To search the entire book, enter a term or phrase in the form below
Bacterial Endotoxin
© Kenneth Todar, PhD
Endotoxins are part of the outer membrane of the cell wall of
Gram-negative bacteria. Endotoxin is invariably associated with
Gram-negative
bacteria whether the organisms are pathogenic or not. Although the term
"endotoxin" is occasionally used to refer to any cell-associated
bacterial
toxin, in bacteriology it is properly reserved to refer to the lipopolysaccharide
complex associated with the outer membrane of Gram-negative
pathogens such
as Escherichia coli, Salmonella, Shigella, Pseudomonas,
Neisseria,
Haemophilus influenzae, Bordetella pertussis and Vibrio
cholerae.
The relationship of endotoxin (lipopolysaccharide) to the bacterial
cell surface is
illustrated
in Figure 1 below.
Figure
1. Structure of the
cell envelope of a Gram-negative bacterium (e.g. E. coli). The peptidoglycan sheet
is actually in the periplasm, the space between the inner (plasma) and
outer membranes. The inner face of the outer membrane is composed of
phospholipid, the same as in the plasma membrane. The outer face of the
outer membrane contains some phospholipid but primarily it is composed
of lipopolysaccharide which has the amphipathic qualities of a
phospholipid. Lipopolysaccharide is endotoxin. Imbedded in the outer
membrane are various outer membrane proteins, lipoprotein and porin
proteins, which sometimes play a role in virulence of a Gram-negative
bacterium, but they are not considered endotoxins.
The biological activity of endotoxin is associated with the
lipopolysaccharide
(LPS). Toxicity is associated with the lipid component (Lipid
A) and immunogenicity is associated with the polysaccharide
components. The cell wall antigens (O antigens) of Gram-negative
bacteria are components of LPS. LPS elicits a variety of inflammatory
responses
in an animal and it activates complement by the alternative
(properdin)
pathway, so it may be a part of the pathology of Gram-negative
bacterial
infections.
In vivo, Gram-negative
bacteria probably release minute amounts of
endotoxin
while growing. This may be important in the stimulation of natural
immunity. It is known that small amounts of endotoxin may be
released
in a soluble form by young cultures grown in the laboratory. But
for the most
part, endotoxins remain associated with the cell wall until
disintegration
of the organisms. In vivo,
this results from autolysis,
external lysis mediated by complement and lysozyme, and phagocytic
digestion
of bacterial cells.
Compared to the classic exotoxins of bacteria, endotoxins are less
potent
and less specific in their action, since they do not act enzymatically.
Endotoxins are heat stable (boiling for 30 minutes does not destabilize
endotoxin), but certain powerful oxidizing agents such as superoxide,
peroxide
and hypochlorite, have been reported to neutralize them. Endotoxins,
although antigenic, cannot
be converted to toxoids. A comparison of the properties of bacterial
endotoxins
and classic exotoxins is shown in Table 1.
Table 1. Characteristics of
bacterial
endotoxins and classic exotoxin
| PROPERTY |
ENDOTOXIN |
EXOTOXIN |
| CHEMICAL NATURE |
Lipopolysaccharide
(mw = 10kDa) |
Protein (mw = 50-1000kDa) |
| RELATIONSHIP TO CELL |
Part of outer membrane |
Extracellular, diffusible |
| DENATURED BY BOILING |
No |
Usually |
| ANTIGENIC |
Yes |
Yes |
| FORM TOXOID |
No |
Yes |
| POTENCY |
Relatively low (>100ug) |
Relatively high (1 ug) |
| SPECIFICITY |
Low degree |
High degree |
| ENZYMATIC ACTIVITY |
No |
Often
|
| PYROGENICITY |
Yes |
Occasionally |
The Role of LPS in the Outer Membrane of
Gram-negative
Bacteria
The function of the outer membrane of Gram-negative bacteria is to
act
as a protective permeability barrier. The outer membrane is impermeable
to large
molecules
and hydrophobic compounds from the environment.
LPS is essential to the function of the outer membrane. First, it
establishes a permeability barrier
that is permeable only to low molecular weight, hydrophilic molecules.
In the E. coli the ompF and ompC porins exclude passage of all
hydrophobic
molecules and any hydrophilic molecules greater than a molecular weight
of about 700 daltons. This prevents penetration of the bacteria by bile
salts and other toxic molecules from the GI tract. It also a barrier to
lysozyme and many antimicrobial agents. Second, in an animal host, it
may impede destruction
of the bacterial cells by serum components and phagocytic cells. Third,
LPS may play a role as an adhesin used in colonization of the host.
Lastly, variations in LPS structure provide for the existence of
different antigenic strains of a pathogen that may be able to bypass a
previous immunological response to a related strain.
Chemical Nature of Endotoxin
Most of the work on the chemical structure of endotoxin has been
done
with species of Salmonella and E. coli. LPS can be
extracted
from whole cells by treatment with 45% phenol at 90o. Mild
hydrolysis
of LPS yields Lipid A plus polysaccharide.
Lipopolysaccharides are complex amphiphilic molecules with a mw of
about
10kDa, that vary widely in chemical composition both between and among
bacterial species The general architecture of LPS is shown in Figure 2.
The general structure of Salmonella LPS is shown in Figure 3
and
the complete structure of Salmonella lipid A is illustrated in
Figure
4.
Figure
2. General architecture
of Lipopolysaccharide
Figure
3. General Structure
of Salmonella LPS. Glc = glucose; GlcNac =
N-acetyl-
glucosamine; Gal = galactose; Hep = heptose; P = phosphate; Etn =
ethanolamine;
R1 and R2 = phoshoethanolamine or aminoarabinose. Ra to Re indicate
incomplete
forms of LPS. The Rd2 phenotype (not shown) would have only a single
heptose
unit. The Rc, Rd2, and Rd1 mutants lack the phosphate group attached to
Hep.
Figure
4. Complete structure
of the Lipid A Moiety of LPS of S. typhimurium, S. minnesota,
and E. coli
LPS consists of three components or regions: Lipid A, an R
polysaccharide and an O polysaccharide.
Region I. Lipid A is the lipid component of LPS. It contains
the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of
a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty
acids
(FA) attached. Usually 6 FA are found. All FA in Lipid A are saturated.
Some FA are attached directly to the NAG dimer and others are
esterified
to the 3-hydroxy fatty acids that are characteristically present. The
structure
of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae
Lipid A is virtually constant.
The primary structure of Lipid A has been elucidated and Lipid A has
been chemically synthesized. Its biological activity appears to depend
on a peculiar conformation that is determined by the glucosamine
disaccharide,
the PO4 groups, the acyl chains, and also the KDO-containing
inner core.
Region II. Core (R) antigen or R polysaccharide is attached
to
the 6 position of one NAG. The R antigen consists of a short chain of
sugars.
For example: KDO - Hep - Hep - Glu - Gal - Glu - GluNAc -
Two unusual sugars, heptose and
2-keto-3-deoxyoctonoic
acid (KDO), are usually present, in the core polysaccharide. KDO is
unique and invariably
present
in LPS and so it has been used as an indicator in assays for LPS
(endotoxin).
With minor variations, the core polysaccharide is common to all
members
of a bacterial genus (e.g. Salmonella), but it is structurally
distinct
in other genera of Gram-negative bacteria. Salmonella, Shigella
and Escherichia have similar but not identical cores.
Region III. Somatic (O) antigen or O polysaccharide is
attached to the core polysaccharide. It consists of repeating
oligosaccharide
subunits made up of 3 - 5 sugars. The individual chains vary in length
ranging up to 40 repeat units. The O polysaccharide is much longer than
the core polysaccharide, and it maintains the hydrophilic domain of the
LPS molecule. A major antigenic determinant (antibody-combining site)
of
the Gram-negative cell wall resides in the O polysaccharide.
Great variation occurs in the composition of the sugars in the O
side
chain between species and even strains of Gram-negative bacteria. At
least
20 different sugars are known to occur and many of these sugars are
characteristically
unique dideoxyhexoses, which occur in nature only in Gram-negative cell
walls. Variations in sugar content of the O polysaccharide contribute
to
the wide variety of antigenic types of Salmonella and E.
coli
and presumably other strains of Gram-negative species. Particular
sugars
in the structure, especially the terminal ones, confer immunological
specificity
of the O antigen, in addition to "smoothness" (colony morphology) of
the
strain. Loss of the O specific region by mutation results in the strain
becoming a "rough" (colony morphology) or R strain.
The elucidation of the structure of LPS (Figure 3) relied heavily on
the availability of mutants each blocked at a particular step in LPS
synthesis.
The biosynthesis of LPS is strictly sequential. The core sugars are
added
sequentially to Lipid A by successive additions, and the O side chain
is
added last, one preassembled subunit at a time. The properties of
mutants
producing incomplete LPS molecules suggests the nature and biological
functions
performed by various parts of the LPS molecule.
In E. coli and Salmonella, loss of the O antigen
results in partial loss of virulence, suggesting that
this
portion of LPS is important during a host-parasite interaction. It is
known
that
such "rough" mutants are more susceptible to phagocytosis and serum
bactericidal
reactions.
Loss of the more proximal parts of the core, as in "deep rough"
mutants
(i.e. in Rd1, Rd2, and Re mutants in Figure 3) makes the strains
sensitive
to a range of hydrophobic compounds, including antibiotics, detergents,
bile salts and mutagens. This area contains a large number of charged
groups
and is thought to be important in maintaining the permeability
properties
of the outer membrane.
Mutants in the assembly of Lipid A cannot be isolated except as
conditional
lethal mutants and this region must therefore be essential for cell
viability.
The innermost region of LPS, consisting of Lipid A and three residues
of
KDO, appears to be essential for viability, presumably for assembling
the
outer membrane.
LPS and virulence of Gram-negative Bacteria
Both Lipid A (the toxic component of LPS) and the polysaccharide
side
chains (the nontoxic but immunogenic portion of LPS) act as
determinants
of virulence in Gram-negative bacteria.
The O polysaccharide and virulence
Virulence, and the property of "smoothness", is associated
with an
intact
O
polysaccharide, The involvement of the polysaccharide
chain in virulence is shown
by the fact that small changes in the sugar sequences in the side
chains
of LPS result in major changes in virulence. How are the polysaccharide
side chains involved in the expression of virulence? There are a number
of possibilities:
1. O-specific antigens could allow organisms to adhere
specifically
to certain tissues, especially epithelial tissues.
2. Smooth antigens probably allow resistance to phagocytes,
since
rough mutants are more readily engulfed and destroyed by phagocytes.
3. The hydrophilic O polysaccharides could act as water-solubilizing
carriers
for toxic Lipid A. It is known that the exact structure of the
polysaccharide
can greatly influence water binding capacity at the cell surface.
4. The O antigens could provide protection from damaging
reactions
with antibody and complement. Rough strains of Gram-negative
bacteria
derived from virulent strains are generally non virulent. Smooth
strains
have polysaccharide "whiskers" which bear O antigens projecting from
the
cell surface. The O antigens are the key targets for the action of host
antibody and complement, but when the reaction takes place at the tips
of the polysaccharide chains, a significant distance external to the
general
bacterial cell surface, complement fails to have its normal lytic
effect.
Such bacteria are virulent because of this resistance to immune forces
of the host. If the projecting polysaccharide chains are shortened or
removed,
antibody reacts with antigens on the general bacterial surface, or very
close to it, and complement can lyse the bacteria. This contributes to
the loss of virulence in "rough" colonial strains.
5. The O-polysaccharide or O antigen is the basis of antigenic
variation among many important Gram-negative pathogens including E.
coli, Salmonella and Vibrio cholerae. Antigenic
variation
guarantees the existence of multiple serotypes of the bacterium, so
that
it is afforded multiple opportunities to infect its host if it can
bypass
the immune response against a different serotype. Furthermore, even
though
the O polysaccharides are strong antigens, they seldom elicit immune
responses
which give full protection to the host against secondary challenge with
specific endotoxin.
Lipid A and virulence
The physiological activities of LPS are mediated mainly by
the Lipid
A component of LPS. Lipid A is a powerful biological response
modifier
that can stimulate the mammalian immune system. During infectious
disease
caused by Gram-negative bacteria, endotoxins released from, or part of,
multiplying cells have similar effects on animals and significantly
contribute
to the symptoms and pathology of the disease encountered.
Since Lipid A is embedded in the outer membrane of bacterial
cells,
it probably only exerts its toxic effects when released from
multiplying
cells in a soluble form, or when the bacteria are lysed as a result of
autolysis, complement and the membrane attack complex (MAC), ingestion
and killing by phagocytes, or killing with certain types of
antibiotics.
The injection of living or killed Gram-negative cells or purified
LPS
into experimental animals causes a wide spectrum of nonspecific
pathophysiological
reactions, such as fever, changes in white blood cell counts,
disseminated
intravascular coagulation, hypotension, shock and death.
Injection of fairly small doses of endotoxin results in death in most
mammals.
The sequence of events follows a regular pattern: (1) latent period;
(2)
physiological distress (diarrhea, prostration, shock); (3) death. How
soon
death occurs varies on the dose of the endotoxin, route of
administration,
and species of animal. Animals vary in their susceptibility to
endotoxin.
The mechanism is complex.
In humans, LPS binds to a lipid
binding protein (LBP) in the serum
which transfers it to CD14 on
the cell membrane, which in turn
transfers it to another non-anchored protein, MD2, which associates
with Toll-like receptor-4 (TLR4).
This triggers the signaling cascade
for
macrophage/endothelial cells to secrete pro-inflammatory cytokines and
nitric oxide that lead to characteristic "endotoxic shock". CD14 and
TLR4 are present on several cells of the immunological system cells,
including
macrophages and dendritic cells. In monocytes and macrophages, three
types of events are
triggered
during their interaction with LPS:
1. Production of cytokines, including IL-1, IL-6, IL-8,
tumor
necrosis factor (TNF) and platelet-activating factor. These, in turn,
stimulate
production of prostaglandins and leukotrienes. These are powerful
mediators
of inflammation and septic shock that accompanies endotoxin toxemia.
LPS
activates macrophages to enhanced phagocytosis and cytotoxicity.
Macrophages
are stimulated to produce and release lysosomal enzymes, IL-1
("endogenous
pyrogen"), and tumor necrosis factor (TNFalpha), as well as other
cytokines
and mediators.
2. Activation of the complement cascade. C3a and C5a cause
histamine
release (leading to vasodilation) and affect neutrophil chemotaxis and
accumulation. The result is inflammation.
3. Activation of the coagulation cascade. Initial
activation
of Hageman factor (blood-clotting Factor XII) can activate
several
humoral systems resulting in
a. coagulation: a blood clotting cascade that leads
to coagulation, thrombosis, acute disseminated intravascular
coagulation,
which depletes platelets and various clotting factors resulting in
internal
bleeding.
b. activation of the complement alternative pathway
(as above, which leads to inflammation)
c. plasmin activation which leads to fibrinolysis
and hemorrhaging.
d. kinin activation releases bradykinins and other
vasoactive peptides which causes hypotension.
The net effect is to induce inflammation, intravascular coagulation,
hemorrhage and shock.
LPS also acts as a B cell mitogen, stimulating the polyclonal
differentiation and multiplication of B-cells and the secretion of
immunoglobulins,
especially IgG and IgM.
Textbook of Bacteriology Index
