In vitro activation of Escherichia coli prohaemolysin to the mature membrane-targeted toxin requires HlyC and a low molecular-weight cytosolic polypeptide

The c. 110 kDa haemolysin toxin secreted by Escherichia coli and other pathogenic Gram-negative bacteria is synthesized as the non-toxic precursor, prohaemolysin (proHlyA), which is unable to target mammalian cell membranes until activated intracellularly by an unknown mechanism dependent upon the coexpressed c. 20 kDa protein, HlyC. We have established in vitro post-translational activation of proHlyA in membrane-depleted cell extract fractions from E. coli recombinant strains containing (separately) the proHlyA and HlyC proteins. In vitro activation was calcium-independent and effective over a pH range of 6 to 9 and at temperatures from 42 degrees C to 4 degrees C. HlyC cell extract was also able to activate proHlyA which had been secreted out of cells containing the export proteins HlyB and HlyD. Fractionation of HlyC cell extracts by sucrose gradient centrifugation and molecular weight chromatography revealed activating fractions as having a molecular mass of 40 kDa, suggesting that the HlyC activator is present physiologically in a multimeric form. Cell extracts containing activation-competent HlyC and proHlyA were inactive following dialysis, but activity was restored by complementation with a cell extract lacking both proteins. HlyC and proHlyA proteins which were overproduced separately from recombinant expression plasmids were inactive following purification, but activity could again be restored with a Hly-negative cell extract. These experiments demonstrated that HlyC is not sufficient for activation; an additional cellular factor is required. The cellular factor was found in enterobacteria but not other bacteria or eukaryotic cells. It was cytosolic, protease-sensitive, and behaved as a c. 10 kDa polypeptide in a number of assays including dialysis, sucrose gradient centrifugation, and gel filtration chromatography. Thus activation was possible in a defined in vitro reaction containing only purified proHlyA, HlyC, and the cellular factor. Kinetic studies in which the relative concentrations of the three components of proHlyA activation were varied suggested that neither HlyC nor the cellular factor acts as a conventional enzyme, with each participating in a finite number of activation events.     

Thermodynamics of a protein acylation: activation of Escherichia coli hemolysin toxin

HlyC, hemolysin-activating lysine-acyltransferase, catalyses the acylation (from acyl-acyl carrier protein [ACP]) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, 564 and 690 of the 1024 amino acid primary structure, to form hemolysin (HlyA). Isothermal titration calorimetry was used to measure the thermodynamic properties of the protein acylation of proHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site directed mutation analyses of acylation sites. Acylation of proHlyA-derived proteins catalyzed by HlyC was overall an exothermic reaction driven by a negative enthalpy. The reaction, whose kinetics are compatible to a ping-pong mechanism, is composed of two partial reactions. The first, the formation of an acyl-HlyC intermediate, was entropically driven, most likely by noncovalent complex formation between acyl-ACP and HlyC; enthalpy-driven acyl transfer followed, resulting in acyl-HlyC and ACPSH product formation. The second partial reaction was an energetically unfavorable acyl transfer from acyl-enzyme intermediate to the final acyl acceptor, a proHlyA derivative. Overall the acylation of proHlyA-derived proteins catalyzed by HlyC was driven by the energetics of the acyl enzyme intermediate reaction. Of the two acylation sites, intactness of the site equivalent to proHlyA K564 was more important for acylation reaction thermodynamic stability.     

Insights into the catalytic mechanism of HlyC, the internal protein acyltransferase that activates Escherichia coli hemolysin toxin.

Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is converted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC. Acyl-acyl carrier protein (ACP) is the essential acyl donor. The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemistry 38, 9541-9548]. The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions. Assessments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs. Mutations of a single conserved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue. Unlike numerous other His23 mutants, however, the H23K enzyme showed significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA.     

Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli.

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.     

An ordered reaction mechanism for bacterial toxin acylation by the specialized acyltransferase HlyC: formation of a ternary complex with acylACP and protoxin substrates

The 110 kDa haemolysin protoxin (proHlyA) is activated in the Escherichia coli cytosol by acyl carrier protein-dependent fatty acylation of two internal lysine residues, directed by the co-synthesized protein HlyC. Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis-Menten analysis. The Vmax of HlyC at saturating levels of both substrates was approximately 115 nmol acyl group min-1 mg-1 with KMacylACP of 260 nM and KMproHlyA of 27 nM, kinetic parameters sufficient to explain why in vivo HlyC is required at a concentration equimolar to proHlyA. HlyC bound the fatty acyl group from acylACP to generate an acylated HlyC intermediate that was depleted in the presence of proHlyA, but enriched in the presence of proHlyA derivatives lacking acylation target sites. HlyC was also able to bind in vivo 4'-phosphopantetheine. Substitution of conserved amino acids that could act as putative covalent attachment sites did not prevent binding of the fatty acyl or 4'-phosphopantetheine groups. These data and substrate variation analyses suggest that the unique acylation reaction does not involve covalent attachment of fatty acid to the acyltransferase, but rather that it proceeds via a sequential ordered Bi-Bi reaction mechanism, requiring the formation of a non-covalent ternary acylACP-HlyC-proHlyA complex.     

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.     

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.     

The UPEC pore-forming toxin α-hemolysin triggers proteolysis of host proteins to disrupt cell adhesion, inflammatory, and survival pathways

Uropathogenic Escherichia coli (UPEC), which are the leading cause of both acute and chronic urinary tract infections, often secrete a labile pore-forming toxin known as α-hemolysin (HlyA). We show that stable insertion of HlyA into epithelial cell and macrophage membranes triggers degradation of the cytoskeletal scaffolding protein paxillin and other host regulatory proteins, as well as components of the proinflammatory NFκB signaling cascade. Proteolysis of these factors requires host serine proteases, and paxillin degradation specifically involves the serine protease mesotrypsin. The induced activation of mesotrypsin by HlyA is preceded by redistribution of mesotrypsin precursors from the cytosol into foci along microtubules and within nuclei. HlyA intoxication also stimulated caspase activation, which occurred independently of effects on host serine proteases. HlyA-induced proteolysis of host proteins likely allows UPEC to not only modulate epithelial cell?functions, but also disable macrophages and suppress inflammatory responses.     

Activation of hemolysin toxin: relationship between two internal protein sites of acylation

HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined. ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured. The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites. Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate. In a variety of constructs, the two acylation sites had similar K(m) values, but their V(max) values and catalytic efficiencies as substrates differed. Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.     

Fatty acids covalently bound to alpha-hemolysin of Escherichia coli are involved in the molten globule conformation: implication of disordered regions in binding promiscuity

Alpha-hemolysin (HlyA) is a pore-forming toxin secreted by pathogenic strains of Escherichia coli. The toxin is synthesized as a protoxin, ProHlyA, which is matured in the cytosol to the active form by acylation at two internal lysines, K563 and K689 (HlyA). It is widely known that the presence of fatty acids is crucial for the hemolytic and cytotoxic effects of the toxin. However, no detailed physicochemical characterization of the structural changes produced by fatty acids in the soluble protein prior to membrane binding has been carried out to date. The effects of chemical denaturants, the ANS binding parameters (Kd and n) and the sensitivity to proteases were compared between the acylated and unacylated protein forms HlyA and ProHlyA. Our results are consistent with a molten globular form of the acylated protein. Moreover, because molten globule proteins are intrinsically disordered proteins, using disorder prediction analyses, we show that HlyA contains 9 regions composed of 10-30 natively disordered amino acids. We propose that this conformation induced by covalently bound fatty acids might provide HlyA with the ability to bind to a variety of molecules during its action mechanism.     

Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes.

We have studied the C-terminal signal which directs the complete export of the 1024-amino-acid hemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Isolation and sequencing of homologous hlyA genes from the related bacteria Proteus vulgaris and Morganella morganii revealed high primary sequence divergence in the three HlyA C-termini and highlighted within the extreme terminal 53 amino acids the conservation of three contiguous sequences, a potential 18-amino-acid amphiphilic alpha-helix, a cluster of charged residues, and a weakly hydrophobic terminal sequence rich in hydroxylated residues. Fusion of the C-terminal 53 amino acid sequence to non-exported truncated Hly A directed wild-type export but export was radically reduced following independent disruption or progressive truncation of the three C-terminal features by in-frame deletion and the introduction of translation stop codons within the 3' hlyA sequence. The data indicate that the HlyA C-terminal export signal comprises multiple components and suggest possible analogies with the mitochondrial import signal. Hemolysis assays and immunoblotting confirmed the intracellular accumulation of non-exported HlyA proteins and supported the view that export proceeds without a periplasmic intermediate. Comparison of cytoplasmic and extracellular forms of an independently exported extreme C-terminal 194 residue peptide showed that the signal was not removed during export.     

Relevance of Fatty Acid Covalently Bound to Escherichia coli ��-Hemolysin and Membrane Microdomains in the Oligomerization Process

α-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.Escherichia coli α-hemolysin, HlyA,⁴ is an exotoxin that elicits a number of responses from mammalian target cells and also alters the membrane permeability of host cells, causing lysis and death (1, 2). Synthesis, maturation, and secretion of E. coli HlyA are determined by the hlyCABD operon (3). The gene A product is a 110-kDa polypeptide corresponding to protoxin (Pro-HlyA), which is matured in bacterial cytosol to the active form (HlyA) by HlyC-directed acylation. This post-translational modification involves a covalent amide linkage of fatty acids at two internal lysine residues (Lys-563 and Lys-689) for activation (4). HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures (two acylation sites and three possible modifying groups in each site, C14:0 (68%), C15:0 (26%) and C17:0 (6%) (5)). Although these fatty acids are not required for the binding of the toxin to membranes, they are essential for the hemolytic process, inducing a molten globule conformation and promoting the irreversibility of the binding (6, 7).It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes. However, these experiments have yielded conflicting results. Although a group of researchers is in favor of a monomer as the active species of the toxin in membranes, other groups postulate that an oligomerization process is involved. Based on experiments with lipid bilayers, Menestrina et al. (8) have suggested that one single HlyA molecule is responsible for the formation of the channel. HlyA has also been recovered from deoxycholate-solubilized erythrocyte membranes as a monomer, indicating either that oligomerization is not required for pore formation or that oligomers are dissociated in the detergent (1).On the other hand, Benz et al. (9) have found that small variations of toxin concentration have had a considerable effect on the specific membrane conductance. An increase in HlyA concentration, by a factor of 5, results in about 40–100-fold higher membrane conductance. This means that several HlyA molecules could be involved in channel formation (9). Besides, they have found that the active channel-forming oligomer and inactive monomer are in an association-dissociation equilibrium (10). In addition, the complementation of inactive deleted mutant proteins of HlyA with the corresponding wild type toxin produces hemolytic activity, suggesting that two or more toxin molecules aggregate before pore formation (11). All of the evidence suggests the formation of an oligomer.Experiments employing erythrocytes and model membranes have shown that the lesion created by HlyA is perhaps a more complicated event than the creation of a simple, static protein-lined pore. We have recently found that addition of nanomolar concentrations of toxin to planar lipid membranes have resulted in a decrease in membrane lifetime up to 3 orders of magnitude in a voltage-dependent manner, a typical behavior of proteolipidic pores (12). Moayeri and Welch (13) have previously demonstrated that osmotic protection of erythrocytes by sugars of different sizes is a function of toxin concentration and assay time. It appears that HlyA induces heterogeneous erythrocyte lesions that increase in size over time and that the rate of the putative growth in the size of HlyA-mediated lesions is temperature-dependent (13).On the other hand, it has been recognized that a variety of pathogens and toxins interacts with microdomains in the plasma membrane. These microdomains are enriched in cholesterol and sphingolipids and probably exist in a liquid-ordered phase, in which lipid acyl chains are extended and ordered (14). Many proteins are targeted to these membrane microdomains by their favorable association with ordered lipids. Interestingly, these proteins are linked to saturated acyl chains, which partition well into these domains (15).In this context, and in view of the fact that acyl chains covalently bound to proteins are determinant of specific protein-protein interactions, this research presents a study of HlyA oligomerization on sheep erythrocytes, as well as the implication of fatty acids and cholesterol-enriched microdomains in this process.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis

HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.     

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.     

Translocation and compartmentalization of Escherichia coli hemolysin (HlyA).

Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.     

Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins.

Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells.     

Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion

An early event in Salmonella infection is the invasion of non-phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact-dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full-length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N-terminal protease-resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli-derived liposomes into the cytosol of living mammalian cells.     

Inactivation of host Akt/protein kinase B signaling by bacterial pore-forming toxins

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin alpha-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and alpha-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.     

Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.     

A heterologous membrane protein domain fused to the C-terminal ATP-binding domain of HlyB can export Escherichia coli hemolysin.

The hydrophobic-rich NH2-terminal 34 amino acids of a tetracycline resistance determinant (TetC) were fused to the COOH-terminal 240 amino acids of the hemolysin transporter, HlyB, which contains a putative ATP-binding domain. This hybrid protein replaced the NH2-terminal 467-amino-acid portion of HlyB and could still export the Escherichia coli hemolysin (HlyA). Export by the hybrid protein was approximately 10% as efficient as transport by HlyB. Extracellular secretion of HlyA by the TetC-HlyB hybrid required HlyD and TolC. The extracellular and periplasmic levels of beta-galactosidase and beta-lactamase in strains that produced the hybrid were similar to the levels in controls. Thus, HlyA transport was specific and did not appear to be due to leakage of cytoplasmic contents alone. Antibodies raised against the COOH terminus of HlyB reacted with the hybrid protein, as well as HlyB. HlyB was associated with membrane fractions, while the hybrid protein was found mainly in soluble extracts. Cellular fractionation studies were performed to determine whether transport by the hybrid occurred simultaneously across both membranes like wild-type HlyA secretion. However, we found that HlyA was present in the periplasm of strains that expressed the TetC-HlyB hybrid. HlyA remained in the periplasm unless the hlyD and tolC gene products were present in addition to the hybrid.