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THE BACTERIA
Structure-Function-Pathogenicity Relationships
MM 1-16
Table of Contents
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Educational Objectives
Structure -Function-Pathogenicity Relationships
Capsule
Cell Wall
Protoplasmic Membrane
Pili
Flagella
Summary
Educational Objectives
General
1. To compare and contrast the Gram-positive and the Gram-negative bacterial cells
2. To develop an understanding of the relationships between cell components and clinical
features of disease
3.
To explain the bacterial growth curve
4.
To familiarize you with immune reactions induced by the bacterial cell
Specific (terms and concepts upon which you will be tested)
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Adhesion
Alternate complement pathway
Bacterial growth curve
Capsule
Cell wall structure
Cytoplasmic membrane
Disseminated intravascular coagulation
Endospore
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Endotoxin
Exponential phase
Fimbriae
Flagellum
Functions/effects of cell wall component
Glycan
H Antigen
K Antigen
Lag phase
Lipid A
Lipopolysaccharide (LPS)
Lipoteichoic acid
Logarithmic phase
M, R and T proteins
Murein
N-acetyl-D-glucosamine
N-acetyl-D-muramic acid
O-antigen
Outer membrane
Peptidoglycan
Periplasmic space
Phase of decline
Pili
Plasmid
Protein A
S-R variation
Shwartzman reaction
Stationary phase
Teichoic acid
Tumor necrosis factor
Structure-Function-Pathogenicity Relationships
The bacteria are approximately ten times the size of viruses, ranging from 0.4 um to 2.0 um in
size. They assume one of three morphological forms, spheres (cocci), rods (bacilli) or spirals
although there is much variation in each group. The morphology of a bacterium is maintained
by a rigid cell wall and it is the nature of this cell wall that allows us to divide bacteria into two
basic groups, Gram-positive bacteria and Gram-negative bacteria.
It is important to note the differences between the human (eukaryotic) cell and the bacterial
(prokaryotic) cell because many of these differences account for disease pathogenesis and it
has also been possible to exploit these differences in developing a chemotherapy regimen. In
contrast to the human cell, the bacterial cell:
1. May have a capsule. Not all bacterial cells have a capsule but when it is present it is a
major virulence factor. The capsule
includes the K-antigen.
2. May have anouter membrane which is the outer surface of the cell or, in the case of
encapsulated strains, lies just
underneath the capsule. This has a trilaminar appearance. It contains
lipopolysaccharides (LPS). These are known
as endotoxins. They are also the somatic or O-antigen and are used in serological typing
of species. These occur only in
Gram-negative bacteria.
3. May have a periplasmic space which lies between the outer membrane and the plasma
membrane. This is filled with the
periplasmic gel which contains various enzymes. Again, this occurs only in the Gramnegative bacteria.
4. Has a rigid cell wall made of peptidoglycan (except for the mycoplasma). This cell wall is
thick in Gram-positive bacteria and
thin in Gram-negative bacteria. It is the thickness of the peptidoglycan that accounts for
the ability/lack of ability to retain the
crystal violet used in the Gram stain.
5. Has a cytoplasmic membrane lacking sterols (except for the mycoplasma). Up to 90%
of the ribosomes are attached to
this membrane. It also contains:
a.
The energy-producing cytochrome and oxidative phosphorylation system.
b.
The membrane permeability (transport) systems.
c.
Various polymer-synthesizing systems.
d.
An ATPase.
6. Has a cytoplasmic membrane invagination termed the mesosome. This controls septa
formation in the dividing cell and is the
attachment site for the chromosome.
7. May have a flagellum which arises from the plasma membrane and protrudes through the
cell wall. This is the source of the H
antigen which is used in serologic diagnosis. It is also the motility organ and possibly an
organ for attachment to a human
cell. It is considered a virulence factor.
8. Has hairlike microfibrils, termed fimbriae or pili, which originate in the plasma membrane
and protrude through the cell
wall. They are straighter, thinner and shorter than flagella. The pili contain chemical
compounds called adhesins which
allow the cell to bind to specific receptors on various human tissues. This binding gives
rise to organ specificity of some
bacterial strains. Fimbriae/pili are major virulence factors.
9. Has ribosomes attached to the plasma membrane and also free in the cytoplasm which
have a mass of 70S (the human
ribosome has a mass of 80S). The protein and RNA species in the bacterial ribosome
differ from
those in the human ribosome.
10. May have an endospore within the cytoplasm. This is a body that allows the organism
to resist adverse conditions.
11.
Has a nucleus lacking a nuclear membrane.
12. May have a circular plasmid. This is a small (relative to the chromosome) piece of DNA
that often codes for virulence
factors.
13.
Has a haploid (single) chromosome.
There are many common themes in bacterial pathogenicity related to cell structure of the
species. These are based on the presentation to the human body of the bacteria, its parts and
its metabolites. When an organism, or more commonly a number of organisms of the same
species, enters the human body and encounters no host defenses, it will exhibit a growth
curve like the one depicted below for a closed system.
In the lag phase there is an increase in cell size at a time when little or no cell division is
occurring. During this phase, there is a marked increase in macromolecular components
(many of which are toxic to the human cell), metabolic activity and susceptibility to physical
and chemical agents. The lag phase is a period of adjustment necessary for the
replenishment of the cell's pool of metabolites to a level commensurate with maximum cell
synthesis.
In the exponential orlogarithmic phase, the cells are in a state of balanced growth. During
this state, the mass and the volume of the cell increase by the same factor in such a manner
that the average composition of the cells and the relative concentrations of the metabolites
remain constant. During this period of balanced growth, the rate of increase can be expressed
by a natural exponential function.
The accumulation of waste products, exhaustion of nutrients, change in pH, induction of host
immune mechanisms and other obscure factors exert a deleterious effect on the culture,
resulting in a decreased growth rate. During the stationary phase, the viable cell count
remains constant. The formation of new organisms equals the death of organisms in the
system.
As the amount of the factors detrimental to the bacteria within the body increase, more
bacteria are killed than are formed. During the phase of decline there is a negative
exponential phase which results in a decrease in the numbers of bacteria within the system.
During all phases of the bacterial growth cycle, the host is exposed to the components of the
bacterial cell. This exposure results in the induction of pathology as well as of immune
mechanisms. The outcome is either life or death of the human, depending on the relative
rates of induction of these phenomena.
Capsule
(K-antigen)
A fundamental requirement for most pathogenic bacteria that enter the human body is to
escape phagocytosis by macrophages or polymorphonuclear phagocytes. The most common
means utilized by bacteria to avoid phagocytosis is an antiphagocytic capsule. The capsule is
a major virulence factor, e.g. all of the principal pathogens which cause pneumonia and
meningitis, including Haemophilus influenzae, Neisseria meningitidis, Escherichia coli,
Streptococcus pneumoniae, Klebsiella pneumoniae and group B streptococci have
polysaccharide capsules on their surface. Nonencapsulated mutants of these organisms are
avirulent.
The chemical nature of the capsule is important in the functions the capsule plays in the
infection process. The capsules of bacteria are chemically diverse but the majority of them
are polysaccharide in nature. These polymers are composed of repeating oligosaccharide
units of two to four monosaccharides. Some may contain acetic acid, pyruvic acid and/or the
methyl esters of hexoses. At least two species of pathogenic bacteria produce protein
capsules; Bacillus anthracis produces a capsule of pure D-glutamic acid and Yersinia pestis
produces a capsule of mixed amino acids. Capsules may be weakly antigenic to strongly
antigenic, depending on their chemical complexity. Capsules may be covalently linked to the
underlying cell wall or just loosely bound to it. Not all bacteria form capsules but in those that
do the capsule is the interface between the bacterial cell and the external environment. As
such it may serve a diversity of functions in disease including:
1. Antiphagocytosis - the smooth nature of the capsule prevents the phagocyte from
adhering to and engulfing the bacterial
cell. Furthermore, opsonins are prevented from binding to the cell and the
process of opsonization is hindered.
2. Prevention of neutrophil killing of engulfed bacteria - lysosome contents do not
have direct access to the interior of the
bacterial cell and thus cannot kill the cell.
3.
Prevention of complement-mediated bacterial cell lysis.
4. Prevention of polymorphonuclear leukocyte migration to the site of infection Bacteroides fragilis produces a
polysaccharide capsule high in succinic acid. Succinic acid is released from the
capsule and paralyzes
the pmn leukocyte.
5. Toxicity to the host cell - this takes many forms depending on the chemical
nature of the capsule. One example is the
capsule of B. fragilis which induces abscess formation.
6.
Adhesion to the host cell.
7.
Protection of anaerobes from oxygen toxicity.
8. Determination of colonial type - bacteria with capsules form smooth (S) colonies
while those without capsules form rough
(R) colonies. A given species may undergo a phenomenon called S-R variation
whereby the cell loses the ability to
form a capsule. Some capsules are very large and absorb water; bacteria with
this type of capsule (e.g., Klebsiella
pneumoniae) form mucoid (M) colonies.
9.
Enhancement of the pathogenicity of other species in a mixed infection.
10.
Receptors for bacteriophage.
11.
Induction of antibody synthesis - this is the basis for:
a.
Serological diagnosis.
b. Vaccine production. A polyvalent (23 serotypes) polysaccharide vaccine
of Streptococcus pneumoniae capsule is
available for high risk patients. There is also a polyvalent (4 serotypes)
vaccine of Neisseria meningitidis capsule
available. A monovalent vaccine made up of capsular material from
Haemophilus influenzae is also
available.
c.
Quellung reaction
It should be kept in mind that a given species of bacteria may give rise to several serotypes
based on the capsular antigen. For example, Streptococcus pneumoniae produces over 70
capsular serotypes which have the structure of teichoic acid-like polymers.
The capsule of bacteria may be penetrated by structures arising from the cell wall or plasma
membrane such as cell wall specific polysaccharide, cell wall teichoic acid, plasma membrane
lipoteichoic acid, flagella and pili.
Cell Wall
Gram-positive bacteria
The cell wall lies immediately external to the plasma membrane; it is the interface with the
external environment in those organisms lacking a capsule, otherwise it is overlaid with the
capsule. The rigid cell wall is a single bag-shaped structure composed of a network of
repeating, cross-linked peptidoglycan, also called murein.
The glycan component is constituted of the two amino sugars, glucosamine and muramic
acid. They occur as alternate ß-1, 4-linked N-acetyl-D-glucosamine and N-acetyl Dmuramic acid residues. The glycan and peptide units are linked through the lactic acid
carboxyl group of N-acetylmuramic acid to the amino terminus of a tetrapeptide. The
glycotetrapeptides are cross-linked through the tetrapeptide units, forming a continuous 3dimensional framework. While the tetrapeptide unit may vary with the species, the invariant
feature of the tetrapeptide component is the presence of D-alanine, which is always the
linkage unit between peptidoglycan chains.
Thus, the cell wall can be several layers thick, each layer being a sheet of linked
peptidoglycan units. The Gram-positive bacterial cell wall is distinguished by having multiple
layers of peptidoglycan sheets and is thus up to ten times the thickness of a Gram-negative
bacterial cell wall.
Attached to the rigid peptidoglycan framework of the cell wall are various polysaccharides
which are covalently linked to the peptidoglycan. These fall into two groups:
A. Cell wall teichoic acids - these are polymers of phosphodiester-linked polyols.
They usually contain ribitol, or
occasionally glycerol, and are covalently linked to peptidoglycan through
substituted phosphodiester groups on the C-6
hydroxyl of N-acetylmuramic acid residues. Teichoic acids are specifically
modified in different bacteria by addition to
the polyol units of ester linked D-alanine, D-lysine or O-glycoside linked glucose,
galactose or N-acetyl-hexosamines.
B. Cell wall specific polysaccharides. These are polymers of mono- and disaccharides which may be linear or branched.
They contain no phosphate.
C. In some cases the cell wall of Gram-positive bacteria may contain proteins of
special significance. Examples of these
are:
1.
The M, T and R proteins of the group A streptococci
2. Protein A of Staphylococcus aureus
A composite of the cell wall of Gram-positive bacteria is diagrammed below.
Gram-negative bacteria
In contrast to the Gram-positive bacterial cell wall, the Gram-negative bacterial cell wall is
much more complex. It consists of a rigid peptidoglycan layer, that is much thinner than that
found in the Gram-positive cells, overlaid by an outer membrane containing a diversity of
structures.
 O-antigen = somatic antigen
 LPS = lipopolysaccharide
 KDO = 2-keto-3-deoxyoctonic acid
Between the cytoplasmic membrane and the outer membrane is the periplasmic space
containing a gel-like periplasm in which resides the cell wall peptidoglycan as well as various
enzymes.
In addition to phospholipids, the outer membrane contains unique Gram-negative
lipopolysaccharides (LPS) and various proteins (porons) and lipoproteins. Each of these
types of compounds is antigenic and is used to speciate and subspeciate organisms
serologically. Of these compounds the LPS is the most important.
LPS is an amphiphile composed of three regions: O-polysaccharide (the O- or somaticantigen), the core polysaccharide and lipid A. Lipid A is anchored in the outer membrane.
LPS is also known as endotoxin.
The peptidoglycan of the Gram-negative cell is chemically similar to but not identical with the
peptidoglycan of the Gram-positive cell. The major difference between the two cell types is in
the thickness of the peptidoglycan rather than the chemical makeup.
When the bacterial cell wall is placed in the environment of the human body as part of a
viable microorganism, there is a diversity of functions/effects that can be noted. Some of
these are specific for Gram-negative organisms (due to the relative complexity of their cell
walls) and some are general. The functions/effects of the cell wall include:
1.
Maintenance of the morphology of the organism
2. Enhancement of the immune response to various cell metabolites by
muramyldipeptide
(N-acetylmuramyl-L-alanyl-D-isoglutamine), i.e., it is an adjuvant
3.
Induction of fever by muramyldipeptide (i.e., its a pyrogen)
4.
Induction of sleep by muramyldipeptide (i.e., its a somnogen)
5. Competition of muramyldipeptide with serotonin (5-hydroxytryptamine) for
receptors on macrophages. Serotonin, when
bound to the macrophage, enhances the chemotactic response of the
macrophage. Thus, muramyldipeptide blocks
this response in the inflammatory reaction.
6. Induction of inflammatory arthritic joint disease by peptidoglycan-linked
polysaccharides (e.g., the polysaccharide of
group A streptococci linked to peptidoglycan)
7. Induction of granulomatous liver disease by peptidoglycan-linked
polysaccharides
8.
Stimulation of hemopoietic stem cells by peptidoglycan-linked polysaccharides
9. Induction of chronic inflammatory bowel disease (i.e. Crohn's disease) by
peptidoglycan-linked polysaccharides,
especially those of Mycobacterium paratuberculosis.
10. Induction of the immune response by the teichoic acids of Gram-positive
bacteria. This response is used in the
serological identification of Gram-positive bacteria.
11. Induction of the immune response by the O-polysaccharide (somatic antigen)
portion of the lipopolysaccharide of the
outer membrane of Gram-negative bacteria. This response is used in the
serological identification of the Gram-negative
bacteria.
12.
Endotoxin (LPS) induction of:
A. Fever-Leukocytes take up Lipid A which induces the synthesis and
secretion of interleukin 1. Interleukin 1 acts
on the heat regulation centers in the brain to cause fever.
B. Shwartzman reaction - hemorrhagic necrosis at the site of infection
following exposure of another part of the
body to a relatively small amount of Lipid A. This is due to the clearing
of fibrin polymers at the inflammation
site.
C. Disseminated intravascular coagulation - this can lead to lethal shock.
For this reason, it is especially
important in patients (e.g., with carcinoma) who suffer chronic
disseminated intravascular coagulation (defined
as a 10-20% decrease in circulating platelets and clotting factors).
D. Macrophage production of tumor necrosis factor which results in
various effects including:
(1) Endothelial cell loss of their usually anticoagulant properties
(thus enhanced fibrin deposition and
increased disseminated intravascular coagulation).
(2) Adherence of polymorphonuclear leukocytes to the vascular
endothelium, causing them to degranulate
and form reactive oxygen intermediates such as superoxide
anion and hydrogen peroxide. This promotes
tissue necrosis and circulatory collapse.
The overall effects of tumor necrosis factor are depicted below.
E. Activation of complement via the alternative pathway whereby the activator
surface (Lipid A) of the
Gram-negative cell facilitates the combination of Factor B and C3b.
The final phase in the activation of the alternative complement cascade is the
formation of the membrane attack complex which is initiated by the C4 convertase
cleavage of C5.
The subsequent formation of the membrane attack complex is non-enzymatic and
follows the pathway diagrammed below.
Although a small amount of lysis occurs when C8 binds to C5b67, it is polymerized
C9 that forms pores in the cell membrane that causes most lysis.
F.
Stimulation of bone marrow cell proliferation.
G.
Nonspecific enhancement of immune responses (i.e., action as adjuvants).
H.
Enhancement of radiation resistance
I.
Clotting of horseshoe crab amebocyte lysates (Limulus lysate reaction).
J.
Engender hypersensitivity reactions
13.
Functioning of the outer membrane of the Gram-negative cell wall as:
A. A barrier to noxious environmental compounds. The barrier effect is
seen most clearly in enteric bacteria that
must cope with bile salts and digestive enzymes such as
phospholipases and lysins. In enteric bacteria the
tightly fitting hydrophilic lipopolysaccharides, metal ligands, and proteins
of the outer membrane outer surface
form a hydrophilic barrier to lipophilic molecules. Excluded are many
antibiotics.
B.
A molecular sieve for small water-soluble molecules.
C.
An absorption site for bacteriophage
D.
An absorption site for cellular conjugation
E.
A reservoir for proteases, other enzymes and toxins
Protoplasmic Membrane
The protoplasmic membrane lies underneath the pepticloglycan layer of the cell wall and
encloses the cytoplasm. It does not play a major role in disease pathogenesis. However it
plays a vital role as an osmotic barrier, the site of initiation of cell wall synthesis, the site of
attachment of the chromosome, the site of the cytochrome system and the location of the
various transport enzymes. The only known role of the plasma membrane in pathogenesis is
that it is the source of lipoteichoic acid which protrudes through the peptidoglycan of the
Gram-positive cell and presents as a surface marker. As such it acts in a similar, but weaker,
fashion as the lipid A of the Gram-negative cell. Specifically the lipoteichoic acid, during the
disease process, causes:
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Dermal necrosis (Shwartzman reaction)
Induction of cell mitosis at the site of infection
Stimulation of specific immunity
Stimulation of non-specific immunity
Adhesion to the human cell
Complement activation
Induction of hypersensitivity (anaphylaxis)
Pili
The plasma membrane is the structure that anchors the pili. While they arise from the plasma
membrane, the pili are not considered part of the plasma membrane. They are organelles that
are anchored in the membrane and protrude through the cell wall to the outside of the cell.
They are termed adhesins because their major function is adhesion to other cells, both
bacterial and human.
1. F-pili are produced by male bacteria and allow them to bind to female bacteria to
promote sexual conjugation. This
allows bacteria to spread antibiotic-resistant genes through a population at a
fairly high frequency.
2.
Type I and type II pili promote adhesion to human cells with these results:
a. Binding of platelets and fibrin around the bacterial cell to evade
phagocytosis, promote fibrin deposition on heart
valves and promote blood clots.
b. Binding of bacterial cells to epithelial adhesion receptors which results in
interactions which may kill the human
cell. For example, Neisseria gonorrhoeae is avirulent if it lacks pili.
Flagella
Flagella are organs of locomotion which are also anchored in the membrane and protrude
through the cell wall to the external part of the cell. They are considered virulence factors
because they allow the bacterial cell to evade phagocytes in viscous material by swimming
away from them and secondly they allow the bacterial cell to come into close contact with the
adhesion receptors on the human cell. Flagella are the source of the H-antigen used in
serotyping many motile species of bacteria.
Summary
1.
Bacteria occur as spheres (cocci), rods (bacilli) or spirals.
2. All bacteria are classified as Gram-positive (retain the gram stain) or Gram-negative (do
not retain the gram stain).
3. Structural features of bacteria that are not seen in the human cell, or differ from those in
the human cell, include a capsule, an
outer membrane, a periplasmic space, a rigid cell wall, a cytoplasmic membrane lacking
sterols, the mesosome, flagellum,
fibrae (pili), 70S ribosomes, endospore, lack of a nuclear membrane, plasmids and a
haploid chromosome.
4. The major antigens of the bacterial cell are the capsule (K-antigen), the
lipopolysaccharide (O-antigen) and the flagellum
(H-antigen).
5. The growth cycle of a culture of bacteria is divided into four phases: lag phase,
exponential phase, stationary phase, decline
phase.
6. The capsule of bacteria is most commonly polysaccharide in nature but proteinaceous in
at least two species, Bacillus
anthracis and Yersinea pestis.
7. The capsule is a major virulence factor that allows bacteria to evade phagocytosis, avoid
the killing effects of lysosomal
enzymes, avoid complement-mediated cell lysis, paralyze leukocytes, induce pathology
in the host tissue, adhere to the host
cell, protect anaerobic cells from oxygen toxicity, produce a unique colony type, enhance
its pathogenicity, adsorb
bacteriophage and induce antibody synthesis.
8. Bacteria with capsules from smooth (S) colones; those without a capsule from rough (R)
colonies; those with hydrophilic
capsules from mucoid (M) colonies.
9. Serologically, the capsule is important in diagnosis, vaccine production and as the basis
for the Quellung reaction.
10. The cell wall of bacteria is made up sheets of cross-linked repeating units of
peptidoglycan. In Gram-positive cells this is
relatively thick as compared to Gram-negative cells.
11. Linked to the cell wall of bacteria are teichoic acids, cell wall specific polysaccharides
and, in some cases, proteins of special
significance.
12. Gram-negative bacterial cells contain lipopolysaccharide (LPS) in their outer membrane.
This is the source of the O-antigen
and endotoxin.
13. The functions/effects of the cell wall include maintenance of the morphology or the
bacterial cell, action as an adjuvant,
induction of fever, induction of sleep, competition with serotonin for receptors on
macrophages, induction of inflammation,
induction of liver granuloma, stimulation of hemopoietic stem cells, induction of bowel
inflammation, induction of antibody
synthesis.
14. Endotoxin induces fever, hemorrhagic necrosis (Shwartzman reaction), disseminated
intravascular coagulation, production of
tumor necrosis factor, activation of the alternate complement pathway, stimulation of
bone marrow cell proliferation,
enhancement of the immune and the Limulus lysate reaction.
15. The lipoteichoic acid of Gram-positive bacteria acts similar to the endotoxin of Gramnegative bacteria.
16.
Pili contain adhesins which allow the bacterial cell to bind to human cells.
17.
Flagella are organs of locomotion that are used in serotyping strains of bacteria.
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