Bacteriology

Dr. Salleh; Medical Microbiology; Phase II; MBBS-ims, MSU; Bacteriology 1

BACTERIILOGY

Lecture notes (2):

Bacteria: Structure-Function-Pathogenicity Relationships

Table of Contents

 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)

• Adhesion
• Alternate complement pathway
• Bacterial growth curve
• Capsule
• Cell wall structure
• Cytoplasmic membrane
• Disseminated intravascular coagulation
• Endospore
• 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 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).
• Macrophage production of tumor necrosis factor which results in various
effects including:
• 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.
• 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.

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