A simple in vitro acylation assay based on optimized HlyA and HlyC purification

HlyA is a toxin secreted by uropathogenic Escherichia coli strains. HlyA belongs to the repeats in the toxin protein family and needs (i) a posttranslational, fatty acylation at two internal lysines by the acyltransferase HlyC and (ii) extracellular ion binding to achieve its active conformation. Both processes are not fully understood and experiments are often limited due to the low amounts of protein available. Here, we present an optimized purification protocol for the proteins involved in HlyA activation as well as a quick and nonradioactive assay for in vitro HlyA acylation. These may simplify future experiments, e.g., activity scanning and characterization of HlyA or HlyC mutants as demonstrated with single and double HlyA lysine mutants.     

嗜虫耶尔森氏菌HlyA及HasA外分泌表达系统的构建

【目的】构建一株具备外分泌蛋白功能的工程菌,解决杀虫毒素无法由胞内分泌至胞外,无法直接作用于虫体等问题,为松墨天牛防治提供新思路。【方法】本研究先测定从松墨天牛肠道及其生境中分离出的嗜虫耶尔森氏菌(CSLH88)的生长特性及抗性,进而对其进行分子改造。构建HlyA (pGHKW2)以及HasA (pGHKW4)外分泌表达载体,利用电穿孔法将其转入CSLH88菌株,获得能够表达绿色荧光蛋白的工程菌。利用稀释涂板及荧光体式镜检测技术对两个质粒进行遗传稳定性检测,并采用SDS-PAGE及Western blotting技术验证蛋白外分泌功能。【结果】CSLH88菌株培养2–4 h能够进入对数生长期,并对卡那霉素(Kan)敏感。成功构建了含有Kan抗性基因的pGHKW2(GenBank:MK562405)和pGHKW4(GenBank:MK562404)两个外分泌表达载体的CSLH88工程菌株。其中,发现pGHKW4质粒更加适合在嗜虫耶尔森氏菌中稳定遗传。SDS-PAGE及Western blotting检测结果表明HlyA系统无法在CSLH88菌株中将目的蛋白分泌到胞外,而HasA系统则可以有效地发挥外分泌表达功能。【结论】通过对HlyA及HasA两个外分泌表达系统进行研究,从中筛选出HasA型血红素转运系统作为CSLH88菌株的外分泌表达系统,为后续外分泌杀虫毒素蛋白菌株构建以及CSLH88菌株的致病性研究奠定基础。     

Cloning of the phycobilisome rod linker genes from the cyanobacterium Synechococcus sp. PCC 6301 and their inactivation in Synechococcus sp. PCC 7942

Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.     

Conversion of an extracellular cytolysin into a phagosome-specific lysin which supports the growth of an intracellular pathogen

Listeria monocytogenes is a facultative intracellular pathogen which secretes a pore-forming cytolysin, listeriolysin O (LLO), necessary for intracellular growth. Clostridium perfringens is an extracellular pathogen which secretes a related cytolysin, perfrlngolysln O (PFO). When PFO is secreted by intracellular L. monocytogenes, it is toxic to the infected host cell. PFO-mediated toxicity renders the infected host cell permeable to gentamicin and leads to the death of the intracellular bacteria. In this study, we selected for L. monocytogenes mutants in which PFO supported the intracellular growth of L. monocytogenes. Six independent mutants were isolated, each containing a single amino acid change within the PFO protein. Three classes of PFO mutations were identified, all capable of mediating lysis of the vacuole but without a toxic effect upon the infected host cell. The first class had a severe defect in haemolytic activity. The second class had a change in the pH optimum of PFO. The third class had nearly wild-type levels of haemolytic activity, but had a decrease in protein half-life in the host-cell cytosol. Acquisition of single amino acid changes in PFO were sufficient to convert an extracellular cytolysin into a vacuole-specific lysin which mediated growth of L. monocytogenes in cultured cells.     

The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes

Summary The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca²⁺ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca²⁺ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca²⁺ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca²⁺. These data indicate that the repeat domain of haemolysin is responsible for Ca²⁺-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca²⁺ in haemolysis is presented.     

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli α-hemolysin membrane translocation system

Summary Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli -haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a -sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system. Correspondence to: H. Nakano     

The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin fromEscherichia coli

Summary As a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains within haemolysin A (HlyA) that are essential for its secretion. For this purpose we examined the properties of a deletion and Tn5 insertions into the region of theHlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate. We showed that removal of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide is still haemolytically active. Cellular fractionation studies showed that haemolytic activity does not accumulate in large amounts within the periplasmic space during normal secretion. More significantly, activity does not appear to accumulate within this compartment when the export functionshlyB andhlyD are removed. These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium.     

Acyl chains are responsible for the irreversibility in the Escherichia coli alpha-hemolysin binding to membranes

Hemolysin (HlyA) is an extracellular protein secreted by uropathogenic strains of Escherichia coli. The mature HlyA is able to bind to mammalian target cell membranes including those of the immune system, causing lysis. The lytic activity is absolutely dependent upon the Hlyc-dependent acylation of Prohemolysin. In this paper we show, through Trp fluorescence studies and denaturation in Guanidine hydrochloride, that the acylation is responsible for the loose conformation of the active protein, necessary to transform it from soluble to membrane-bound form. Previous studies showed that toxin binding to the bilayers occurs in, at least two ways, a reversible adsorption and irreversible insertion. We demonstrated that the irreversibility is due to the acyl chains in the HlyA, as shown by the protein transfer from multilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) to large unilamellar vesicles containing POPC-doxyl as protein fluorescence quencher.     

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.     

Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin

HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.     

Mutations affecting activity and transport of haemolysin in Escherichia coli

Summary Temperature-sensitive mutants that exhibit an altered haemolytic phenotype were isolated from Escherichia coli harbouring the plasmid pHly152. Complementation with recombinant plasmids carrying one of the four hly genes (C, A, B or D) allowed localization of the hly ^ts mutations. A ts mutation in hlyC leads to a proleu exchange in amino acid position 53 of HlyC. Two ts mutations in HlyA were found in positions 312 (serpro) and 315 (thrile). Both amino acid exchanges are located in the same hydrophobic domain of HlyA which extends from amino acids 299 to 327. Two different mutations were introduced by site-specific mutagenesis in this hlyA domain: one by an exchange of ala, val to asp, glu (positions 313, 314) altering the hydrophobicity of this region and another which removes most of this hydrophobic portion. Both mutants have entirely lost the haemolytic activity but the mutant haemolysins are still efficiently transported across both membranes when hlyB and hlyD are provided. Functional HlyC is not required for the transport of the mutant haemolysins. Two site-specific mutations at the N-terminal end of hlyA (one at amino acid position 2 leading to a thrpro exchange and another deleting ile and thr at positions 4 and 5) also do not affect the transport of the altered haemolysins. The thrpro exchange enhances the haemolytic activity of the corresponding mutant, whereas the ile, thr deletion exhibits little or no effect on the haemolytic activity. Removal of the last 37 amino acids from the C-terminal end of HlyA leads to a truncated haemolysin which retains its haemolytic activity but is not secreted by the HlyB and HlyD transport system.     

Enhancing functional expression of heterologous lipase B in Escherichia coli by extracellular secretion

Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli has been technically problematic due to protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis. To overcome these problems, an alternative approach was explored in this study by extracellular secretion of PalB via two Sec-independent secretion systems, i.e., the α-hemolysin (type I) and the modified flagellar (type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Both shaker flask and bioreactor cultivations were performed to characterize the developed PalB expression/secretion systems. Bioactive PalB was expressed and secreted extracellularly either as a HlyA fusion (i.e., PalB-HlyA via type I system) or an intact protein (via type III system). However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. Also importantly, the secretion strategy appeared to have a minimum impact on cell physiology. PalB secretion via the type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via the type III system was slow with lower specific PalB activities but effective cell growth.     

Inactivation of haemolysin production in Escherichia coli by transposon insertion results in loss of virulence

A haemolytic E. coli strain is nephropathogenic for mice. Mutagenesis by transposon insertion resulted in mutants with altered haemolytic activity.Reduction or elimination of the haemolytic activity is accompanied with loss of virulence. It is shown that this loss of virulence is due to altered haemolytic activity and not caused by the transposon insertion itself.     

Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae

A new toxin of Enterobacter cloacae able to lyse erythrocytes and leukocytes was found. Purification of the toxin was performed by salt precipitation, gel filtration, ion exchange and HPLC in C₈ column. SDS-PAGE electrophoresis showed more than one bank corresponding to the leukotoxin able to form polymers and aggregate like some pore-forming cytotoxins (RTX). In culture supernatant the toxin showed 1 HU/ml (hemolytic unit) and 1.5 LU/ml (leukotoxic unit); after purification it reached 15 HU/ml and 20 LU/ml. The ratio between HU and percentage red cells affected the lytic capacity. E. cloacae toxin stimulated the oxidative metabolism of neutrophils, but over 50 μg toxin/ml the stimulus ceased as it was shown by NBT assay due to cell death. Chemiluminescence evidenced an increase in superoxide anion generation, but an excess of toxin interfered with this stimulus, as was previously observed in HlyA Escherichia coli toxin. Cross-reaction was found by immunoblotting with this HlyA. E. cloacae toxin presented higher amounts of proline, valine, aspartic and glutamic acids than HlyA. E. cloacae toxin was similar to HlyA in the prescence of a glycine-rich DNA sequence and in the observed effect of calcium on toxin activity. E. cloacae toxin did not cross-react by immunoblotting with hemolysin HmpA of Proteus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes

alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.     

Leukotoxin from Aggregatibacter actinomycetemcomitans causes shrinkage and P2X receptor‐dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat‐in‐toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α‐haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA‐mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA‐mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS‐exposure is preceded by increments in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Interestingly, LtxA‐mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA‐induced [Ca²⁺]_i increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2‐mediated auto‐ and paracrine signalling may prevent LtxA‐mediated cell damage.     

Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein

The human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac⁺), all of which produce the 87kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac⁻) also carry the gene, Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33-amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C-termlnal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease-type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.