′ end‐sequence encoding ω‐2 to the C‐terminus with the corresponding C‐terminal sequences of nine other human GPI‐anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS–PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out‐performing the others in GPI‐anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at ω→ω + 2. J. Cell. Biochem. 84: 68–83, 2002. © 2001 Wiley‐Liss, Inc.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH‐enhanced proteolysis of the regulator Spx by ClpXP

The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP‐dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C‐terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C‐terminus showed that a 28‐residue C‐terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH‐targeted 28 residue segment of the Spx C‐terminus bears a ClpXP‐recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C‐terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild‐type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of helix α6, exposes the C‐terminus of Spx for recognition and proteolysis by ClpXP.     

Pyrazole amino acids: hydrogen bonding directed conformations of 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue

A series of model compounds containing 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue with N‐terminal amide/urethane and C‐terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single‐crystal X‐ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ? and ψ close to ±180°. The studied 1H‐pyrazole with N‐terminal amide/urethane and C‐terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C‐terminal ester group is present, the second conformation with torsion angles ? and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Sequential unfolding of the hemolysin two‐partner secretion domain from Proteus mirabilis

Protein secretion is a major contributor to Gram‐negative bacterial virulence. Type Vb or two‐partner secretion (TPS) pathways utilize a membrane bound β‐barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C‐proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N‐proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site‐specific mutants of HpmA265. By correlating the effect of individual site‐specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C‐terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C‐terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N‐proximal polar core subdomain.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C‐terminal stable core

Autotransporter (AT) proteins are a broad class of virulence factors from Gram‐negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT ‘passenger’ across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by a ～ 20 kDa stable core at the C‐terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference between the passenger N‐ and C‐terminus represents a truly common mechanistic feature. Yersinia pestis YapV is homologous to Shigella flexneri IcsA, and like IcsA, YapV recruits mammalian neural Wiskott–Aldrich syndrome protein (N‐WASP). In vitro, the purified YapV passenger is functional and rich in β‐sheet structure, but lacks a ～ 20 kDa C‐terminal stable core. However, the N‐terminal 49 residues of the YapV passenger globally destabilize the entire YapV passenger, enhancing its OM secretion efficiency. These results indicate that the contributions of AT passenger sequences to OM secretion efficiency extend beyond a C‐terminal stable core, and highlight a role of the passenger N‐terminus in reducing passenger stability in order to facilitate OM secretion of some AT proteins.     

Conformational analysis of the full‐length M2 protein of the influenza A virus using solid‐state NMR

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N‐terminal ectodomain and the C‐terminal cytoplasmic tail remains largely unknown. Using two‐dimensional (2D) magic‐angle‐spinning solid‐state NMR, we have investigated the secondary structure and dynamics of full‐length M2 (M2FL) and found them to depend on the membrane composition. In 2D ¹³C DARR correlation spectra, 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)‐bound M2FL exhibits several peaks at β‐sheet chemical shifts, which result from water‐exposed extramembrane residues. In contrast, M2FL bound to cholesterol‐containing membranes gives predominantly α‐helical chemical shifts. Two‐dimensional J‐INADEQUATE spectra and variable‐temperature ¹³C spectra indicate that DMPC‐bound M2FL is highly dynamic while the cholesterol‐containing membranes significantly immobilize the protein at physiological temperature. Chemical‐shift prediction for various secondary‐structure models suggests that the β‐strand is located at the N‐terminus of the DMPC‐bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain‐truncated M2(21–97), which no longer exhibits β‐sheet chemical shifts in the DMPC‐bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol‐rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β‐strand location suggests that chemical‐shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.