Portals and Pathways: Principles ofBacterial Toxin Entry into Host CellsMany pathogenic bacteria generate potent toxins that breach themembrane barrier of host cells to access their intracellular targets

Steven R. Blanke

During infection, pathogenic microbesactively remodel host cells and tis-sues to create a more suitable nichefor withstanding the rigors of thehost environment. One of the most

effective remodeling strategies is the genera-tion of protein toxins that modulate importantfunctions of both immune and nonimmunecells. The central importance of toxins to thevirulence strategies of pathogens is most poi-gnantly illustrated in diseases such as diphtheriaor botulism, where essentially all of the symp-toms can be attributed to toxins acting upon theirhost cells.

Many of the most potent bacterial toxins actinside host cells (see table). Their site of action isperhaps not surprising, if one considers thatfrom the perspective of pathogenic microbes,the eukaryotic intracellular environment isa treasure trove of regulatory pathways andnetworks that are ripe for manipulation.The potency of intracellularly acting toxinsis derived, in part, from their mode of ac-tion; most are enzymes that catalyze thecovalent modification of specific moleculartargets. To be successful, however, intracel-lularly acting toxins must access their sub-strates inside target cells. This is no smallfeat, as the eukaryotic plasma membrane isa formidable gatekeeper that effectively re-stricts macromolecules such as toxins frompassing freely into cells. To overcome themembrane barrier, intracellularly actingtoxins are either injected directly into hostcells by pathogenic microbes or, alterna-tively, enter cells in a manner that is mi-crobe-independent (Fig. 1).

Overall Paradigms of

Toxin Entry into Cells

Some gram-negative pathogens inject their tox-ins into the cytosol of host cells through bacte-rial transport machines that function as macro-molecular syringes (Fig. 1). The syringes, whichresemble either bacterial flagella or conjugativepili, facilitate the direct passage of toxin effec-tors from bacterial donor cells into eukaryoticcells by processes referred to as Type III or TypeIV secretion mechanisms, respectively. TheType III and Type IV secretion machines do notallow the free exchange of proteins betweenprokaryotic and eukaryotic cells, but, instead,appear to selectively regulate which toxin effec-tors can pass through the syringe needle. Whilegram-positive bacteria do not appear to use

• Many bacterial pathogens generate toxins thatact inside host cells, as a mechanism for remod-eling the host environment.

• Pathogens directly inject toxins into host cellsor, alternatively, release AB toxins, which mustthen enter host cells independent of the bacte-rium.

• AB toxins are “hard-wired” with propertiesthat exploit cellular entry pathways, and thesetoxins co-opt host mechanisms for taking upmolecules from their surfaces.

• Several questions surrounding entry of AB tox-ins into host cells remain unresolved, includinghow B fragments help to move A fragments ofAB toxins across membranes and also how spe-cific toxins are routed into cells by differentmechanisms.

Steven R. Blanke isAssociate Professorin the Departmentof Microbiology andthe Institute forGenomic Biology atthe University ofIllinois, Urbana.sblanke@life.uiuc.edu

26 Y Microbe / Volume 1, Number 1, 2006

Type III and IV secretion mechanisms, somespecies apparently construct large poreswithin the plasma membrane of target cellsthat function as portals for direct effectordelivery.

The direct delivery of toxin effectors intohost cells has several implications for viru-lence. On one hand, direct injection is bothefficient and selective, as the intimate inter-action between microbes and host cellsguarantees that most or all of the toxineffectors generated by a microbe can ulti-mately access their substrates. On the otherhand, injection mechanisms limit the extentto which a pathogen can remodel the host,because a bacterium can directly affect onlya single target cell at any given time. Patho-genic microbes exert a broader sphere ofinfluence by releasing a bolus of toxin thatcan act upon many cells within a giventissue and/or diffuse away to modulate oneor more types of cells at multiple locationswithin the host. For example, the lethal andedema toxins of Bacillus anthracis dissemi-nate within the host bloodstream and actupon both immune and nonimmune cells. Inthis and similar cases, intracellularly acting,or AB, toxins are not injected into host cellsby the bacterium, but must facilitate theirown entry to access their targets (Fig. 1).

Principles of AB Toxin Entry into Cells

As the first step in cellular entry, AB toxins bindto one or more plasma membrane surface recep-tors (Fig. 1), which can be proteins, glycopro-teins, or glycolipids. Host cells lacking suchreceptors are generally resistant to intoxication,underscoring the importance of toxin-receptorcomplexes. Some receptor-bound toxins (e.g.,diphtheria toxin, Pseudomonas exotoxin A, andShiga toxin) are targeted for clathrin-dependentendocytosis, which is how eukaryotic cells takeup proteins and lipids from their surfaces. Othertoxins, including cholera and tetanus toxins,enter cells by one of several alternative “clath-rin-independent” endocytic pathways. Ultimately,AB toxins are trafficked to translocation portals,where they cross the membrane barrier into thecytosol. Collectively, these observations illus-trate an important principle about AB toxins—namely, to access their intracellular substrates,

they coopt existing mechanisms for taking up molec-ular cargo from the host cell surface.

However, there is a second important principle,which is that AB toxins are not simply delivered intocells as passive cargo, but, instead, must be “hard-wired” to engage and exploit cellular entry path-ways. AB toxins incorporate two discrete and essen-tial functional components that vary considerably intheir physical arrangement but are generally con-served in terms of function (Fig. 2). Thus, toxin “Afragments” are the active moiety that can modifyintracellular target molecules by one of several iden-tified enzymatic activities, including ADP-ribosyla-tion, UDP-glucosylation, or proteolysis. Meanwhile,the “B fragments” serve as delivery vehicles for theirA components by binding to plasma membrane sur-face receptors and facilitating translocation of theA components into the cytosol through available por-tals.

Actions of intracellularly acting toxins on target cells

Toxin Pathogen Activities

ExoS Pseudomonas aeruginosa ADP-ribosylation, GTPase activationExoT Pseudomonas aeruginosa ADP-ribosylation, GTPase activationAdenylate

cyclaseBordetella pertussis Adenylate cyclase, hemolysis

Cholera toxin Vibrio cholerae ADP-ribosylation, immunomodula-tory effects

Pertussis toxin Bordetella pertussis ADP-ribosylation, T cell mitogen,hemagglutination, signal trans-duction through inositol phos-phate

Shiga toxin Shigella dysenteriae Inhibition of protein synthesis, in-duction of cytokine expression

Toxin A Clostridium difficile Actin depolymerization, mitochon-drial alterations and apoptosis

Lethal toxin Bacillus anthracis Alterations in dendritic cell func-tion, apoptosis of various celltypes, Hypoxic tissue injury, re-pression of glucocorticoid recep-tor transactivation, lysis of mac-rophages

PMT Pasteurella multocida Cytotoxicity of various cell types,mitogenesis of various cell types,immunomodulatory effects

Aerolysin Aeromonas hydrophila Hemolysis, cell vacuolationAlpha toxin Staphylococcus aureus Hemolysis, apoptosisEnterotoxin Clostridium perfringens Cytotoxicity, loosening of tight

junctionsPneumolysin Streptococcus pneumonia Cytolysis, complement activationRTX toxin Vibrio cholerae Cell rounding, actin cross-linkingVacA Helicobacter pylori Alterations in late endosomes, al-

terations in mitochondrial mem-brane permeability, inhibition ofT-cell proliferation

Volume 1, Number 1, 2006 / Microbe Y 27

Toxins Exploit the Acidic Environment of

Endosomal Compartments

To transport their A fragments into the cytosol,some AB toxins, including diphtheria, anthrax,and the botulinum neurotoxins, exploit the dropin pH to between 5.0 and 6.0 as endocyticvesicles are trafficked from the plasma mem-brane into the cell (Fig. 3). Endosome acidifica-tion triggers profound structural changes inthese toxins, resulting in the insertion of B frag-ments into the membrane and the formation ofion-conducting channels. Partially unfolded Afragments are generally thought to use these Bfragment-derived channels as conduits into the

cytosol, although it has been challeng-ing to prove this model.

While toxin B fragments sense andrespond to the relatively low pH of hostendosomes, toxin A fragments alsoembody properties that promote trans-location. For instance, they must beable to reversibly unfold at pH 5–6,engage the translocation machinery,pass through the endosomal mem-brane, and then functionally refoldwithin the cytosol. Recently, host fac-tors have been implicated in this pro-cess, as suggested by the discovery thatin vitro translocation from endosomesrequires both a source of energy (ATP)and a host cell cytosolic translocationfactor (CTF) complex consisting ofchaperonin heat shock protein (Hsp) 90and thioredoxin reductase. Thus, bacte-rial toxins engage host-cell componentsthat are ordinarily involved in proteinfolding.

Toxins Exploit the Sec61

Retro-Translocon in the

Endoplasmic Reticulum

Members of a second group of AB tox-ins access cytosolic substrates by ex-ploiting membrane transport com-plexes located in the endoplasmicreticulum (ER) (Fig. 3). Upon enteringtheir target cells, these toxins exploitone of several “retrograde” traffickingpathways destined for the ER. Withinthe lumen of the ER, the A fragmentsare transported through an existing

membrane complex whose primary player is aprotein called Sec61. The Sec61 complex ordi-narily functions as a “retrotranslocon”—send-ing improperly folded proteins back into thecytosol to be degraded in a proteosome-depen-dent manner.

How is it then that some bacterial toxins cansuccessfully exploit a cellular system whose pri-mary function is to send misfolded proteins totheir destruction? The answer to this questionmay again derive from several intriguing prop-erties hardwired into the cargo itself. First, thecrystal structures of several of these A fragmentsreveal regions of poorly defined electron densi-ties, suggesting that they may engage the Sec61

F I G U R E 1

Intracellularly acting bacterial toxins access their substrates within host cells by one ofseveral mechanisms. Some gram-negative pathogens directly inject toxin effectorsthrough flagellar- or pilus-adapted transport machines into eukaryotic cells by Type III orIV secretion systems, respectively. Alternatively, bacteria release intracellular-actingtoxins, also called AB toxins, into the host environment where they act locally or diffuseto act distally to the site of colonization. AB toxins commonly exploit endocytic pathwaysthat eukaryotic cells use for importing proteins. Finally, Bordetella adenylate cyclasetoxins directly enter the cytosol from the plasma membrane.

28 Y Microbe / Volume 1, Number 1, 2006

retrotranslocon by masquerading as partiallymisfolded proteins.

How do transported toxin fragments escapeproteosome-mediated degradation? Proteinsdestined for degradation typically are posttrans-lationally decorated with ubiquitin on their ly-sine residues. However, toxin catalytic frag-ments transported to the cytosol through theSec61 retrotranslocon contain remarkably fewlysine residues, thereby escaping ubiquitination-mediated targeting to host proteosomes. Thesetoxins must have idiosyncratic properties allow-ing them to enter the cytosol through the ERretrotranslocon.

Applying the Lessons Learned from

Intracellularly Acting Bacterial Toxins

There has been an enormous outlay of intellec-tual and financial capital in the development ofsystems and vectors for the delivery of macro-molecular-based therapeutics into cells. How-ever, the pharmaceutical industry has beenlargely unable to emulate the success of patho-genic microbes in sending proteins into mamma-lian cells.

One of the most important lessons learnedfrom the study of AB toxins is that the intendedprotein cargo must not only possess desiredintracellular modulating activities, but mustalso be hard-wired with innate properties fordelivery. For example, toxin A fragments par-tially unfold to pass through either toxin-medi-

ated or Sec61 retrotranslocon-mediated trans-port channels. However, many potential proteincargos, including those that are stabilized withextensive disulfide linkages, may not be capableof being readily unfolded (and refolded) duringtranslocation. In addition, toxin A fragmentsthat pass through the Sec61 retrotransloconhave additional primary sequence requirements(e.g., lysine paucity within the primary struc-ture) that, again, may be too restrictive for mostproteins. Finally, despite the apparent success ofAB toxins in modulating host cells, there isconsiderable evidence that the translocationprocess is relatively inefficient, implying thatprotein delivery into cells may be restricted toenzymes, so that by catalytic turnover, the ef-fects of even a few successfully transported mol-ecules may be amplified throughout the cell.

Despite inherent difficulties in delivering ex-ogenous proteins into cells, there are successstories. By swapping the normal toxin receptor-binding domain with antibodies or portions ofantibodies targeting surface receptors, immuno-toxins have been selectively sent into malignantcells to kill them (Fig. 4). Moreover, the anthraxtoxin has been engineered to deliver T-cytotoxiclymphocyte-stimulating epitopes into cells forprocessing and MHC class I presentation thatstimulates a T-cell response with the capacity tobe protective.

Exciting new ideas are also emerging frommany areas of chemistry and bioengineeringthat may circumvent restrictions on protein de-

F I G U R E 2

The diverse architectures of AB intracellular acting toxins. AB intracellular-interacting toxins are classified according to their architectures.In general, the B fragments are responsible for transporting the catalytic A fragments into cells. For single chain AB toxins, includingdiphtheria toxin and Pseudomonas aeruginosa exotoxin A, the A and B fragments are contiguous, until separated by a host cell protease(furin) and reduction of a disulfide linkage. Among the AB5 toxins, including cholera toxin, the E. coli heat-labile toxins, pertussis toxin, andthe Shiga toxins, discrete genes encode the A and B fragments, which assemble as non-covalent complexes in which the A fragment restsupon a donut-shaped pentamer of B fragments. The A fragment may be cleaved into two polypeptides, with the catalytic A1 fragmentreleased from the shorter A2 fragment that extends down into the hole of the B-fragment-derived pentamer. For the binary toxins, includinganthrax toxins and the Clostridium perfringens iota toxin, discrete genes encode the A and B fragments, which are released as separatepolypeptides that interact at the surface of the host cell following activation of the B fragment. The tripartite AB toxins, represented by thecytolethal distending toxins, are encoded as three separate fragments, with two of them combining as the B fragment to deliver the singleA fragment into cells.

Volume 1, Number 1, 2006 / Microbe Y 29

livery. For example, “smart polymers” are beingtargeted to endosomes that, upon sensing thelowering of pH, interact with and destabilizeendosomal membranes to promote the con-trolled release of endosomal contents into thecytosol. Such a system would potentially in-crease the repertoire of molecules that could bedelivered into the cytosol by removing the con-straints of having to be translocated through themembrane. A striking feature is that many sys-tems under development exploit some of thesame innate properties of target cells used by AB

toxins, such as the capacity to target proteins forinternalization through existing endocyticmechanisms.

Future Prospects

Despite intensive study, many exciting issuessurrounding the entry of AB toxins into hostcells remain unresolved. The mechanisms un-derlying the “routing” of toxins into cells, in-cluding the extent to which receptor bindingdetermines both the trafficking patterns and ul-timate destination, may not be as clear-cut as

F I G U R E 3

Entry of AB toxins into target cells. Some toxins use host endocytic pathways that cells use ordinarily for degrading exogenous proteinswithin lysosomes. However, several AB toxins, including diphtheria, anthrax, and the botulinum neurotoxins, translocate their A fragmentsinto the cytosol by exploiting the acidic environment of early endosomes. Endosome acidification triggers conformational changes, withtoxins inserting their B fragments into the membrane and forming ion-conducting channels. Partially unfolded A fragments use thesetoxin-derived channels as conduits into the cytosol. Host factors are also involved. Other AB toxins exploit the degradation pathway formisfolded proteins. Nascent proteins destined for secretion are transported from the endoplasmic reticulum (ER), via the Golgi stack andthe trans-Golgi network (TGN) to secretory vesicles that fuse with the plasma membrane to release the proteins. The secretion pathway isat least partially reversible, enabling several bacterial toxins to travel this “retrograde” pathway from the plasma membrane to the ER lumenthrough pores to the cytosol. Several AB toxins, including cholera toxin, shiga toxin, and Pseudomonas aeruginosa exotoxin A, enter thecytosol through the ER complex whose primary protein is Sec61. The B fragments of these toxins bind to receptors to facilitate thetrafficking of catalytic A fragments to the ER. Toxin A fragments pass through the Sec61 retrotranslocon, but escape ubiquitination andproteosome-mediated degradation.

30 Y Microbe / Volume 1, Number 1, 2006

previously thought. Indeed, several toxins, in-cluding cholera toxin, have the capacity to entercells by several different mechanisms mediatedby the same receptor. The issue is further con-founded by the demonstration that some toxins,such as the vacuolating cytotoxin (VacA) fromHelicobacter pylori, exert differential effectsupon multiple cell types. Because VacA binds tomultiple components on the plasma membranesurface of a single cell type, an exciting possibilityis that it might engage alternative entry pathwaysto access multiple targets within the same cell.

The fate of toxin A fragments subsequent totheir translocation into the cytosol is also poorlyunderstood. While some catalytic domains maysimply diffuse to their intracellular substrates,signals for intracellular targeting may also behard-wired in some toxins, as recently shownfor the Pseudomonas aeruginosa Type III effec-tor ExoS and the catalytic fragment of the Clos-

tridium botulinum neurotoxin B. The details ofhow these targeting signals function, as well aswhether other toxins require additional mecha-nisms to locate their targets within the highlycompartmentalized intracellular milieu, are notunderstood.

Probably the least understood step in the en-try process of AB toxins remains membranetranslocation, especially the exact mechanism(s)by which B fragments facilitate the movement ofthe A fragment across the membrane.

Finally, it remains to be seen whether all theportals and pathways used by bacterial toxins toenter the cell have been identified. The discoveryof additional cellular entry pathways and mech-anisms would not only provide novel insightsinto eukaryotic cell biology, but also benefitongoing efforts to design protein-based thera-peutics that act upon intracellular moleculartargets.

ACKNOWLEDGMENTS

This article is based, in part, on a symposium at the 2004 National ASM Meeting in New Orleans, La.,, called “Portals andPathways: Entry of Virulence Factors into Host Cells.” The author acknowledges support by the National Institutes of Health,AI45928, AI53287, AI55883, AI57156, and AI59095.

F I G U R E 4

Efforts to develop macromolecule-based therapeutics based on attributes of bacterial toxins. Some investigators are seeking to exploitattributes of toxins in protein-based therapeutics. (A) Magic bullets—by linking toxin catalytic A fragments to the receptor binding domainsof eukaryotic proteins, the goal is to selectively deliver the catalytic domains of some toxins into malignant cells. (B) Molecular syringes—thegoal is to use toxin B fragments to deliver heterologous cargo into target cells.

Volume 1, Number 1, 2006 / Microbe Y 31

SUGGESTED READING

Ding, Z., K. Atmakuri, and P. J. Christie. 2003. The outs and ins of bacterial type IV secretion substrates. Trends Microbiol.11:527–535.Falnes, P. O., and K. Sandvig. 2000. Penetration of protein toxins into cells. Curr. Opin. Cell Biol. 12:407–413.Ghosh, P. 2004. Process of protein transport by the type III secretion system. Microbiol Mol. Biol. Rev. 68:771–795.Ladant, D., and A. Ullmann. 1999. Bordetella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol.7:172–176.Lencer, W. I., and B. Tsai. 2003. The intracellular voyage of cholera toxin: going retro. Trends Biochem. Sci. 28:639–645.Madden, J. C., N. Ruiz, and M. Caparon. 2001. Cytolysin-mediated translocation (CMT): a functional equivalent of type IIIsecretion in gram-positive bacteria. Cell 104:143–152.Montecucco, C., E. Papini, and G. Schiavo. 1994. Bacterial protein toxins penetrate cells via a four-step mechanism. FEBSLett. 346:92–98.Murthy, N., J. Campbell, N. Fausto, A. S. Hoffman, and P. S. Stayton. 2003. Bioinspired pH-responsive polymers for theintracellular delivery of biomolecular drugs.Bioconjug Chem. 14:412–419.Ratts, R., H. Zeng, E. A. Berg, C. Blue, M. E. McComb, C. E. Costello, J. C. vanderSpek, and J. R. Murphy. 2003. Cytosolicentry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex. J. Cell Biol. 160:1139–1150.

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