Flying under the radar: The non-canonical biochemistry and molecular biology of petrobactin from Bacillus anthracis.

The dramatic, rapid growth of Bacillus anthracis that occurs during systemic anthrax implies a crucial requirement for the efficient acquisition of iron. While recent advances in our understanding of B. anthracis iron acquisition systems indicate the use of strategies similar to other pathogens, this review focuses on unique features of the major siderophore system, petrobactin. Ways that petrobactin differs from other siderophores include: A. unique ferric iron binding moieties that allow petrobactin to evade host immune proteins; B. a biosynthetic operon that encodes enzymes from both major siderophore biosynthesis classes; C. redundancy in membrane transport systems for acquisition of Fe-petrobactin holo-complexes; and, D. regulation that appears to be controlled predominately by sensing the host-like environmental signals of temperature, CO2 levels and oxidative stress, as opposed to canonical sensing of intracellular iron levels. We argue that these differences contribute in meaningful ways to B. anthracis pathogenesis. This review will also outline current major gaps in our understanding of the petrobactin iron acquisition system, some projected means for exploiting current knowledge, and potential future research directions.

Anthrax: a motor protein determines anthrax susceptibility.

A new study has found that polymorphisms in the host gene kif1C, which encodes a kinesin-like motor protein, determine whether mouse macrophages are resistant or sensitive to anthrax lethal toxin. These findings may lead the way to discovering how both germ and host factors might contribute to a lethal infection.

Biochemical and physiological changes induced by anthrax lethal toxin in J774 macrophage-like cells.

Experiments were performed to probe the mechanism by which Bacillus anthracis Lethal Toxin (LeTx) causes lysis of J774 macrophage-like cells. After incubation of cells with saturating concentrations of the toxin, two categories of effects were found, which were distinguishable on the basis of chronology, Ca(2+)-dependence, and sensitivity to osmolarity. The earliest events (category I), beginning 45 min postchallenge, were an increase in permeability to 22Na and 86Rb and a rapid conversion of ATP to ADP and AMP. Later events (category II) included alterations in membrane permeability to 45Ca, 51Cr, 36Cl, 35SO4, 3H-amino acids, and 3H-uridine, beginning at 60 min; inhibition of macromolecular synthesis, leakage of cellular lactate dehydrogenase and onset of gross morphological changes, at approximately 75 min; and cell lysis, beginning at 90 min. Category II events exhibited an absolute requirement for extracellular Ca2+ and were blocked by addition of 0.3 M sucrose to the medium, whereas category I events were attenuated, but not blocked, by either of these conditions. On the other hand, both ATP depletion and the category II events were blocked in osmotically stabilized medium that was also isoionic for Na+ and K+. This suggests that permeabilization of the plasma membrane to monovalent cations and water may be the earliest of the physiological changes described here. The resulting influx of Na+ and efflux of K+ would be expected to cause depletion of ATP, via increased activity of the Na+/K+ pump. Subsequently the influx of Ca2+, induced by depletion of ATP, imbalances in monovalent cautions, and/or more dramatic changes in permeability due to influx of water, would be expected to trigger widespread changes leading ultimately to cytolysis.

Petrobactin Is Exported from Bacillus anthracis by the RND-Type Exporter ApeX.

Bacillus anthracis-a Gram-positive, spore-forming bacterium-causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen.IMPORTANCEBacillus anthracis requires iron for growth and employs the siderophore petrobactin to scavenge this trace metal during infections. While we understand much about petrobactin biosynthesis and ferric petrobactin import, how apo-petrobactin (iron free) is exported remains unknown. This study used a combination of bioinformatics, genetics, and mass spectrometry to identify the petrobactin exporter. After screening 17 mutants with mutations of candidate exporter genes, we identified the apo-petrobactin exporter (termed ApeX) as a member of the resistance-nodulation-cell division (RND) family of transporters. In the absence of ApeX, petrobactin accumulates inside the cell while continuing to export petrobactin components that are capable of transporting iron. Thus, the loss of ApeX does not affect the ability of B. anthracis to cause disease in mice. This has implications for treatment strategies designed to target and control pathogenicity of B. anthracis in humans.

A recombinant C-terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity.

Clostridium perfringens enterotoxin (CPE) is believed to be involved in several important gastrointestinal illnesses. Recent studies have identified a number of distinct molecular events which occur after CPE treatment of eukaryotic cells or isolated membranes. Additional studies are underway to determine the temporal order and intrinsic importance of each CPE event for cytotoxicity. We now demonstrate that a truncated CPE fragment binds to membranes, but is unable to insert into membranes or cause any other subsequent post-insertion event. This is the first experimental evidence supporting the importance of membrane insertion for CPE cytotoxicity. Binding of the CPE fragment is also shown to be irreversible, strongly suggesting that the irreversible binding of wild-type CPE is not due solely to insertion of CPE into membranes.

Lethal factor active-site mutations affect catalytic activity in vitro.

The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

On the role of macrophages in anthrax.

Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin. This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells. We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin. Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested. These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host. Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro. Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection. These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin.

Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli.

A complete copy of the gene (cpe) encoding Clostridium perfringens enterotoxin (CPE), an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses, has been cloned, sequenced, and expressed in Escherichia coli. The cpe gene was shown to encode a 319-amino-acid polypeptide with a deduced molecular weight of 35,317. There was no consensus sequence for a typical signal peptide present in the 5' region of cpe. Cell lysates from recombinant cpe-positive E. coli were shown by quantitative immunoblot analysis to contain moderate amounts of CPE, and this recombinant CPE was equal to native CPE in cytotoxicity for mammalian Vero cells. CPE expression in recombinant E. coli appeared to be largely driven from a clostridial promoter. Immunoblot analysis also demonstrated very low levels of CPE in vegetative cell lysates of enterotoxin-positive C. perfringens. However, when the same C. perfringens strain was induced to sporulate, much stronger CPE expression was detected in these sporulating cells than in either vegetative C. perfringens cells or recombinant E. coli. Collectively, these results strongly suggest that sporulation is not essential for cpe expression, but sporulation does facilitate high-level cpe expression.

Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity.

Clostridium perfringens type A enterotoxin (CPE) causes the symptoms associated with C. perfringens food poisoning. To determine whether the C-terminal half of CPE contains receptor-binding activity, the 3' half of the cpe structural gene was cloned with an Escherichia coli expression vector system. E. coli lysates containing the expressed C-terminal CPE fragment (CPEfrag) were then assayed for CPE-like serologic, receptor-binding, and cytotoxic activities. CPEfrag was shown to contain an epitope located at or near the receptor-binding domain of the CPE molecule. Competitive-binding studies showed specific competition for CPE receptors between CPE and CPEfrag lysates. CPEfrag lysates did not cause cytotoxicity in Vero (African green monkey kidney) cells. However, preincubation with CPEfrag lysates specifically protected Vero cells from subsequent CPE challenge. This indicates that CPEfrag recognizes the physiologic receptor which mediates CPE cytotoxicity. Collectively, these studies indicate that the C-terminal half of CPE contains a receptor-binding domain but additional amino acid sequences appear to be required for CPE cytotoxicity.

Clostridium perfringens enterotoxin.

Current knowledge of CPE action is briefly summarized in Figure 1. After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr. Almost simultaneously with insertion, there is a sudden change in ion fluxes. The molecular events behind the induction of ion flux changes remain undefined, but might involve either direct formation of membrane pores by CPE or activation of pre-existing membrane pores. As intracellular ion levels change, cellular metabolism is affected and processes such as macromolecular syntheses are inhibited. One of the ion flux effects resulting from CPE treatment involves increased Ca2+ influx; as more Ca2+ enters the cell, morphologic damage and permeability alterations for larger molecules occur. It remains to be determined if both morphologic damage and larger permeability alterations are necessarily linked but, for example, it could be envisioned that CPE-induced Ca2+ influx causes a cytoskeletal collapse leading to altered membrane permeability. The cytoskeleton has been shown to be sensitive to intracellular Ca2+ levels and is important in normal membrane structure/function relationships. As the cumulative effects of CPE inhibit cellular metabolism, cell death occurs. The precise irreversible CPE lethal action still must be identified. As CPE-treated intestinal epithelial cells die in vivo, histopathologic damage appears. This damage results in loss of normal intestinal function causing secretion of fluids and electrolytes. This effect is clinically manifested as diarrhea. The strongly cytotoxic action of CPE clearly distinguished the action enterotoxin from STa or CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus.

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.

Anthrax protective antigen forms oligomers during intoxication of mammalian cells.

The protective antigen component (PA) of anthrax toxin binds to receptors on target cells and conveys the toxin's edema factor (EF) and lethal factor (LF) components into the cytoplasm. PA (83 kDa) is processed by a cellular protease, yielding a 63-kDa fragment (PA63), which binds EF and/or LF. When exposed to acidic pH, PA63 inserts into membranes and forms ion-conductive channels. By electron microscopy, a significant fraction of purified PA63 was found to be in the form of a multi-subunit ring-shaped oligomer (outer diameter, 10.4 nm). The rings are heptameric, as judged by inspection and by rotational power spectra. Purified PA63 showed a high M(r) band, apparently corresponding to the oligomer, on SDS-polyacrylamide gels, and oligomer of similar size was formed in cells in a time-dependent manner after addition of full-length PA. Inhibitors of internalization and endosome acidification blocked conversion of cell-associated PA to a high molecular weight species, and medium at pH 5.0 induced oligomer formation in the presence or absence of the inhibitors. These results correlate PA63 oligomerization with conditions required for translocation of EF and LF across lipid bilayers, implying that the PA63 oligomer may function in translocation.

A conjugated synthetic peptide corresponding to the C-terminal region of Clostridium perfringens type A enterotoxin elicits an enterotoxin-neutralizing antibody response in mice.

A synthetic peptide homolog corresponding to the C-terminal 30 amino acids of Clostridium perfringens type A enterotoxin (CPE) was conjugated to a thyroglobulin carrier and used to immunize mice. Conjugate-immunized mice produced antibodies which neutralized native CPE cytotoxicity, at least in part, by blocking enterotoxin binding. This peptide may be useful for the development of a vaccine to protect against CPE-mediated disease.

Mapping of functional regions of Clostridium perfringens type A enterotoxin.

Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE). Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding. CPE sequences required for at least four epitopes were also identified.

Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent.

Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin. There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids. Further support for the existence of these early steps and further characterization of these events are presented in this report. We now report that these early steps in CPE action are largely independent of extracellular divalent cations. It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent. A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps.

Localization of the receptor-binding region of Clostridium perfringens enterotoxin utilizing cloned toxin fragments and synthetic peptides. The 30 C-terminal amino acids define a functional binding region.

In this study a short sequence encoding the receptor-binding activity of the much larger 35-kDa enterotoxin elaborated by Clostridium perfringens was localized by recombinant DNA techniques. Defined fragments corresponding to portions of the enterotoxin gene were cloned into an Escherichia coli expression vector system, and these lysates were analyzed for their ability to compete for binding with native C. perfringens enterotoxin (CPE). The lysate containing CPE290-319 (CPE sequence encompassing residues 290-319) was shown to compete with 125I-CPE for specific binding sites on rabbit intestinal brush border membranes. To confirm this finding, a peptide corresponding to the CPE amino acid sequence 290-319 was synthesized and found to completely block CPE specific binding. To demonstrate directly that CPE290-319 can act as a competitive antagonist of CPE cytotoxicity for physiologic receptors, Vero cells were preincubated with either E. coli lysates containing CPE290-319 or the synthetic peptide corresponding to this sequence. Preincubation of Vero cells with either the lysate or the peptide completely protected these cells from CPE challenge. This information localizes the C-terminal 30 residues of CPE (CPE290-319) as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor.

Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape.

This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mphi). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mphi and replicate within the cytoplasm of these cells. Release from the Mphi occurs 4-6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mphi, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mphi release of anthrax bacilli is atxA regulated. The putative 'escape' genes may be located on the chromosome and/or on pXO1.

Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host.

We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotics resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltransferase (cat) and lacZY genes. A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with intraperitoneal injections of chloramphenicol. Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium. These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids. The pIVET8-based selection system was also used to identify S. typhimurium genes that are induced in cultured macrophages. The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.