**7. References**

128 Biochemistry

**OMV vaccines**: Balsalobre *et al* (2006) demonstrated that physiologically active HlyA is associated with the OMVs produced from *E. coli* laboratory strains and also from natural and clinical isolates. In our laboratory, we found that the unacylated toxin (proHlyA) can also be associated with OMVs (Herlax *et al.*, 2010). On the basis of this finding, OMV vaccines can be designed by effecting a fusion of the desired antigen with ProHlyA. In this way, ProHlyA would direct the exposure of the antigen on the surface of the OMVs without inducing any cytotoxic response. An advantage of OMV vaccines is that, because of their size and lipopolysaccharide content, they are able to induce an adequate immune response. Finally, mention must be made that these hypotheses are just speculative on the basis of what is known about the structure and function of HlyA, whose application in toxin-based therapy still has to be exhaustively investigated and especially the immune response the

*E. coli* is one of the predominant species of facultative anaerobes in the human gut and in the majority of the cases is harmless to the host. These strains are mostly commensals but also contain a group called the extraintestinal pathogenic *E. coli* (ExPEC). Usually the ExPEC are also harmless colonizers but under certain circumstances can translocate and cause infection. The main virulence factor responsible for this translocation is the HlyA toxin, which pathogen is mainly associated with severe UTI but in addition with bacteremia and extraintestinal infections. In this chapter an exhaustive description of the toxin has been delineated; including its synthesis, maturation, and export from the bacteria. Effects produced by HlyA in different target organs have also been discussed. The significance of the maturation process for the toxin cannot be understated. The acylation of the protein at two internal lysines gives the toxin its virulence, by exposing intrinsic disordered regions that are essential to different steps of the toxin's mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsible for the permeability induced in all the target cells, despite the intracellular signal pathway the toxin induces in each specific

Based on the already known structural and functional characteristics of HlyA, we might speculate about its use in toxin-based therapy. Such therapy is a versatile and dynamic research area with a great potential application. Further investigation, however, is required in order to improve the efficiency and safety of toxin-based agents. Investments in the development of delivery and targeting techniques are definitely needed in order to achieve this goal, though the basic research on the structure and mechanism of natural toxins should nevertheless not be abandoned. Topics related to HlyA have still to be clarified concerning the existence of a toxin-specific receptor in target cells and the domains of the toxin involved in its interaction with those putative binding sites. The deeper our knowledge becomes about this unique family of secreted polypeptides, the more easily will we be able to harness

We thank Prof. Norma Tedesco for revising the English grammar; Mario Ramos for the graphic designs; and Dr. Donald F. Haggerty, a retired career biochemist and native English

organ. This activation is unique to prokaryotic proteins.

speaker, for editing the final version of the manuscript.

their great potential for our own benefit.

**6. Acknowledgements** 

toxin might evoke.

**5. Conclusion** 


Bauman, S. J. and M. J. Kuehn (2006). "Purification of outer membrane vesicles from

Bernardes, N., R. Seruca, A. Chakrabarty and A. M. Fialho (2010). "Microbial-based therapy of cancer: Current progress and future prospects" *Bioengineered Bugs* 1(3): 178-190 Beveridge, T. J. (1999). "Structures of Gram-negative cell walls and their derived membrane

Biggers, K. and N. Scheinfeld (2008). "VB4-845, a conjugated recombinant antibody and

Boehm, D., R. Welch and I. Snyder (1990). "Domains of *Escherichia coli* hemolysin (HlyA)

Bours, P. H., R. Polak, A. I. Hoepelman, E. Delgado, A. Jarquin and A. J. Matute (2010). "

Brown, D. A. and E. London (1998). "Structure and origin of ordered lipid domains in

Butcher, E. C. and L. J. Picker (1996). "Lymphocyte homing and homeostasis." *Science* 272:

Cavalieri, S., G. A. Bohach and I. Synder (1984). "*Escherichia coli* \_-hemolysin characteristics

Cavalieri, S. J. and I. S. Snyder (1982). "Effect of *Escherichia coli* alpha-hemolysin on human

Chen, M., R. Tofighi, W. Bao, O. Aspevall, T. Jahnukainen, L. E. Gustafsson, S. Ceccatelli and

Chena, D. J., N. Osterriederb, S. M. Metzgerb, E. Bucklesd, A. M. Doodye, M. P. DeLisaa and

Cheng, E., L. Cardenas-Freytag and J. D. Clements (1999). "The role of cAMP in mucosal adjuvanticity of *Escherichia coli* heat-labile enterotoxin (LT)." *Vaccine* 18: 38–49. Cherukuri, A., T. Shoham, H. W. Sohn, S. Levy, S. Brooks, R. Carter and S. K. Pierce (2004).

Chow, M., C. J. Der and J. E. Buss (1992). "Structure and biological effects of lipid

Claassen, I., J. Meylis, P. van der Ley, C. Peeters, H. Brons, J. Robert, D. Borsboom, A. van

membrane protein containing vesicle vaccine." *Vaccine* 14: 1001–1008. Copland, M. J., T. Rades, N. M. Davies and M. A. Baird (2005). "Lipid based particulate formulations for the delivery of antigen." *Immunol Cell Biol* 83: 97–105

modifications on proteins." *Curr Opin Cell Biol* 4(4): 629-36

G. Celsi (2006). "Carbon monoxide prevents apoptosis induced by uropathogenic

D. Putnam (2010). "Delivery of foreign antigens by engineered outer membrane

"The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts." *J. Immunol.* 172: 370–

der Ark, I. van Straaten, P. Roholl, B. Kuipers and J. Poolman (1996). "Production, characterization and control of a *Neisseria meningitidis* hexavalent class 1 outer

and probable role in pathogenicity." *Microbiol. Rev* 48: 326-343

peripheral leukocyte viability in vitro." *Infect Immun* 36(2): 455-61

2400–2408.

176-186

1959-1964

60-66.

380

vesicles." *J .Bacteriol* 181: 4725-4733

*Int. J. Infect. Dis.* 4: e770–e774

biological membranes." *J Membr Biol* 164: 103-14

*Escherichia coli* toxins." *Pediatr Nephrol* 21(3): 382-9

vesicle vaccines." *PNAS* 107( 7): 3099–3104

*Pseudomonas aeruginosa* and their activation of an IL-8 response." *Microbes Infect* 8:

immunotoxin for head and neck cancer and bladder cancer." *Curr Opin Mol Ther* 10:

involved in binding of calcium and erythrocyte membranes." *Infect Immunity* 58(6):

Increasing resistance in community-acquired urinary tract infections in Latin America, five years after the implementation of national therapeutic guidelines."


Forster, T. (1959). "Transfer mechanisms of electronic excitation." *Discuss. Faraday Soc.* 27: 7-

Foxman, B. (2002.). "Epidemiology of urinary tract infections: incidence, morbidity, and

Foxman, B. and P. Brown (2003). "Epidemiology of urinary tract infections: transmission and risk factors, incidence, and costs." *Infect Dis Clin North Am* 17(2): 227-41 Foxman, B., S. D. Manning, P. Tallman, R. Bauer, L. Zhang, J. S. Koopman, B. Gillespie, J. D.

Fragoso, R., D. Ren, X. Zhang, M. W. Su, S. J. Burakoff and Y. J. Jin (2003). "Lipid raft

Gentschev, I., G. Dietrich and W. Goebel (2002). "The *E. coli* \_-hemolysin secretion system

Gentschev, I., H. Mollenkopf, Z. Sokolovic, J. Hess, S. H. E. Kaufmann and W. Goebel (1996).

Goebel, W. and J. Hedgpeth (1982). "Cloning and functional characterization of the plasmidencoded hemolysin determinant of Escherichia coli." *J Bacteriol* 151(3): 1290-8 Goluszko, P., E. Goluszko, B. Nowicki, S. Nowicki, V. Popov and e. al. (2005). "Vaccination

Griebling, T. L. (2005). "Urologic diseases in America project: trends in resource use for

Guyer, D. M., S. Radulovic, F. E. Jones and H. L. Mobley (2002). "Sat, the secreted

Hackett, M., C. Walker, L. Guo, M. C. Gray, S. V. Cuyk, U. Ullmann, J. Shabanowitz, D. F.

Hall, P. D., G. Virella, T. Willoughby, D. H. Atchley, R. J. Kreitman and A. E. Frankel (2001).

Hamon, M. A., E. Batsche, B. Regnault, T. N. Tham, S. Seveau, C. Muchardt and P. Cossart

for bladder and kidney epithelial cells." *Infect Immun* 70(8): 4539-46

Freytag, L. C. and J. D. Clements (2005). "Mucosal adjuvants." *Vaccine.* 23: 1804-1813.

and its use in vaccine development." *Trends Microbiol.* 10(1): 39-45

Sobel and C. F. Marrs ( 2002). "Uropathogenic *Escherichia coli* are more likely than commensal *E. coli* to be shared between heterosexual sex partners." *Am. J. Epidemiol.*

distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to

"Development of antigen-delivery systems, based on the *Escherichia coli* haemolysin

with purified Dr Fimbriae reduces mortality associated with chronic urinary tract infection due to *Escherichia coli* bearing Dr adhesin." *Infect Immun* 73: 627–631. Griebling, T. L. (2005). "Urologic diseases in america project: trends in resource use for

autotransporter toxin of uropathogenic *Escherichia coli*, is a vacuolating cytotoxin

Hunt, E. L. Hewlett and P. Sebo (1995). "Hemolytic, but not cell-invasive activity of adenylate cyclase toxin is selectively affected by differential fatty acylation in

"Antibody response to DT-GM, a novel fusion toxin consisting of truncated diphtheria toxin (DT) linked to human granulocyte-macrophage colonystimulating factor (GM), during a phase I trial of patients with relapsed or refractory acute

(2007). "Histone modifications induced by a family of bacterial toxins." *Proc Natl* 

economic costs." *Am. J. Med.* 113: 5S–13S

enhance CD3 signaling." *J. Immunol.* 170: 913–921

urinary tract infections in men." *J Urol* 173: 1288–1294.

*Escherichia coli*." *J Biol Chem* 270: 20250-20253

myeloid leukemia." *Clin Immunol* 100: 191–197.

*Acad Sci U S A* 104(33): 13467-72

urinary tract infections in women." *J Urol* 173: 1281–1287.

secretion pathway." *Genes* 179: 133-140

17

156: 1133–1140.


Kiyono, H., J. Bienenstock, J. R. McGhee and P. B. Ernst (1992). "The mucosal immune

Koronakis, E., C. Hughes, I. Milisav and V. Koronakis (1995). "Protein exporter function and

Koronakis, V., C. Hughes and E. Koronakis (1991). "Energetically distinct early and late

Koronakis, V., E. Koronakis and C. Hughes (1989). "Isolation and analysis of the C-terminal

Koronakis, V., J. Li, E. Koronakis and K. Stauffer (1997). "Structure of TolC, the outer

Koronakis, V., A. Sharff, E. Koronakis, B. Luisi and C. Hughes (2000). "Crystal structure of

Koschinski, A., H. Repp, H. Unver, F. Dreyer, D. Brockmeier, A. Valeva, S. Bhakdi and I.

Kreitman, R. J., D. R. Squires, M. Stetler-Stevenson, P. Noel, D. J. FitzGerald, W. H. Wilson

(BL22) in patients with B-cell malignancies." *J Clin Oncol* 23: 6719-6729. Kreitman, R. J., W. H. Wilson, K. Bergeron, M. Raggio, M. Stetler-Stevenson and D. J.

chemotherapy-resistant hairy-cell leukemia." *N Engl J Med* 345: 241-247 Kuehn, M. J. and N. C. Kesty (2005). "Bacterial outer membrane vesicles and host-pathogen

Lakowicz, J. R., I. Gryczynski, W. Wiczk, G. Laczko, F. C. Prendergast and M. L. Johnson

Lally, E. T., I. R. Kieba, A. Sato, C. L. Green, J. Rosenbloom, J. Korostoff, J. F. Wang, B. J.

Langston, K. G., L. M. Worsham, L. Earls and M. L. Ernst-Fonberg (2004). "Activation of

Lee, C. K. (2001). "Vaccination against Helicobacter pylori in non-human primate models

Lee, C. K., R. Weltzin, W. D. Thomas and et. al. (1995). "Oral immunization with

immunization and vaccine development." *Reg Immunol* 4: 54-62.

*Mol.Microbiol* 16: 87-96

*EMBO J.* 10: 3263-3272

membranes." *EMBO J* 8(2): 595-605

export." *Nature* 405: 914-919

36: 99-115

272(48): 30463-9

*Biochemistry* 43(14): 4338-46

and humans." *Scand J Immunol* 53: 437–442.

dimensional crystals." *Mol Microbiol.* 23: 617-626

interaction." *Genes and Development* 19: 2645-2655

mammalian cells–the pore is on its own." *FASEB J* E80-E87

system: features of inductive and effector sites to consider in mucosal

*in vivo* ATPase activity are correleted in ABC-domain mutants of HlyB."

stages of HlyB/HlyD-dependent secretion across both *Escherichia coli* membranes."

signal directing export of *Escherichia coli* hemolysin protein across both bacterial

membrane component of the bacterial type I efflux system, derived from two-

the bacterial membrane protein TolC central to multidrug efflux and protein

Walev (2006). " Why Escherichia coli \_-hemolysin induces calcium oscillations in

and et. al (2005). " Phase I trial of recombinant immunotoxin RFB4 (dsFv)-PE38

FitzGerald (2001). "Efficacy of the anti-CD22 recombinant immunotoxin BL22 in

(1990). "Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer." *Biophys. Chem.*

Shenker, S. Ortlepp, M. K. Robinson and P. C. Billings (1997). "RTX toxins recognize a beta2 integrin on the surface of human target cells." *J Biol Chem.*

hemolysin toxin: relationship between two internal protein sites of acylation."

recombinant *Helicobacter pylori* urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis." *J Infect Dis* 172: 161–171.


Moolten, F., S. Zajdel and S. Cooperband (1976). "Immunotherapy of experimental animal

Moolten, F. L. and S. R. Cooperband (1970). "Selective destruction of target cells by

Morein, B., B. Sundquist, S. Höglund, K. Dalsgaard and A. Osterhaus (1984). " Iscom, a novel

Morova, J., R. Osicka, J. Masin and P. Sebo (2008). "RTX cytotoxins recognize beta2 integrin

Muller, D., C. Hughes and W. Goebel (1983). "Relationship between plasmid and chromosomal hemolysin determinants of *Escherichia coli*." *J .Bacteriol.* 153: 846-851 Murphy, J. R. (1996). *Corynebacterium Diphtheriae.* University of Texas Medical Branch at

Nicaud, J. M., N. Mackman, L. Gray and I. B. Holland (1986). "The C-terminal, 23 kDa

polysaccharide and DNA transfer determinants." *Mol.Microbiol* 19: 705-714 O'Hanley, P., G. Lalonde and G. Ji (1991). "Alpha-hemolysin contributes to the pathogenicity

Oster, P., D. Lennon, J. O'Hallahan, K. Mulholland, S. Reid and D. Martin (2005). "MeNZB:

Ostolaza, H., B. Bartolome, I. Ortiz de Zarate, F. de la Cruz and F. M. Goñi (1993). "Release

Ostolaza, H. and F. M. Goñi (1995). "Interaction of the bacterial protein toxin alpha-

Parsons, M., B. Vojnovic and S. Ameer-Beg (2004). "Imaging proteinprotein interactions in

Pastan, I., R. Hassan, D. J. FitzGerald and R. J. Kreitman (2007). "Immunotoxin treatment of

Pellett, S., D. Boehm, I. Snyder, G. Rowe and R. Welch (1990). "Characterization of

pyelonephritis." *Infect Immun* 59 1153–1161.

*Acad. Sci.* 277: 690–699.

*Science* 169: 68–70

*Nature* 308: 457–460

*Biol.* 158(2): 137-145

*Trans.* 32: 431-433

58(3): 822-827

*Biophys Acta* 1147(1): 81-88

activity." *FEBS Lett.* 371(3): 303-306

cancer." *Annu.Rev. Med.* 58: 221–237.

Galveston.

tumors with antitumor antibodies conjugated to diphtheria toxin or ricin." *Ann. NY* 

diphtheria toxin conjugated to antibody directed against antigens on the cells."

structure for antigenic presentation of membrane proteins from enveloped viruses."

receptors through N-linked oligosaccharides." *P Natl Acad Sci USA* 105: 5355–5360.

peptide of *E. coli* haemolysin 2001 contains all the information necessary for its secretion by the haemolysin (Hly) export machinery." *FEBS Lett* 204(2): 331-5 Nieto, J., C. Hughes, M. Bailey and V. Koronakis (1996). "Suppression of transcription

polarity in the *E.coli* hemolysin operon by a short upstream element shared by

of piliated digalactoside-binding *Escherichia coli* in the kidney: efficacy of an alphahemolysin vaccine in preventing renal injury in the BALB/c mouse model of

A safe and highly immunogenic tailor-made vaccine against the New Zealand *Neisseria meningitidis* serogroup B disease epidemic strain." *Vaccine* 23: 2191–2196. Ostolaza, H., L. Bakas and F. Goñi (1997). "Balance of electrostatic and hydrophobic

interactions in the lysis of model membranes by *E. coli* alpha-haemolysin." *J Membr* 

of lipid vesicle contents by the bacterial protein toxin alpha-haemolysin." *Biochim* 

haemolysin with model membranes: protein binding does not always lead to lytic

cell motility using fluorescence resonance energy transfer (FRET)." *Biochem. Soc.* 

monoclonal anibodies against the *Escherichia coli* hemolysin." *Infection and immunity*


Soloaga, A., H. Ostolaza, F. Goñi and F. De la Cruz (1996). "Purification of *Escherichia coli*

Soloaga, A., P. Veiga, L. García Segura, H. Ostolaza, R. Brasseur and G. F (1999). "Insertion

Stanley, P., V. Koronakis and C. Hughes (1991). "Mutational analysis supports a role for

Stanley, P., V. Koronakis and C. Hughes (1998). "Acylation of *Escherichia coli* hemolysin: A

Stanley, P., L. Packman, V. Koronakis and C. Hughes (1994). "Fatty acylation of two internal

Thanabalu, T., E. Koronakis, C. Hughes and V. Koronakis (1998). "Substrate-induced

Tran Van Nhieu, G., C. Clair, G. Grompone and P. Sansonetti (2004). "Calcium signalling during cell interaction with bacterial pathogens." *Biology of the cell* 96: 93-101 Trent, M. S., L. M. Worsham and M. L. Ernst-Fonberg (1998). "The biochemistry of

Troeger, H., J. F. Richter, L. Beutin, D. Gunzel, U. Dobrindt, H. J. Epple, A. H. Gitter, M.

Tumpey, T. M., M. Renshaw, J. D. Clements and J. M. Katz (2001). "Mucosal delivery of

Ulmer, J. B., U. Valley and R. Rappuoli (2006). "Vaccine manufacturing: Challenges and

Valeva, A., I. Walev, H. Kemmer, S. Weis, I. Siegel, F. Boukhallouk, T. Wassenaar, T.

protein:prediction and experiment." *Molecular Microbiology* 31: 1013-1024 Stanley, P., V. Koronakis, K. Hardie and C. Hughes (1996). "Independent interaction of the

*Eur. J. Biochem.* 238: 418-422

HlyA." *Mol Microbiol* 20(4): 813-22

haemolysin." *Mol Microbiol* 5(10): 2391-403

*Molecular Biology Reviews* 62: 309-333

*EMBO Journal* 17(22): 6487-6496

acyltransferase." *Biochemistry* 37(13): 4644-52

translocation." *Cell Microbiol* 9(10): 2530-40

solutions." *Nat Biotechnol* 24: 1377–1383

266: 1992-1996

694–697

36663

pro-haemolysin, and a comparison with the properties of mature \_-haemolysin."

of *Escherichia coli* \_-haemolysin in lipid bilayer as a non-transmembrane integral

acyltransferase HlyC with two maturation domains of the *Escherichia coli* toxin

multiple structural features in the C-terminal secretion signal of *Escherichia coli* 

unique protein lipidation mechanism underlying toxin fuction." *Microbiology and* 

lysine residues required for the toxic activity of *Escherichia coli* hemolysin." *Science*

assembly of a contiguous channel for protein export from *E.coli* : reversible bridging of an inner-membrane translocase to an outer membrane exit pore." *The* 

hemolysin toxin activation: characterization of HlyC, an internal protein

Zeitz, M. Fromm and J. D. Schulzke (2007). "*Escherichia coli* alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial

inactivated influenza vaccine induces B-cell-dependent heterosubtypic crossprotection against lethal influenza A H5N1 virus infection." *J Virol* 75: 5141–5150 Uhlen, P., A. Laestadius, T. Jahnukainen, T. Soderblom, F. Backhed, G. Celsi, H. Brisman, S.

Normark, A. Aperia and A. Richter- Dahlfors (2000). "Alpha-haemolysin of uropathogenic *E. coli* induces Ca+2 oscillations in renal epithelial cells." *Nature* 405:

Chavakis and S. Bhakdi (2005). "Binding of *Escherichia coli* Hemolysin and Activation of the Target Cells is Not Receptor-dependent." *J Biol Chem* 280: 36657-

