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W Goebel

Publications and source records attributed to W Goebel.

At least 91 records · Page 5Linked to original sources
PubMed1997

Isolation of catalase-negative Listeria monocytogenes strains from listeriosis patients and their rapid identification by anti-p60 antibodies and/or PCR.

Two catalase-negative Listeria monocytogenes serovar 1/2b strains were isolated from listeriosis patients in 1995 in Germany. The infections appeared in individuals from different cities at different seasons and were caused by L. monocytogenes strains of different clonal types. In particular, the catalase reaction of one strain isolated from blood was consistently negative, whereas this reaction was only reversibly blocked when the strain was freshly isolated from ascitic fluid. After subculturing, the catalase-positive reaction was restored. Initially, identification of these isolates was difficult to achieve not only because of the lack of a catalase reaction, which generally distinguishes L. monocytogenes from other morphologically similar pathogenic gram-positive bacteria, but also because other routinely used biochemical tests such as CAMP and the commercial API test gave unclear results. However, rapid and unequivocal identification of these strains was possible by analyzing secretions of the p60 protein in culture supernatants by enzyme-linked immunosorbent assay or Western blot (immunoblot) analysis with our recently developed Listeria- and L. monocytogenes-specific anti-p60 antibodies. Additionally, the identifications were confirmed by Listeria- and L. monocytogenes-specific PCR analyses with primers derived from the iap, hly, and prfA genes. Immunoanalyses also allowed for the differentiation of these two strains, whereas no differentiation was possible by PCR when the internal, variable repetitive iap gene portion was analyzed. However, size variations of the PCR products comprising these gene portions which were obtained from a number of L. monocytogenes strains belonging to the same serotypes indicated that this type of PCR is not only useful for specific identifications but may be used in parallel as an additional marker for epidemiological studies. In conclusion, the data suggest that catalase production should not be taken as a strict criterion for the identification of listeriae. Furthermore, at least the infection caused by the stably catalase-negative strain supports the notion that catalase does not seem to be necessary for the intracellular growth of L. monocytogenes.

Aged↗
PubMed1997

The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella.

The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.

Animals↗
PubMed1996

Development of antigen-delivery systems, based on the Escherichia coli hemolysin secretion pathway.

We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyAs). In general, the fused genes, carrying the hlyAs sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyAs-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.

Animals↗
PubMed1996

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins.

A simple and efficient procedure for the construction of secreted fusion proteins in Escherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) of E. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we used lacZ, encoding the cytoplasmic beta-galactosidase (beta-Gal), and phoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all beta-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in a secA mutant strain only under SecA-deficient conditions.

Alkaline Phosphatase↗
PubMed1996

The effects of calcium and other polyvalent cations on channel formation by Escherichia coli alpha-hemolysin in red blood cells and lipid bilayer membranes.

Channel formation by Escherichia coli alpha-hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations. Hemolysin showed full channel-forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E. coli cells were grown in calcium-depleted media (< 1 microM Ca2+). The addition of divalent cations decreased the single-channel conductance by about 50% with half-saturation constants of 5 mM and less, while the mean lifetime of the HlyA channel was not affected. The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half-saturation constant was 1 microM or below. These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition. When cells were grown in calcium-depleted media, the supernatants showed absolutely no hemolytic activity. Addition of small amounts of Ca2+ to the supernatant led to toxin-mediated hemolysis. Its half-saturation constant was 120 microM. Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a K(m) of 1.5 mM. Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect. These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.

Bacterial Proteins↗
PubMed1996

Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis.

Vaccination provides the most potent measure against infectious disease, and recombinant (r) viable vaccines expressing defined pathogen-derived antigens represent powerful candidates for future vaccination strategies. In a new approach we constructed r-aroA- Salmonella typhimurium displaying p60 or listeriolysin (Hly) antigen of Listeria monocytogenes in secreted or somatic form in the host cell. Vaccination of mice with r-aroA- S. typhimurium induced protection against the intracellular pathogen L. monocytogenes only with secreted and not with somatic antigen. Secreted Hly was slightly more potent in inducing protective immunity than secreted p60. Both r-aroA- S. typhimurium secreting p60 in the endosome and r-aroA- S. typhimurium secreting Hly in the cytosol induced protective CD4+ and CD8+ T-cells suggesting CD8+ T-cell stimulation independent from intracellular residence of r-aroA- S. typhimurium carriers. Hence, not only the type of antigen but also its display by the r-carrier within the host cell critically influences vaccine efficacy.

Amino Acid Sequence↗
PubMed1996

Extracellular PagC-HlyAs fusion protein for the generation and identification of Salmonella-specific antibodies.

The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyAs) to the mature PagC peptide. This PagC-HlyAs fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyAs fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.

Amino Acid Sequence↗
PubMed1996

Protein p60 participates in intestinal host invasion by Listeria monocytogenes.

The role of p60 in intestinal invasion by Listeria monocytogenes was assessed after oral infection of mice with the p60 low-expressing mutant RIII, or with anti-p60 antibody coated wild-type EGD. Invasion by L. monocytogenes RIII bacteria has been unimpaired suggesting that a low density of p60 suffices for entry. Up to 24 h post infection (p.i.), intestinal penetration by L. monocytogenes EGD bacteria was markedly reduced by coating with anti-p60 antibodies. In histological sections, anti-p60 antibody-treated L. monocytogenes EGD, but not uncoated listeriae were still detectable 24 h p.i. at the apical surface of enterocytes in the intestine. We conclude that p60 contributes to host invasion through the natural port of listerial entry, the intestinal epithelium.

Animals↗
PubMed1996

A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins.

A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene InlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position -40. In contrast to the transcription of the InlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain-heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, InlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An InlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.

Amino Acid Sequence↗
PubMed1996

Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron.

The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility-shift assays (EMSAs) using cell-free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA-specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA-specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain-heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA-free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA-free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA-containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.

Bacterial Proteins↗
PubMed1996

Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization.

Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod-shaped Gram-negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus- and species-specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the gamma-subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the gamma-subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co-evolution of the symbiotic partners and a vertical transmission of the bacteria.

Animals↗
PubMed1996

Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA.

The two Listeria monocytogenes strains EGD and NCTC 7973 display a different regulation pattern of their PrfA-dependent genes. All PrfA-dependent genes from L. monocytogenes NCTC 7973 are much more efficiently transcribed in brain-heart infusion medium than those from strain EGD. Transcription of these genes in EGD is, however, induced after shift into Minimal Essential Medium (MEM) to a level that is comparable to that of strain NCTC 7973. Expression of the internalin gene (inlA) is also influenced by PrfA, but only one (P2) out of three mapped promoters is strictly dependent on PrfA. In contrast to the other PrfA-regulated genes, transcription of inlA even from the P2 promoter is reduced in both strains after shift to MEM. The prfA deletion mutant SLCC 53 complemented with multiple copies of prfA synthesizes large amounts of monocistronic prfA transcript, but there is no concomitant increase in the transcripts of the PrfA-dependent genes. However, upon a shift into MEM, transcription of the PrfA-dependent genes (with the exception of the inlA gene) is highly induced even in the absence of de novo protein synthesis. The PrfA proteins of the two studied L. monocytogenes strains differ in their ability to activate PrfA-dependent genes. This difference is probably the result of amino acid exchange(s) in the C-terminal part of these proteins. Strain EGD supplemented with multiple copies of prfA-7973 shows a similar regulation of the PrfA-dependent genes as strain NCTC 7973, whereas multiple copies of prfA-EGD introduced into strain EGD hardly change the rate of transcription of the PrfA-dependent genes. Our data thus suggest that PrfA-mediated gene expression depends not only on the amount of the PrfA protein and the 'quality' of the putative PrfA-binding sites, but also on the 'quality' of the PrfA protein which seems to be influenced by its amino acid composition and by environmental parameters.

Bacterial Proteins↗
PubMed1996

Detection and characterization by differential PCR of host eukaryotic cell genes differentially transcribed following uptake of intracellular bacteria.

Host eukaryotic cell genes that are differentially transcribed after phagocytosis of various pathogenic and nonpathogenic bacterial cells were identified by a differential PCR (DPCR) system. This DPCR procedure favors detection and isolation of host genes affected at the transcriptional level by selecting for poly(A) tails but differs substantially from reverse transcription-PCR. Several unidentified macrophage gene fragments from genes that were either transcriptionally activated or downregulated following uptake of Listeria monocytogenes into J774 mouse macrophage cells were initially defined by this DPCR procedure. Because of the sensitivity of the DPCR technique, all of the genes exhibited less than a 10-fold difference in transcription compared with noninfected cells as measured by limiting-dilution PCR. One of the gene fragments has a very high level of homology with a mitogen-activated protein kinase phosphatase (MKP-1), whereas the other affected fragments showed no homologies to known gene sequences. In addition, one of the gene fragments (WS30-B2/1) was specifically downregulated after L. monocytogenes uptake and another gene was repressed by uptake of either Shigella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activated following general phagocytosis (i.e., following uptake of any species of bacterium tested). Further characterization of the affected genes was conducted by using mutants of L. monocytogenes. A hemolysin-negative mutant of L. monocytogenes failed to elicit transcriptional regulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosome may be required to initiate these responses. Furthermore, mutants with mutations in mpl and actA, two genes whose gene products are involved in actin polymerization and intrahost spread, also did not induce regulation of WS10-B4/14. These results demonstrate that (i) DPCR can identify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these genes may be new or may never before have been linked to interactions between hosts and pathogens.

Animals↗
PubMed1996

Differences in virulence and in expression of PrfA and PrfA-regulated virulence genes of Listeria monocytogenes strains belonging to serogroup 4.

Listeria monocytogenes isolates belonging to serogroup 4 (subtypes 4a, 4ab, 4b, 4c, 4d, and 4e) exhibit different levels of virulence in mice. Molecular studies indicate that in comparison with the control strain EGD (serotype 1/2a), these strains differ in the expression of the PrfA-regulated virulence genes, including prfA itself. Strains of serotypes 4c, 4d, 4e, and especially 4a show a low level of invasiveness in Caco-2 cells, which correlates in part with the low level of expression of the inlA gene. All serotypes reach the cytoplasm, at the latest, 2 h postinfection and become surrounded by polymerized actin within the next hour, but actin tail formation by serotype 4a, 4c, 4d, and 4e strains is drastically reduced. The actA genes in these serogroup 4 strains are expressed in minimum essential medium and within the phagocytic cell line J774. However, the amounts and (in part) the sizes of the ActA proteins in these strains differ under these conditions. The reduced actin tail formation by serotype 4a, 4c, 4d, and 4e strains may be due to the low level of in vivo expression of ActA. In addition, the loss of one repeat unit in the ActA proteins of serotype 4a and 4e strains may also contribute to the less efficient actin tail formation observed with these strains.

Animals↗
PubMed1996

The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine.

Recent studies have shown that Salmonella typhimurium invades the M cells of Peyer's patches (PP) of the murine ileum. The slyA gene of S. typhimurium has also recently been reported to affect virulence of this pathogen in mice and survival in macrophages. We therefore compared the effect on PP tissue of four strains of S. typhimurium: a wild-type strain, two slyA insertion mutants, and a recombinant S. typhimurium derivative carrying multiple copies of slyA. Invasion assays performed 2 and 7 days after orogastric infection revealed significantly lower numbers of bacteria of the slyA mutants and of the SlyA-overproducing strain in PP than of the wild type. However, similar numbers of bacteria of all strains were still present in the lumen of the small intestine after these times. Invasion assays of PP tissue after 90-min ileal loop infection yielded comparable numbers of bacteria of all strains in PP. Transmission and scanning electron microscopy of PP tissue after ileal loop infection demonstrated that the two slyA mutants and the SlyA-overproducing strain were able to attach to, induce membrane ruffling of, and invade M cells in a way morphologically and quantitatively similar to that of the wild type. In contrast to the wild type, both slyA mutants and, to a lesser extent, the SlyA-overproducing strain were significantly impaired in their ability to destroy M cells and adjacent enterocytes. Taken together, these data suggest that slyA is involved in intracellular survival and M-cell cytotoxicity but not in the invasion process and that the amount of SlyA needs to be precisely balanced for virulence.

Animals↗
PubMed1996

Differential regulation of cytokine and cytokine receptor mRNA expression upon infection of bone marrow-derived macrophages with Listeria monocytogenes.

Cytokine and cytokine receptor mRNA expression was analyzed by PCR-assisted amplification of RNA extracted from bone marrow-derived macrophages (BMM phi) at different time points after infection with Listeria monocytogenes. The mRNAs for the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) were induced early after infection, whereas IL-6 mRNA appeared later and even nonhemolytic Listeria strains, which are unable to grow inside eukaryotic cells, induced the same cytokine mRNAs at levels similar to those of the wild-type strain. In most cases, the amounts of cytokines determined by various bioassays correlated with the level of mRNA induction. Inhibition of phagocytic uptake of L. monocytogenes by cytochalasin D treatment resulted in adherent bacteria which still induced the proinflammatory cytokines. In BMM phi, the level of IL-1 receptor II mRNA was unaffected, whereas mRNA expression of the two subtypes of tumor necrosis factor receptors (TNF-RI and TNF-RII) was differentially regulated upon infection: transcription of TNF-RI was reduced, and that of TNF-RII mRNA was induced. Similar to the decreased TNF-RI mRNA expression, gamma interferon receptor mRNA was downregulated in L. monocytogenes-infected BMM phi. This dose- and time-dependent induction or downregulation of cytokine receptor mRNA following L. monocytogenes infection of BMM phi was not observed upon infection of established macrophage-like cell lines J774 and P388D1. Induction of IL-6 mRNA as well as IL-1 alpha/beta and TNF-alpha mRNAs upon L. monocytogenes infection of BMM phi occurs independently of autocrine TNF-alpha signaling via TNF-RI or TNF-RII, as shown by infection of TNF-RI- and TNF-RII-deficient macrophages derived from mutant B6 x 129 mice. In contrast to gamma interferon receptor mRNA, both TNF receptor subtype mRNAs were not influenced by L. monocytogenes infection of hybrid (B6 x 129) mouse macrophages. Whereas the proinflammatory cytokine mRNAs were even induced after infection with the nonpathogenic species L. innocua, no alteration of cytokine receptor mRNA expression was observed after challenge of BMM phi with this nonpathogenic species, suggesting that the modulation of cytokine and cytokine receptor expression by L. monocytogenes could be an important way of inhibition of macrophage stimulation.

Animals↗
PubMed1996

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.

Acyl Carrier Protein↗