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By Glen D. Armstrong, PhD
To cause infections, microbial pathogens must first tightly adhere to hostcell surfaces. This allows pathogenic microbes to oppose cleansing action --such as the flushing of urine or tears, peristaltic movement of the gut ormucociliary function of respiratory epithelial cells -- that would otherwisequickly eliminate them from their host's body. Many pathogenic bacteria expressspecialized surface proteins, called adhesins, which mediate their binding tohost cells. Most, but not all, microbial adhesins are long filamentousstructures microbiologists call pili or fimbriae. On the host cell, the complexcarbohydrate sequences of glycoproteins or glycolipids represent receptors towhich many microbial pili are known to bind. It is this binding of pili tocomplex carbohydrate receptors that allows many microbes to successfullycolonize host cell surfaces. With few exceptions, microbial exotoxins bind tohost cell glycolipids, thus mediating exotoxin access to their host celltargets.
In theory, therefore, soluble carbohydrate receptor analogs mightcompetitively antagonize the pathophysiological effects of exotoxins or inhibitbacterial colonization of host cell surfaces.
The continuing rise in microbial resistance to antibiotic drugs is a majorconcern to all healthcare practitioners. It has stimulated infection controlprofessionals and medical microbiologists to seek new ways of preventing ortreating classical and emerging infectious diseases.
The merits of education and the enforcement of infection control measures areundisputed. In many cases, the simplest and most economical of measures,frequent handwashing for example, have been shown to have profound positiveeffects.1 Recent advances in vaccine development have contributedsignificantly to infection control. The application of immunization to aparticular problem is only practical in cases where a subject's immune system isfunctioning normally. In many cases, it is simply not practical to developvaccines for infectious diseases where the annual incidence would be less thanthe anticipated rate of adverse effects in a mass immunization program.Effective drugs will remain a last resort in cases where immunization does notrepresent a viable option or when other control measures fail.
The main problem associated with current antimicrobial drugs is the hugecapacity of microorganisms to become resistant to these drugs, therebyeventually rendering them ineffective. In the presence of such drugs, microbeswhich are unaffected by them soon become the predominant members of thepopulation. The acquisition of microbial resistance mechanisms requires thepharmaceutical industry to search for new derivatives to which microbialresistance has not developed. In most cases, this enterprise involves chemicallyaltering the structures of known drugs or creating completely synthetic agentswith suitable antimicrobial properties. Nonetheless, the principles of drugdiscovery have not changed and continue to involve the perpetual quest foragents designed to prevent the in-vivo replication of pathogenic microbes.Perhaps it is time to rethink the philosophy of antimicrobial drug design andinvestigate the feasibility of producing agents that will not createenvironments favoring the proliferation of genotypically resistant microbes.
Recent advances in the fields of bacterial genomics, proteomics and molecularbiology have allowed us the benefit of a much more thorough understanding ofpathogenic mechanisms and the intricate interplay between microbes and theirhosts.2 These studies reveal potential methods of interfering withthe mechanisms of the infection process in ways that may not promote theproliferation of microbes insensitive to the desired inhibitory effects of theseagents. Such novel agents should probably be referred to as anti-infectiverather than antimicrobial since they will be designed to inhibit the infectionprocess rather than the microbes themselves.
In virtually every example, the attachment of pathogenic microorganisms ortheir toxic exoproducts to host cells represents the first step in the processleading to a clinically apparent condition. My research is based on theobservation that many pathogenic microbes or their exotoxins attach to complexcarbohydrate sequences, also referred to as glycans, displayed on host cellsurfaces.3 These attachment processes are highly specific, usuallyinvolving a microbial protein which recognizes a specific host cell complexcarbohydrate sequence or sequences.4 It is conceivable that solublecarbohydrate receptor-based drugs designed to competitively inhibit thesemicrobial attachment processes may be therapeutically beneficial in cases whereconventional antibiotics are, for one reason or another, ineffective.5The rest of this article will elaborate on how we and others have been testingthis concept.
Enterohemorrhagic E. coli (EHEC) cause a condition called hemorrhagiccolitis (HC) in humans.6 Although several EHEC serotypes have beenidentified, E. coli O157:H7 serotype is isolated from the majority of HCcases in North America, Europe and Japan.7 Patients who develop EHEC-mediatedHC are also at risk of developing a potentially life-threatening post-infectioncomplication known as the hemolytic-uremic syndrome (HUS). This seriouscomplication of HC is directly linked to the production of potent EHECcytotoxins.8 These represent a family of cytotoxins which are relatedto the Shiga toxin (Stx) produced by Shigella dysenteriae Type 1. One ofthe EHEC cytotoxins, Stx1, is virtually identical to the Stx expressed by Shigelladysenteriae9-10 and was formerly called Shiga-like toxin I (SLT-I).This cytotoxin was also called Verotoxin 1 (VT 1) because its cytotoxic activitywas initially described in African green monkey (Vero) kidney cells.11The other EHEC cytotoxin, formerly called SLT-II or VT 2 but now referred to asStx2, is more distantly related to Stx1. In addition, several variant forms ofStx2 have been described in EHEC isolates. Regardless of their relationships,though, all the EHEC Shiga toxins share structural and functional similarities.8
The EHEC Shiga toxins are classical hexameric "A/B5" exotoxinsconsisting of one enzymatically active A subunit and 5 identical B subunits.12-13Although the cytotoxic action of EHEC Shiga toxins is expressed by their Asubunits in the cytoplasm of host cells, absent the B pentamer that binds toglycan receptors on the surface of the host cell,3 the A subunit isatoxic. The Stx B pentamer is critical to the intoxication process because itacts as a mediator for A subunit binding to and translocation into the host cellcytoplasm. Given its critical binding function, it seemed feasible that solubleB pentamer glycan receptor analogs might competitively inhibit Stx binding toand intoxication of host cells. The therapeutic benefits of such Stx receptoranalogs might be to prevent HUS developing in subjects suffering from HC.
Typically, protein-carbohydrate interactions are of the low affinity variety.14Unlike enzymes, which contain a deep cleft or tunnel into which thesubstrate tightly binds, the carbohydrate ligand binding domains of proteinssuch as the EHEC Shiga toxins usually occur as shallow depressions on thesurface of the protein. Much of the binding energy in carbohydrate-proteininteractions is derived from the displacement of disordered water molecules fromthe depression in the surface of the protein by the ordered hydroxyl groupsdisplayed by the glycan sequence.15 Nature compensates for the weakbinding by simply multiplying the number of equivalent ligand binding domains incarbohydrate-binding proteins. This allows these proteins to achieve tightbinding by simultaneously engaging multiple receptor sequences.
We have used the X-ray crystal structure of the Stx1 B pentamer, complexedwith soluble glycan receptor analogs,16 as a template for creating asymmetrical soluble inhibitor, we call Starfish, capable of simultaneouslyembracing the multiple domains found on the receptor binding face of thisprotein.17 The resulting Starfish analog displayed a solid phasebinding inhibition constant that was superior, by 10 million times, to that of amonovalent inhibitor capable of engaging only one of the multiple Stx1 Bpentamer receptor binding domains. In further co-incubation studies,17the polyvalent carbohydrate-based soluble inhibitor protected Vero cells, atconcentrations in the ?M range, from a lethal challenge dose of both Stx1 andStx2.
After we disclosed our multivalent inhibitor in the literature, a group ofJapanese investigators presented data demonstrating that ?M doses of apolyvalent inhibitor they called "Super Twig" protected mice fromlethal challenge doses of both Stx1 and Stx2 [(Nishikawa, K., et al. Developmentof a new type of Shiga toxin neutralizer, carbosilane dendrimer, whichcompletely rescues the lethality of Shiga toxin 2 in mice. 2000. Abstract 415,4th International Symposium and Workshop on Shiga toxin [Verocytotoxin],producing Escherichia coli infections, Kyoto, Japan).] In this report,the inhibitor, like the toxins, was administered intravenously and appeared tobe well tolerated by the mice. Since the Japanese report, we have obtainedcompelling evidence demonstrating the efficacy of symmetrical polyvalentinhibitors in protecting mice from the lethal effects of Stx118 andnow Stx2 (in preparation). The dosages used in our in vivo experiments wereequivalent to a few milligrams of inhibitor per Kg of body weight and so far,we, like the Japanese, have not observed any toxic effects of thesecarbohydrate-based inhibitors in mice.
Others19 have used an approach similar to ours to developeffective inhibitors for two additional bacterial exotoxins in the A/B5classification group; Cholera toxin and the Heat-labile (LT) toxins expressed byenterotoxigenic E. coli (ETEC), for example. It would seem that, at leastfor bacterial exotoxins that display symmetrical arrays of multiple ligand-bindingsites, it is generally feasible to produce carbohydrate-based soluble inhibitorsthat have exceedingly beneficial effects at pharmacological dosages. Theserecent findings bode well for potentially commercializing polyvalentcarbohydrate-based receptor analogs for treating diseases caused by EHEC, ETEC,and, possibly, Vibrio cholerae.
Not all bacterial carbohydrate-binding proteins display the same five-foldrotational symmetry as the A/B5 family of exotoxins. Some bacterial exotoxins,those expressed by Clostridium difficile, the organisms responsible forantibiotic-associated colitis in hospitalized patients or residents ofextended-care facilities, for example, contain a linear array of multiplecarbohydrate-binding domains.20 Filamentous bacterial adhesions mayalso display linear arrays of multiple carbohydrate-binding domains.21Alternately, the pili or fimbriae expressed by some bacterial pathogens maydisplay carbohydrate-binding domains only at their distal tips. In these"tip domain" situations, polyvalency may be achieved not only by thesymmetrical arrangement of multiple carbohydrate binding sites at the tips ofthe individual pili but also by expressing multiple pili per organism.21
The situation presents an even greater challenge to developing effectiveinhibitor analogs because the flexibility of these filaments contributes to arelative lack of symmetry in these systems. Once an organism has attached itselfto a host cell, it may be impossible for a soluble inhibitor to reverse thisprocess. We22, however, have demonstrated that a syntheticglycoconjugate consisting of bovine serum albumin (BSA) containing covalentlybound N-acetyllacosaminyl glycoside sequences inhibited bundle-formingpili (BFP) -- mediated attachment of enteropathogenic E. coli (EPEC) tocultured human epithelial cells. This inhibitory activity appeared to depend onthe average number, i.e., potential valency, of N-acetyllacosaminylglycosides attached to each BSA molecule and was specific for this particularcarbohydrate sequence. In EPEC, at least, the relative absence of a symmetricalarrangement of the BFP-localized carbohydrate-binding domains did not hinder theinhibitory activity of a BSA glycoconjugate containing multiple glycan ligands.
Further investigation into the EPEC host cell binding inhibition mechanism ofBSA glycoconjugates revealed the process was more complex than we originallybelieved. These studies demonstrated that inhibition likely involved twomechanisms, one involving simple competitive inhibition, and the other involvinga time-dependent glycoconjugate-mediated loss of BFP from the surface of thebacteria. At a glance, this would appear to be counterintuitive to the role ofBFP in EPEC attachment to host cells. However, EPEC colonize host cells by acomplex multi-step process. BFP-mediated attachment represents only the firststep in EPEC colonization of host cells. Once this first step has successfullyoccurred, the continuing presence of BFP on the surface of EPEC may interferewith the subsequent colonization steps. We are speculating, therefore, that EPECBFP binding to specific carbohydrate receptors may produce an as yet unknownsignal informing the bacterium that it has made initial contact with anaccommodating host cell and may now discard its BFP in preparation for the nextphase of colonization.
Once EPEC have colonized a specific site in the host, it may be necessary forsome organisms to be released from this primary colonization site. In theabsence of any host response, these released organisms would then be free tocolonize additional, perhaps downstream host sites, thereby perpetuating theinfection process. Once they have been released from the influence of their hostcell glycan receptors, these planktonic organisms may once again produce BFPthat would enable them to colonize the new sites in the host. It is during thissecondary phase of the infection process, the colonization of additional hostsites, that the administration of a multivalent carbohydrate receptor analog maybe of benefit in a subject suffering from EPEC-mediated diarrhea.
Others have also noted that EPEC alter the expression of their adhesins inresponse to contacting host cell surfaces and there is some speculation thatglycan receptors may be generally involved in these sensing reactions.23Many pathogenic microbes, including EPEC, possess the ability to assemble acomplex multimeric organelle, called a Type III secretory apparatus, when theycontact host cell surfaces.6 This Type III secretory apparatus isnecessary for injecting virulence factors into the cytoplasm of host cells.These factors cause host cells to modify their physiological behavior conduciveto the pathogenic process. In light of what is known about the possible role ofglycan receptors in regulating adhesin expression in EPEC, it is possible thatspecific host cell surface carbohydrate sequences may also influence thefunction of microbial Type III secretory systems. Should this assumption proveto be valid, soluble carbohydrate receptor analogs may also eventually be usefulfor disrupting the function of Type III secretory systems. Such analogs might,for example, trigger Type III-mediated secretion of virulence factors fromplanktonic bacteria, thereby causing these factors to disperse harmlessly to theexterior of the host cells.
It is unlikely microorganisms will rapidly develop resistance tocarbohydrate-based anti-infective drugs because this would involve alteringtheir ability to bind such agents. Organisms that alter their ability to bind tosoluble host cell carbohydrate receptor analogs would not gain an advantage overtheir wild-type siblings because these mutants would also lose their ability tobind or respond properly to host cells. Alternatively, microorganisms mayacquire the capacity to express adhesins with a specificity for different hostcell receptors. However, although it is quite possible for microbes to acquirethe genetic capacity to express complex multi-protein virulence factors such aspili or Type III secretory systems, en block6, this mayrequire a sustained environment which highly favors organisms displaying the newphenotype. It is unlikely the use of carbohydrate-based anti-infective drugsonly during the acute phase of an infection would create such a sustainedenvironment. To be of any use to the microbes, the newly acquired adhesins wouldalso have to display a specificity for other unique carbohydrate sequencesalready present on host cells.
Glen D. Armstrong is a professor in the Department of Medical Microbiologyand Immunology at the University of Alberta, Edmonton, AB, Canada.