Bacteria:
Structure-Function-Pathogenicity Relationships
Contents
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)
·
Adhesion
·
Capsule
·
Fimbriae
·
Glycan
·
Lipid A
·
Murein
·
Pili
·
Plasmid
Structure-Function-Pathogenicity Relationships
The bacteria are approximately ten times the size of viruses,
ranging from 0.4 µm to 2.0 µm in size. They assume one of three morphological
forms:
·
spheres
(cocci),
·
rods
(bacilli) or
·
spirals
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 an outer 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) 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 Gram-negative 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 or
logarithmic 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
D-muramic 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 3-dimensional 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 di-saccharides
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 somatic-antigen),
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, uramyldipeptide 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.
·
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.
·
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).
·
Endothelial
cell loss of their usually anticoagulant properties (thus enhanced fibrin
deposition and increased disseminated intravascular coagulation).
·
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.
·
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.
·
Stimulation
of bone marrow cell proliferation.
·
Nonspecific
enhancement of immune responses (i.e., action as adjuvants).
·
Enhancement
of radiation resistance
·
Clotting
of horseshoe crab amebocyte lysates (Limulus lysate reaction).
·
Engender
hypersensitivity reactions
13.
Functioning
of the outer membrane of the Gram-negative cell wall as:
·
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.
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.
·
A
molecular sieve for small water-soluble molecules.
·
An
absorption site for bacteriophage.
·
An
absorption site for cellular conjugation.
·
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:
·
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:
·
Binding
of platelets and fibrin around the bacterial cell to evade phagocytosis,
promote fibrin deposition on heart valves and promote blood clots.
·
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 owel 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
Gram-negative 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.
