Bacterial protein toxins

1. BACTERIAL PROTEIN TOXINS
Shams-ud-Duha Syed
Mphil 1st
Advances in microbiology
Instructor: Dr. Qismat Shakeela

2. TOXINS & TOXIGENESIS:
“Toxins are the special class of bacterial macromolecular substances that when produced during
natural or experimental infection of the host or introduced parenterally, orally (bacterial food
poisoning), or by any other route in the organism results in the impairment of physiological functions
or in overt damage to tissues.”
“Toxigenesis is the ability to produce toxins, is an underlying mechanism by which many bacterial
pathogens produce disease.”

3. TYPES OF BACTERIAL TOXINS:
Bacterial toxins are differentiated into two major classes on the basis of their
chemical nature, regardless of their cellular location and the staining features of
the bacteria that produce them:
Bacterial Protein Toxins
Toxic lipopolysaccharide Complexes

4. EXOTOXIN VERSUS ENDOTOXIN

5. 2.2. STRUCTURAL AND GENETIC ASPECTS

6. 2.2.1. MOLECULAR TOPOLOGY
• A striking feature of bacterial protein toxins is the broad variety of molecular size
and topological features in contrast to the more homogeneous structures of
protein effectors of eukaryotic origin (e.g., hormones, neuropeptides, cytokines,
growth factors).
1.Single-Chain Molecules Oligomeric Molecules
Macromolecular Complexes of Toxins Associated to Non-Toxic Moieties
Protoxin Forms Three Dimensional Crystal Structure

7. 1. SINGLE-CHAIN MOLECULES
• Most protein toxins occur as single-chain holoproteins varying in size.
• Common single-chain protein toxins are enlisted in the table.
Protein toxin Bacteria
1. Thermostable enterotoxin E. coli
2. Tetanus neurotoxin Clostridium tetani
3. Botulinum neurotoxin Clostridium botulinum
4. Toxin A & B Clostridium deficile

8. 2. OLIGOMERIC MOLECULES:
• Several toxins occur as multimolecular complexes comprising two or more
noncovalently bonded different subunits.
• Common oligomeric protein toxins alongwith bacterial source are enlisted:
Protein Toxin Bacteria
1. Cholera toxin Vibrio cholera
2. E. coli thermolabile enterotoxin I & II E. coli
3. Shiga Toxin Shigella
4. E. coli shiga-like-toxin E. coli
5. Pertussis toxin Bordetella pertussis

9. 3. MACROMOLECULAR COMPLEXES OF TOXINS
ASSOCIATED TO NON-TOXIC MOIETIES
• Sometimes protein toxins are present as macromolecule complexes which are associated to non-toxic
subuits.
• Example:
Botulinum neurotoxins(BoNT) found in bacterial cultures and contaminated foodstuffs. These
complex, comprise three different forms, M toxin (a BoNT molecule), L toxin and LL toxin
• The form of progenitor toxin found varies between the different toxinogenic types, and more than
one form may be produced by a single strain. All three forms have been found in type A Clostridium
botulinum strains.
• C. botulinum type G strains produce the L toxin. The botulinum toxin of type E and F strains is
composed exclusively of M progenitor toxin (8).

10. 4. MULTIFACTOREAL TOXINS:
• A number of toxins designated binary toxins are composed of two independent
single chains not joined by either covalent or noncovalent bonds.
• Each individual protein separately expresses little or no toxicity
Toxin Bacteris
1. Leucocidin S. aureus
2. R-toxin S. Aureus
3. Hemolysin/Bacteriocin Enterococcus faecalis
4. C2 Toxin Clostridium botulinum
5. Iota Toxin Clostridium perfringes
6. Iota-like Toxin Clostridium
spiroforme

11. 5. PROTOXIN FORMS:
• Several protein toxins are secreted in their immature form into the culture
medium as inactive protoxins similarly to several proenzymes (zymogens).
• These protoxins are converted to active toxins by proteolytic enzymes.
• For example,
1. C. perfringens ε- and iota-toxins,
2. the C2-toxin of C. botulinum (component C2-II),
3. aerolysin of Aeromonas hydrophila

12. 2.2.1.6. THREE-DIMENSIONAL CRYSTAL STRUCTURE
• Since the elucidation in 1986 of the three-dimensional structure of P
. aeruginosa
exotoxin A, that of 28 other toxins (among them 10 of the family of Gram-
positive cocci superantigens) has been estab.lished so far

13. 2.2.2. MOLECULAR GENETICS
• The past 15 yr (1983–1998) may be considered as the golden age of the
molecular genetics of bacterial protein.
• More than 150 structural genes have been cloned and sequenced (vs only 10 by
the end of 1982). About 85% of the genes are chromosomal.
• The other genes are located on mobile genetic elements: bacteriophages,
plasmids, and transposons.

14. 1. BACTERIOPHAGIC GENES
Bacteriophagic genes were found to encode:
diphtheria toxin,
cholera toxin
S. pyogenes erythrogenic toxins A and C
S. aureus enterotoxins
C. botulinum toxins C1 and D, and E. coli.
shiga-like toxins I and II

15. 2. PLASMID-BORNE
tetanus toxin
anthrax toxin complex PA, EF and LF
S. aureus enterotoxin D
E. coli heat-labile
heat-stable enterotoxins
3. TRANSPOSON ENCODED:
Heat-stable enterotoxin

16. 3. MECHANISMS OF ACTION OF PROTEIN TOXINS ON
EUKARYOTIC TARGET CELLS

17. Toxin effects on target cells
may be classified
operationally into two
types:
type I effects (toxins strictly
acting at the surface of the
cell [cytoplasmic
membrane] without
penetrating into the cell
cytosol)
type II effects (toxins
ultimately acting on
specific molecular targets
into the cytosol after after
binding on)

18. 3.1. TOXINS STRICTLY ACTING ON THE CELL
SURFACE
• This process concerns two distinct types of toxins with totally different molecular
modes of action:
1. Damage to the cytoplasmic membrane
2. Induce biological effects through signal transduction processes.

19. 3.1.1. TOXINS ELICITING CELL DAMAGE (LYSIS) BY
DISRUPTION OF THE CYTOPLASMIC MEMBRANE
• These toxins, also known as cytolysins (hemolysins when acting on erythrocytes
and leukotoxins when acting on leukocytes)
• They constitute 35% of the entire bacterial toxin repertoire.
• Produced by both Gram-positive and Gram-negative bacteria.
• Two classes of toxins could be differentiated within this group.
1. ENZYMATICALLY ACTIVE TOXINS
2. PORE-FORMING TOXINS

20. Production of
Enzymatically active
toxin
(Phospholipases A,
C and D)
Hydrolyzes the
phospholipids of
Membrane
Destabilization of
Lipid Bilayer
Cell
Lysis
1. ENZYMATICALLY ACTIVE TOXINS :

21. 2. PORE-FORMING TOXINS:
• No known enzymatic activity
• Cell-membrane impairment
Production
of Protein
toxin
Insertion into the
cytoplasmic
phospholipid–
cholesterol bilayer
Creation of
stable pores
of d/f sizes
Oligomerization
(the conversion
of a monomer or
a mixture of
monomers into
an oligomer)

22. 2. TOXINS ACTING ULTIMATELY IN THE CYTOSOL
• Inactivate the molecular targets essential for cell functions.
Initial
Binding
of toxin
to cell
surface
receptor
Endocyt
osis
Translocation into
cytosol through
endocytic vesicular
membrane
Binding to
the Target
cell
Inactivation
of cellular
functions

23. PROTEIN TOXINS RESEMBLE ENZYMES
• The protein toxins resemble enzymes. Like enzymes, bacterial exotoxins are:
• Proteins
• Denatured by heat, acid, proteolytic enzymes
• Have a high biological activity (most act catalytically)
• Bacterial protein toxins are highly specific in the substrate utilized and in their
mode of action.
• Usually, the site of damage caused by the toxin indicates the location of the
substrate for that toxin. Terms such as "enterotoxin", "neurotoxin", "leukocidin" or
"hemolysin" are used to indicate the target site of some well-defined protein toxins.

24. CYTOTOXIC ACTIVITY BY PROTEIN TOXINS
• Certain protein toxins have very specific cytotoxic activity (i.e., they attack specific
cells, for example, tetanus or botulinum toxins)
• Some (as produced by staphylococci, streptococci, clostridia, etc.) have fairly
broad cytotoxic activity and cause nonspecific death of tissues (necrosis).
• Toxins that are phospholipases may be relatively nonspecific in their cytotoxicity.
This is also true of pore-forming "hemolysins" and "leukocidins".
• A few protein toxins cause death of the host and are known as "lethal toxins", (e.g.
anthrax toxin).

25. PROTEIN TOXINS ARE STRONGLY ANTIGENIC.
• In vivo, specific antibody (antitoxin) neutralizes the toxicity of these bacterial
proteins.
• In vitro, specific antitoxin may not fully inhibit their enzymatic activity.
• Protein toxins are inherently unstable: In time they lose their toxic properties but
retain their antigenic ones.

26. TOXOIDS:
Toxoids are detoxified toxins which retain their antigenicity and their immunizing
capacity (first discovered by Ehrlich)
The formation of toxoids can be accelerated by:
• Treating toxins with a variety of reagents including formalin, iodine, pepsin,
ascorbic acid, ketones, etc.
• The mixture is maintained at 37o at pH range 6 to 9 for several weeks.
• Toxoids can be use for artificial immunization against diseases caused by
pathogens where the primary determinant of bacterial virulence is toxin production.
• E.g immunizing against diphtheria and tetanus that are part of the DPT vaccine.

27. EXOTOXINS
• Exotoxins are proteins that can be produced by gram-positive or
gram-negative bacteria and include cytolytic enzymes and receptor-
binding proteins that alter a function or kill the cell.
• In many cases, the toxin gene is encoded on a plasmid (tetanus toxin
of C. tetani, LT(Heat-labile) and ST(Heat-stable) toxins of
enterotoxigenic E. coli) or a lysogenic phage (Corynebacterium
diphtheriae and C. botulinum).

28. SUPERANTIGENS
• Superantigens are a special group of toxins.
• These molecules activate T cells by binding simultaneously to a T-cell receptor and a
major histocompatibility complex class II (MHC II) molecule on an antigen presenting
cell without requiring antigen.
• Superantigens activate large numbers of T cells to release large amounts of interleukins
(cytokine storm), including IL-1, TNF, and IL-2, causing life- threatening autoimmune-
like responses.
• This superantigen stimulation of T cells can also lead to death of the activated T cells,
resulting in the loss of specific T-cell clones and the loss of their immune responses.
• Superantigens include :
 Toxic shock syndrome toxin of S. aureus
 Staphylococcal enterotoxins
 Erythrogenic toxin A or C of S. pyogenes.

29. BIOLOGICALACTIVITY OF ENDOTOXIN
• The biological activity of endotoxin is associated with the lipopolysaccharide (LPS).
• Toxicity is associated with the lipid component (Lipid A)
• Immunogenicity (antigenicity) is associated with the polysaccharide components.
• The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS.
• LPS activates complement by the alternative (properdin) pathway and may be a part of the
pathology of most Gram-negative bacterial infections.

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