Bacterial Toxin and Secretion Machine

YAO Yu-Feng
Laboratory of Bacterial Pathogenesis, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine Email: yfyao@sjtu.edu.cn

ShanghaiMicrobiology for graduate of Medicine Cellular Jiao Tong University School students

Part I

Bacterial Toxin

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Definition of Biotoxin
A biotoxin is a poisonous substance produced by living cells or organisms. Toxins are nearly always proteins that are capable of causing disease on contact or absorption with body tissues by interacting with biological macromolecules such as enzymes or cellular receptors. Toxins vary greatly in their severity, ranging from usually minor and acute (as in a bee sting) to almost immediately deadly (as in botulinum toxin). Biotoxins vary greatly in purpose and mechanism, and can be highly complex or relatively small 3 Shanghai Jiao Tong University School of Medicine protein.

For What?
Biotoxins in nature have two primary functions: Predation (spider, cobra, jellyfish, wasp) Defense (bee, poison dart frog)

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Bacterial Toxin
Bacterial toxins are effective and specific poisons produced by bacteria. They usually consist of an amino acid chain which can vary in molecular weight between a couple of hundred (peptides) and one hundred thousand (proteins).
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Alexandre Yersin (1863-1943)

Emile Roux (1853-1933)

[We now know that production of this toxin is stimulated when the bacteria themselves become infected by a virus. Once infected, diphtheria bacilli emit a powerful neurotoxin that is capable of paralyzing vital muscles like the heart and diaphragm.]
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Outline
Introduction Typical bacterial toxins Toxin structure Biological function Utilization for human being

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Classification
Protein toxin, the toxic lipopolysaccharide complexes Endotoxin and Exotoxin -Lipopolysaccharide &
lipooligosaccharide

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Protein Toxins
148 (46%) from Gram-positive bacteria 175 (54%) from Gram-negative bacteria Extracellular toxins: 75% Intracellular toxins : 25% Membrane damaging/pore-forming cytolysins: 110 (approx 35%)

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Toxin Producing Bacteria

Escherichia coli Vibrio cholera Staphylococcus aureus Yersinia pestis Streptococcus pyogenes Shigella dysenteriae Clostridium botulinum Bordetella pertussis Corynebacterium diphtheriae
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Bacterial Toxins: Methods and Protocols, 2000

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Bacterial Toxins: Methods and Protocols, 2000

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Bacterial Toxins: Methods and Protocols, 2000

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Bacterial Toxins: Methods and Protocols. 2000

Bacterial Toxins
LPS Alpha Hemolysin Cytotoxic Necrotizing Factor 1 (CNF1)

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Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS) is a large molecule consisting of a lipid and a polysaccharide joined by a covalent bond. LPS is a major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. LPS is an endotoxin and induces a strong response from normal animal immune systems. The only Grampositive bacteria that possesses LPS is Listeria monocytogenes. LPS binds the CD14/TLR4/MD2 receptor complex, which promotes the secretion of pro-inflammatory cytokines in macrophages.
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Structure of LPS

Schematic structure of lipopolysaccharide (LPS) from Brucella spp.

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LPS Synthesis
Lipid A is assembled in the cell membrane and the core sugars attached sequentially. O-antigen subunits are independently synthesized. The fully synthesized O-antigen is then attached to the lipid A-core (generating lipopolysaccharide) in the cell membrane before passage/insertion into the outer membrane.

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Roles in vivo

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LPS and Signal Transduction

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Lipopolysaccharide of leptospiral outer membrane activates macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans.
Nat Immunol. 2001, 2(4):346
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Hemolysin RTX toxins

RTX (Repeats in Toxin) toxins are important virulence factors produced by a wide range of Gram-negative bacteria. RTX toxins fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins. E. coli -hemolysin (HlyA) is the bestcharacterized RTX protein secreted by type I secretion system. In E. coli, hly operon is located either on chromosome-bound pathogenicity islands or on transmissible plasmids.
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RTX Cytolysins

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RTX Cytolysins Structure

GGXGXDXUX (U is L, I, U, W, Y or F)
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Int. J. Med. Microbiol. 2002, 292:149

Hemolysis Streptococcus

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Alpha Hemolysin
HlyA cause lysis of red blood cells in vitro and associated with pathogenicity. HlyB, belongs to the ATP binding cassette (ABC) superfamily of eukaryotic and prokaryotic protein transporters, provide energy for HlyA export. HlyC is required for HlyA activation. HlyD is a part of export channel.
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HlyA synthesis, maturation, and export

Microbiol Mol Biol Rev. 1998 , 62(2): 309

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HlyC recognition domain

Microbiol Mol Biol Rev. 1998 , 62(2): 309

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Staphylococcal -Hemolysin

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Science 274 (5294): 1859

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Model for HlyA Export

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Trends Microbiol. 2002, 10(1):39

Application of the Hemolysin Secretion System

Secretion of heterologous proteins Live bacterial vaccines based on the hemolysin secretion system

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Nat Biotech. 1996, 14(6):765

The recombinant protein bearing a C-terminal HlyA signal peptide binds the HlyB/HlyD complex
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Biotechnol Adv, 2005, 23(3):177

Live Bacterial Vaccines

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Trends Microbiol. 2002, 10(1):39

Live Vaccines Based on the Hemolysin Secretion System

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Trends Microbiol. 2002, 10(1):39

CNF1
Cytotoxic necrotizing factor 1 (CNF1) 108 kDa A-B protein toxin including 3 domains, no any known signal peptides identified Activating Rho-GTPases with deamidase function

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Three Domains of Cnf1

Toxicon. 2001, 39:1673

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Transcription of cnf1

FEMS Microbiol Rev. 2007, 31:515

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Structure of CNF1-C

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FEMS Microbiol Rev. 2007, 31:515

The Cnf1 and HBMEC

J Biol. Chem, 2002, 277(18):15607

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The cnf1 and Invasion

J Biol. Chem, 2002, 277(18):15607 43

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The Cnf1 and Apoptosis

Lane 2, untreated 5637 cells; 3, treated for 18 h with 100 nM staurosporine; 4, 100 nM staurosporine for 24 h; 5, 24 h with a 1:640 toxin dilution; 6, 48 h with a 1:640 toxin dilution; 7, 72 h with a 1:640 toxin dilution.

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Infect Immun, 2000, 68(10):5869

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Mechanism of CNF1

CNF1 modify glutamine 63 of Rho (61of Rac, Cdc42) by deamidation and thus inhibit GAP activity toward the GTPases which remain in their active state bound to GTP thus able to permanently activate their downstream effectors. GEF, guanine exchange factor; GAP, GTPase activating protein; SW1 switch 1 domain, SW2 switch 2 domain.
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Toxicon. 2001, 39:1673

CNF1 and cancer

FASEB J. 2006, 20(6):606-9

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Before

Botulinum toxin (BOTOX)

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After
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