2. Infections in mankind
Infections in all manner of living
organisms are caused by all sorts of
microorganisms
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Bacteria
Viruses
Single-celled eukaryotes
Etc.
3.
4.
5. Using modern molecular biology
to combat infection
Molecular mechanisms for invading
pathogens best understood for pathogenic
bacteria
◦ Especially those related to E. coli
Bacterial methods are the easiest to
understand
◦ Viruses interact with host cell genome
◦ Single-celled eukaryotic infections are the most
difficult to understand
6. Molecular approaches to
diagnosis
Identification of pathogenic bacteria is often difficult
◦ Bacteria may grow slowly, or not at all outside host cells
Instead of culturing the bacteria, new techniques in
nucleic acid technology are being used.
7. ssu rRNA
Small subunit ribosomal RNA
◦ Each species is different
◦ Bacteria have 16S rRNA
◦ Eukaryotes have 18S rRNA
◦ Diagnosing pathogenic bacteria by ribosomal
RNA sequences is faster than culturing
techniques
8. Ribotyping
Detailed restriction analysis of rRNA genes
DNA from a strain is digested with several
different restriction enzymes
Fragments separated by gel elctrophoresis
Fragments then submitted to Southern Blot test
A probe that recognizes part of the 16S rRNA
sequence is used.
Uses large amounts of DNA
9. PCR
Uses small amounts of DNA
Primers that recognize the conserved region of 16S
rRNA
The fragment is compared to a database of known
organisms
Works well with bacteria that cannot be cultured well.
10. Checkerboard Hybirdization
Allows multiple bacteria to be detected
and identified in one sample
Probes are applied in horizontal lines
across a hybridization membrane
◦ The probes correspond to different bacterial
species
16S genes are amplified by PCR
◦ Fragments are labeled with a fluorescent
dye, and added vertically to the membrane
◦ After hybridization, the membrane is washed
to remove unbound DNA and the hybridized
samples appear as bright dots
12. Virulence segments
Virulence factors are properties that allow
microorganisms cause infections.
◦ Virulence factors can be broken down into three
groups
Those required for invasion of the host
Those required for life inside the host
Those for aggression against the host
13. Mobile virulence segments
In some cases, the DNA that encodes for virulence
factors are borne by virulence plasmids
Some are carried by lysogenic bacteriophages that
are inserted into the bacterial chromosomes of
some strains
Pathenogenicity islands
◦ DNA segments are grouped together and flanked by repeats
May move as a unit by transposition
15. Implications of mobility
Closely related bacterial strains are very different in their
ability to cause disease.
Virulence factors can be transferred to harmless bacteria,
creating novel pathogens
If the harmless strain is a very close relative, we get a new
variant of the old disease
If it isn’t, we run the possibility of having a genuinely new
pathogen that does not act like the old disease.
◦ Yersinia pestis
16.
17. Attachment and entry
Attachment is the first step in many infections
There are two type of adhesions : fimbrial and
nonfimbrial
◦ Pili are thin filaments from the membrane that incorporate
adhesions at the tip
◦ Nonfimbrial adshesions are found on the bacterial cell
surface.
20. The second step : Invasins
Not all bacteria have the ability to enter the host
cell
◦ Some only attach to the outside
◦ Some cells (such as phagocytic cells) absorb the bacterium
but then fail to destroy the bacterium.
◦ Some bacteria utilize invasins, which induce the host cell
into eating them.
22. Turning the tables on bacteria
With the spread of antibacterial
resistance, scientists are considering alternative
approaches
One of these alternatives is to design antiadhesin
drugs that will bind to the adhesin and block
attachment.
◦ Through binding studies and X-ray crystallography, it
has been revealed that pathogenic E. coli adhesins
(FimH) bind to mannose residues on mammalian
glcoproteins
◦ May be blocked by different alkyl- and aryl-mannose
derivatives
23. Decoys
Another approach would be to use genetically engineered gut
bacteria.
◦ Such as nonpathogenic E. coli.
These bacteria would express target oligosaccharides for
adhesins on their cell surfaces, acting as decoys.
Avoid the need of expensive sugar derivatives
One decoy could carry multiple adhesin targets.
24. Inducing non harmful competition
The third possibility may be to equip
nonpathogenic strains with genes for
adhesins and/or invasins from pathogenic
species
These engineered strains would then
compete for receptor sites
By taking away sites from pathogenic
bacteria, the effect of these pathogenic
bacteria may be lessened.
These engineered cells could also be used
for delivering protein pharmacueticals or
segments of DNA for gene therapy
All alternatives are currently in experimental
stages.
25. Iron acquisition
Almost all bacteria need iron
◦ Iron serves as a cofactor for many enzymes
Especially for respiration
Free iron in the body is kept low due to specialized
proteins that tightly bind to it
◦ Surplus iron is bound by transferrin and lactoferrin, two iron
transport molecules
◦ Ferritin, an iron storage protein
26. Siderophores
Siderophores are iron chelators that are
excreted by bacteria, bind iron, and return to
the bacteria cell by specialized transport
systems
The best known siderophore is Enterochelin
(enterobactin).
◦ It is made by E. coli and other enteric bacteria
◦ The FEP transport system transporrts the
enterochelin and FE complex back across the
membrane
◦ Enterochelin bind iron so tightly, it must be
destroyed by Fes protein
◦ Enterochelin is not strong enough to unbind Fe
from transferrin
28. Pathogenic bacteria
Pathogenic bacteria often possess more
potent siderophores that can retrieve iron
from transferrin.
Two examples are mycobactin and
yersinabactin
Yersiniabactin is widespread in the
enteric family, and part of the
pathogenicity island in Yersinia
Other bacteria utilize hemolysin, which
lyses the red blood cells and frees the
hemoglobin (where the iron resides)
30. Bacterial toxins
Bacteria will mount aggressive attacks
against eukaryotic cells by utilizing
toxins.
Toxins
◦ In the broadest sense, anything that
damages eukaryotic cells.
◦ Can be accidental or deliberate
31. Endotoxins
Endotoxins are actually the lipid
components of lipopolysaccharides
◦ LPS forms part of the outer membrane of
gram negative bacteria.
◦ If bacteria are killed, they released LPS
◦ Immune cells attach to LPS by CD14
receptor,
◦ Triggers the release of cytokines
◦ Simultaneous death of massive amounts
of bacteria may result in sepsis.
33. Type I Exotoxin
Do not enter the cell
Bind to a receptor on the cell surface
Stable ( heat stable toxin a) is made
by some strains of e.coli.
◦ Causes overproduction of cyclic GMP
34. Type II Exotoxin
Act on the cell membrane of the target
cell
Some degrade the membrane lipids
themselves or create holes in the
membrane
Hemolysin A disrupts the membrane of
many types of animal cells.
35. Type III Exotoxin
Enter a target cell
Consist of two factors
◦ Toxic protein
◦ Delivery protein
◦ Several interesting examples
36. ADP-Ribosylating toxins
Large family of toxins that hydrolyzes the
cofactor NAD and ADP-ribose
The fragments are transferred to an acceptor
molecule (usually one that binds GTP)
The target becomes locked in a binding
formation, leacing it unable to continue in its
normal processes.
Both cholera and diphtheria toxins use ADPribosylation, but on different targets
◦ Cholera toxins inactivate the G-proteins that
control adenylate cyclase
◦ Diphtheria toxins attack elongation factor EF-2, a
translation factor used for protein synthesis
38. Bacteriophages
Certain other bacteriophages can use
enzymes that utilize NAD and ADPribosylate proteins of their hosts
Usually, it is several bacterial proteins
that are modified so that the target of the
protein is uncertain
◦ Blocking key enzymes can cripple host
metabolism
◦ Modification of host polymerases
Bacteriophage T4, which modifies host E.coli
polymerases, which then loses its ability to
transcribe E.coli genes but not T4 genes.
39. Cholera
Vibrio cholerae does not enter host
tissues
◦ Attaches to the exterior wall of cells lining
small intestine
The bacterium severely damages the
host tissue by excreting cholera toxin
The toxin attacks the epithelial cells,
causing them to lose sodium ions and
water into the intestinal tract
Cholera causes loss of body fluids by
massive diarrhea and then death by
dehydration
40. Virulence proteins of Vibrio
cholerae
Virulence proteins not only include the
cholera toxin, but also pilis and cell-surface
adhesins
The genes for the toxin are carried by a
bacteriophage (CTXphi) that lysogenizes
cholera bacterium
Synthesis of the virulance factors is partially
regulated by the ToxR protein in the wall of
the inner membrane of the bacteium.
◦ This protein ‘senses’ the correct environment and
activates the genes
◦ The internal domain of the protein binds to the
promoters of the virulence genes
42. Cholera toxin
Cholera toxin consists of two protein subunits
◦ Encoded by ctxAB genes
The original A protein is split into two pieces
by a protease and linked by a disulfide bond
The B protein forms a ring like sturctue of five
subunits which surrounds the A subunit
The B protein attaches to the galactose end
of a ganglioside glycolipid.
After attachement, part of the A protein splits
from the protein complex and enters the cell
44. Cholera toxin
After enterin the cell, the toxin splits NAD
into nicotinamide and ADP-ribose
◦ The ADP-ribose is used for ADP-ribosylate
target molecules
The toxin can actually ADP-ribosylate
many acceptors
◦ Free arginine and its derivatives
◦ Many other proteins
◦ Itself, increasing productivity by 50%
The true target is a G-protein, which
regulates adenylate cyclase
45. G-proteins and cholera toxin
Normally, a G-protein will be activatated, bind
to a GTP. And then bind to adenyl cyclase
GTP hydrolysis releases the G-protein and
deactivates it.
ADP ribosylation of an arginine residue
prevents the hydrolysis of the GTP and
results in the G-protein being locked in a
bound state
Causes hyperactivation of adenylate cyclase
and overproduction of cyclic AMP
Loss of sodium and water
GTP analogs that cannot by hydrolyzed show
similar effects.
46. Heat-labile enterotoxins
Cholera toxins and other heat labile
toxins are all variants of the same
toxin
Some enterotoxins in E.coli are
encoded on the Ent-plasmid which
may be transferred
All of these toxins have similar amino
acid sequences and cause the same
symptoms (in varying degrees of
severity)
48. Anthrax toxin
Anthrax is caused by the gram positive
bacterium Bacillus anthracis
In 1877 Rober Koch grew this organism
and demonstrated its ability to grow
spores
There are two important virulence factors
are exotoxins and the capsule, both on
different plasmids
The capsule protects against immune
cells
Thispathogen is very similar to other
Bacillus species
49.
50. Edema factor and Lethal
factor
Anthrax makes two toxins
◦ The edema factor, the first toxin, is an
adenylate cylase
Not toxic in of itself, but intensifies lethal factor
◦ The lethal factor is a protease
Disrupts the domains responsible for proteinprotein signaling
Lyses macrophages
Excessive release of interluekines results in
shock leading to respiratory failure and/or
cardiac failure
51. Antitoxin therapy
Most therapies rely on antibodies against
toxins
But now, more gene related approaches are
beginning to emerge
The dominant-negative mutation is one new
approach
◦ Dominant-negative mutations in the binding
subunit of the toxins
◦ These mutations typically result in inactive
proteins
◦ Occasionally, it will not only inactivate the
proteins themselves, but will also interfere with
functioning proteins
52. Mechanism of dominant-negative
mutations
Involves the binding of a defective subunti to
functional subunits resulting in an inactive
complex
Most of these mutations will affect proteins with
multiple subunits
Multisubunit B Proteins of A and B protein
complexes of cholera and anthrax toxins are a
good example
◦ This type of mutation has been deliberately isolated in
the protective antigen of the anthrax toxin
◦ Mixture of mutant and active subunits resulted in the
binding of A factors which allow the lethal factors to
be built, but not transported into the target cell
◦ Treatment with these modified proteins can protect
humans and mice from lethal doses of anthrax toxin
54. Polyvalent Inhibitor
Phage display is used to isolate
nonnatural peptides
These peptides bind weakly to single
proteins
If several of the these peptides are
attached together on a flexible backbone
(polyvalent inhibitor)
Binding to many target proteins occurs,
causing an increase in affinity
For this to work, the target must be a
multisubunit protein
56. Summary
Bacterial infections for the most part,
may be treated by antibiotics
Plasmids, bacterial viruses and
transposons move genes between
species
Analyzing toxins may allow us to
combat infections