Bacterial toxins with intracellular protease activity

Clinica Chimica Acta 291 (2000) 189–199www.elsevier.com/ locate /clinchim

Bacterial toxins with intracellular protease activity

Ornella Rossetto, Marina de Bernard,, Rossella Pellizzari,Gaetano Vitale, Paola Caccin, Giampietro Schiavo,

*Cesare Montecucco

Department of Biomedical Sciences, University of Padua, Via G. Colombo 3, 35121 Padua, Italy

Abstract

The recent determination of their primary sequence has lead to the discovery of the metallo-proteolytic activity of the bacterial toxins responsible for tetanus, botulism and anthrax. Theprotease domain of these toxins enters into the cytosol where it displays a zinc-dependentendopeptidase activity of remarkable specificity. Tetanus neurotoxin and botulinum neurotoxinstype B, D, F and G cleave VAMP, an integral protein of the neurotransmitter containing synapticvesicles. Botulinum neurotoxins type A and E cleave SNAP-25, while the type C neurotoxincleaves both SNAP-25 and syntaxin, two proteins located on the cytosolic face of the presynapticmembrane. Such specific proteolysis leads to an impaired function of the neuroexocytosismachinery with blockade of neurotransmitter release and consequent paralysis. The lethal factor ofBacillus anthracis is specific for the MAPkinase-kinases which are cleaved within their aminoterminus. In this case, however, such specific biochemical lesion could not be correlated with thepathogenesis of anthrax. The recently determined sequence of the vacuolating cytotoxin ofHelicobacter pylori contains within its amino terminal domain elements related to serine-proteases, but such an activity as well as its cytosolic target remains to be detected. 2000Elsevier Science B.V. All rights reserved.

Keywords: Metallo-proteases; Clostridial neurotoxins; Lethal factor of Bacillus anthracis;Vacuolating cytotoxin of Helicobacter pylori

1. Introduction

Bacteria produce hundreds of different protein toxins, which can be divided

*Corresponding author. Tel.: 1 39-49-827-6058; fax: 1 39-49-827-6049.E-mail address: [email protected] (C. Montecucco)

0009-8981/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0009-8981( 99 )00228-4

190 O. Rossetto et al. / Clinica Chimica Acta 291 (2000) 189 –199

into three main groups depending on the site of action with respect to host cell:(a) toxins acting outside cells by binding to specific proteins or receptors or bydegrading tissue and/or cellular components; (b) toxins altering the permeabilityof the cell membrane; (c) toxins acting inside cells [1]. These latter toxinsconsist of two main protomers, A (active part) and B (binding part) whichenable them to intoxicate cells via a four-step mechanism [2]: (1) they bind tocell surface receptors of proteic or lipidic nature via the B protomer; (2) thetoxin–receptor complex is then internalized inside intracellular compartments ofdifferent nature; (3) they have then the common problem of exiting out thelumen into the cytosol to reach their targets — they do so by translocating the Adomain across the intracellular membrane in a process mediated by the Bprotomer; (4) the A domain displays its enzymatic activity which is generallyvery specific for a selected protein facing the cytosol.

2. Tetanus and botulinum neurotoxins

Tetanus neurotoxin (TeNT) is produced by Clostridium tetani and it is thesole agent responsible for the spastic paralysis of tetanus. At variance, botulinumneurotoxins (BoNT, seven types from A to G) are produced by different speciesof Clostridia and cause botulism, which is characterized by a flaccid paralysisand other symptoms related to inhibition of autonomic cholinergic terminals.TeNT and BoNTs bind selectively to the neuronal presynaptic membrane andare then internalized inside intracellular compartments whence the aminoterminal 50 kDa domain (termed L chain) enters into the cytosol [3,4]. The Lchains of TeNT and BoNTs are zinc-endopeptidases, which cleave specificallythree proteins of the neuroexocytosis apparatus, thereby blocking neurotrans-mitter release [5,6]. TeNT and BoNT/B, /D, /F and /G recognize and cleavespecifically VAMP, a synaptic vesicle associated membrane protein, at differentsingle peptide bonds [4,7–11]. BoNT/A and /E specifically recognize and cutSNAP-25 (synaptosomal-associated protein of 25 kDa) at two different peptidebonds near the COOH terminus [12,13], whereas BoNT/C cleaves syntaxin[14,15] and SNAP-25 [16–18]. Fig. 1 shows the decrease of size of SNAP-25caused by BoNT/A which removes nine residues from the C-terminal end of themolecule (upper panel) and the smaller size of the SNAP-25 fragment generatedby BoNT/E which removes 26 residues. The figure also shows that the peptidebonds cleaved by the two proteases are well conserved among species thusaccounting for the widespread toxicity of these neurotoxins. The fact that thesetoxins are neurospecific and that they are enzymes explains their extremetoxicity. In fact, as long as there is a single copy of toxin active inside thesynapse, the VAMP, or SNAP-25 or syntaxin molecules needed for neuroex-ocytosis, are clipped thereby loosing their functionality.

O. Rossetto et al. / Clinica Chimica Acta 291 (2000) 189 –199 191

Fig. 1. Proteolytic activity of BoNT/A and BoNT/E on SNAP25. Upper panel: 5 mg ofGST-SNAP25b fusion protein were treated with 100 nM BoNT/A or E for 2 h at 378C andsamples were electrophoresed and stained with Comassie Blue. Lower panel: comparison of theprimary structure of SNAP25 from different origins and sites of cleavage with botulinumneurotoxin type A and E.

BoNTs are very useful to human health. BoNT/A is presently the bestavailable treatment for a variety of dystonias and other diseases caused byhyperactivity of cholinergic terminals [19]. The toxin is injected at and aroundthe altered nerve terminals in such an amount as to cause a functional depressionof the release of acetylcholine. The resulting beneficial effects last for severalweeks in the case of the neuromuscular junction and several months in the caseof autonomic terminals. Recently, BoNT/C has been shown to be as effective asBoNT/A in human therapy [20].


3. The anthrax lethal factor

Anthrax is a disease of animals and humans, caused by toxigenic strains ofBacillus anthracis, which secrete three distinct proteins: protective antigen (PA),edema factor (EF) and lethal factor (LF) [21–24]. Both LF and EF bind to PA,which mediates their entry into the cytosol [25–28], where they display theircatalytic activities. EF is a calmodulin-dependent adenylate cyclase [29]. LF is azinc binding protein which contains the His-Glu-Xaa-Xaa-His zinc bindingmotif of zinc-endopeptidases, and was thus suggested to act in the cytosol via ametallo-proteinase activity [30–32]. The injection of the lethal toxin (PA 1 LF)causes the rapid death of laboratory animals [33]. In vitro, LF induces death ofmacrophages and macrophage cell lines via activation of the production ofcytokines and oxygen radicals [34–37].

A yeast two-hybrid screening of a HeLa c-DNA library was performed usingE687Aas a bait an LF allele mutated at the glutamic acid 687 (LF ) of the zinc

binding motif, because a mutation of this residue is known to abolish itsproteolytic activity in metallo-proteases, including clostridial neurotoxins[38,39]. The screen was performed with this mutant to overcome the possibilitythat, following LF proteolysis, putative preys may lose affinity for the bait.Using this approach we identified the MAPKK Mek2 as a LF interacting protein.Fig. 2 shows that incubation of LF with Mek2 causes a loss of immunostainingof an N-terminal epitope recognized by an N-terminal specific antibody. Theprecise site of cleavage was determined by isolation of the clipped Mek2fragment and Edman sequencing (lower panel of Fig. 2). Cleavage of mek2 isaccompanied by cell death in Raw 264.7 cells, a macrophage cell line. We arecurrently trying to determine which is the link between the biochemical, and thecellular activity of LF, which results in death of certain macrophage cell lines inculture.

4. The cytosolic activity of the VacA toxin of Helicobacter pylori

Clear evidence associate prolonged infection of the stomach mucosa withtoxigenic strains of Helicobacter pylori with the development of atrophicgastritis, gastroduodenal ulcers and stomach adenocarcinoma in humans [40–42]. A major virulence factor produced by H. pylori is a cytotoxin, termedVacA, which causes vacuolar degeneration of cells in vitro [43–45]. Oraladministration of purified VacA to mice is sufficient to induce degeneration anderosion of the gastric mucosa with some recruitment of inflammatory cells, twokey events in the process that eventually leads to gastric ulcers. H. pylori strainswith an altered VacA gene are non cytotoxic. These and other evidence implicateVacA in the pathogenesis of human gastroduodenal ulcers.


Fig. 2. LF peptidase activity on Mek2 in vivo. (a) Raw264.7 macrophage cell line was treatedwith PA 200 ng/ml and LF 200 ng/ml for different periods of time. Cell viability was tested withMTT test. In parallel treated cells were lysed and probed by Western blot with antibody against theN-terminal region of Mek2 (N-20). Panel b represents the LF cleavage site and its N-terminalposition in the Mek2 molecule.


VacA is released as a 95-kDa protein and can be cleaved by bacterialproteases into two fragments of 37 kDa (p37) and 58 kDa (p58), with no changeof biological activity [46,47]. p58 at low pH increases the permeability ofliposomes to monovalent cations [48]. This toxin shows the remarkable propertyof being activated by short exposures to very acidic pH values and of being ableto resist to pepsin at pH 2 for prolonged periods of time: conditions mimickingthe intragastric environment [49,50].

Within half an hour from the addition of VacA to cells in culture, vacuolesappear inside the cell. They emerge in the perinuclear area and then grow innumber and size up to several micrometers of diameter to occupy the entirecytosol. The process(es) leading to the formation of such gigantic membraneouscompartments are not known. Massive fusion of smaller compartments promoteddirectly or indirectly by the toxin may be involved, but videomicroscopyanalysis of toxin-treated cells did not reveal such a phenomenon (Montecucco etal., unpublished observations) Alternatively, VacA could inhibit directly orindirectly the fission/maturation of a cell vesicular / tubular /cysternal compart-ment. VacA-induced vacuoles contain fluid phase markers, they are acidic [51]and their membrane is highly enriched in rab7 [52], a small GTP-binding proteinpreviously shown to be associated with late endosomal compartments. Rabproteins are known to regulate extent and specificity of intracellular membranetraffic in eukaryotic cells [53–55]. These proteins are anchored to specificintracellular compartments by geranyl-geranylation at their C-terminal and cyclebetween GDP-bound and GTP-bound form [55], thus absolving a role ofregulators. It was shown that active rab7 is necessary for vacuole formation [56],but VacA does not act via a direct action on rab7. Vacuoles formation causesmis-targeting of acid hydrolases, which are released in the medium and decreasein the degradative capacity of the cell [57,58]. Recently, it was found that VacAform anion channels [59] and the presence of such channel in late endosomeswould increase the turnover of the vacuolar ATPase proton pump. This isbelieved to determine osmotic swelling because it would lead to an increaseduptake of membrane permeant amines, such as the ammonia released byhydrolysis of urea catalysed by the H. pylori urease.

Recently, we have shown that VacA can be expressed in the cytosol of HeLacells, where it causes the formation of vacuoles indistinguishable from thoseinduced by VacA endocytosed by cultured cells from the medium [60]. Takentogether, these results indicate that VacA is a toxin capable of binding to cellsand entering into the cytosol, where it express its toxic activity, as all A-B typetoxins do. These toxins consist of a B domain involved in cell surface bindingand in the cell entry of the catalytically active A protomer [61]. To map theregion of the toxin responsible for the cytosolic activity, we have progressivelyshortened the gene encoding for VacA in such a way that smaller and smallerfragments are produced in the cell cytosol of HeLa cells transfected with the


vacA gene constructs. Since in most di-chain A-B type toxins, the A chain isamino-terminal, we begun with progressive deletions at the C-terminus of VacAand then analysed the effect of N-terminal deletions. We found that more than250 residues could be removed from the C-terminus of the 95-kDa toxin withoutany loss of vacuolating activity. Moreover, deletion of additional 160 residues(VacA1–511) still leaves a toxin with considerable cytosolic activity. On thecontrary, the removal of only six residues from the N-terminus resulted in a highloss of vacuolating activity [62]. Therefore, it appears that the vacuolatingactivity of VacA is confined to the amino terminal portion of the polypeptidechain. The role of the residual amino terminal part of the p58, present in theconstruct 1–672, which is required for activity, is most likely that of assistingthe correct folding of the p37 domain. The amino terminal 24 residues longsegment has an overall hydrophobic character [46] and it could be involved in

Fig. 3. Inhibition of VacA vacuolating activity from serine-proteases inhibitors. HeLa cells wereincubated with 100 mM VacA pre-treated with following inhibitors: (A) 4-Cl-3-(4-F-CH Ph)2

isocoumarine, (B) 7-EtOCONH-4-Cl-3-PrO isocoumarine, (C) 7-PhCH CONH-4-Cl-3-2

(OCH CH Br) isocoumarine, (D) 7-PhNHCSNH-4-Cl-3-(OCH CH Br) isocoumarine, (E) 3-42 2 2 2

Cl isocoumarine, at final concentration of 600 mM. After 9 h, the extent of vacuolation was2

determined by neutral red uptake. Values are expressed as percentage of control represented bycells incubated with VacA alone and are the mean of two experiments run in duplicate. Barsrepresent standard deviations.


mediating the binding of the toxin to the cytosolic face of cell membranes or indirecting the toxin to a selected cytosolic substrate. A large portion of the58-kDa fragment is not necessary for the cytosolic activity of VacA, inagreement with the fact that p58 is membrane active and increases the

1permeability of liposomes to K (48), a property shared by the B protomer ofseveral A-B toxins. The ensemble of these data provides strong evidence thatVacA is an A-B type toxin.

The biochemical nature of the activity of p37 is still unknown. Some featurespresent in the primary sequence are reminiscent of serine proteases. We testedthe possibility that p37 is acting in the cytosol via such an enzymatic activity byusing a series of general inhibitors belonging to the isocumarine family of serineprotease inhibitors. Fig. 3 show that VacA pretreated with isocumarine in-hibitors, dialysed and then added to HeLa cells is inhibited to various extents bythe different inhibitors used. The most potent inhibitor is 3-4,dichloro-iso-cumarine which abolishes the vacuolating activity of the toxin. We are currentlytrying to identify the VacA residue(s) modified by the inhibitors and we cannotexclude the possibility that VacA is not a serine protease and that the inhibitorsis acting as an unspecific chemical modification agent. However, the results arein keeping with the possibility that VacA is a protease. If this were the case, itwould add to the list of bacterial protein toxins with intracellular proteaseactivity, which have been discussed here.

Acknowledgements

Work carried out in the author’s laboratory is supported by Telethon-Italiagrant no. 1068, by MURST 40% Project on Inflammation and by EC grantBiomed-2 BMH4

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Bacterial toxins with intracellular protease activity