Bacterial infections

Bacterial Infections
Amith Reddy
Eastern New Mexico University

Infections in mankind


Infections in all manner of living
organisms are caused by all sorts of
microorganisms
◦
◦
◦
◦

Bacteria
Viruses
Single-celled eukaryotes
Etc.

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

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.

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

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

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.

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

FIGURE 21.1
Checkerboard Hybridization
Probes corresponding to 16S rRNA for each candidate bacterium are attached to a membrane filter in
long horizontal stripes (one candidate per stripe). To quickly identify a group of unknown pathogens,
mixed DNA is extracted from a sample and amplified by PCR using primers for 16S rRNA. The PCR
fragments are tagged with a fluorescent dye and applied in vertical stripes. Each sample is thus
exposed to each probe. Wherever a 16S PCR fragment matches a 16S probe, the two bind, forming a
strong fluorescent signal where the two stripes intersect.
Biotechnology by Clark and Pazdernik
Copyright © 2012 by Academic Press. All rights reserved.

11

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

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

FIGURE 21.2
Pathogenicity Islands of Escherichia coli
Different strains of E. coli vary greatly in their abilities to cause disease. Pathogenic E. coli have
unique regions of DNA that are not found in nonpathogenic strains, called pathogenicity islands (PAI).
The regions are designated I–IV, where I encodes alpha-hemolysin; II encodes alpha-hemolysin and
fimbriae; III encodes fimbriae; and IV encodes the yersiniabactin iron-chelating system.

14

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

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.

FIGURE 21.3
Bacterial Adhesins
(A) The surface of some bacterial cells is covered with pili (fimbriae), composed of helically arranged
pilin protein. At the tip of the pili are adhesins, which recognize the surface glycoproteins of the host
cell. (B) Nonfimbrial adhesins are found on the surface of the bacterial cell.

18

FIGURE 21.4
Assembly of Bacterial Pilus
The pilus has two segments, the tip and the shaft, which are assembled on the outside of the
bacterium. The protein subunits of the pilus are synthesized in the cytoplasm and exported across
both membranes. The proteins are folded in the periplasmic space. The pilus is assembled from the
tip to the base by starting with the adhesin protein and other tip proteins and then adding further layers
of pilin protein beneath.

19

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.

SMBC http://www.smbc-comics.com/index.php?db=comics&id=2331

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

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.

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.

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

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

FIGURE 21.5
Acquisition and Uptake of Iron by Enterochelin
FepA protein is the outer membrane receptor for enterochelin. Energy for crossing the outer
membrane requires the TonB system, which uses the proton motive force. The FepB protein gets
enterochelin from FepA and passes it to the inner membrane permease, consisting of FepG and
FepD. The FepC protein uses ATP to supply energy to FepGD for transport across the inner
membrane.

27

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)

UNN 21.1


29

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

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.

Exotoxins


Most pathogenic bacteria have toxins
that deliberately harm the host.



Secreted by living cells



Mostly exotoxins are proteins.

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

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.


Type III Exotoxin
Enter a target cell
 Consist of two factors


◦ Toxic protein
◦ Delivery protein
◦ Several interesting examples

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

FIGURE 21.6
ADP-Ribosylating Toxins
Nicotinamide adenine dinucleotide (NAD) consists of ADP-ribose linked to nicotinamide. These are
split by some bacterial toxins and the ADP-ribose is attached to a GTP-binding protein, thus
preventing it from splitting GTP.

37

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.

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


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

FIGURE 21.7

Regulation of V. cholerae Virulence Genes
ToxR of V. cholerae sits in the cytoplasmic membrane, where it senses that the cell is in a human host
and directly activates the genes for cholera toxin and for attachment.

41

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

FIGURE 21.8
Structure and Entry of Cholera Toxin
(A) Cholera toxin consists of an A protein plus five copies of B protein. The A protein is split into two
halves (A1 and A2), held together by a disulfide bond. The B protein forms a ring with a central
channel for the A1-S-S-A2 protein. (B) Cholera toxin binds to the host cell when the five B-subunits
recognize ganglioside GM1. The disulfide bond in A1-S-S-A2 breaks, allowing A1 to enter the cell.

43

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

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.

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)


FIGURE 21.9
Mechanism of Action of Cholera Toxin
In their inactive state G proteins bind GDP. When an external signal activates the G protein, the GDP
is exchanged for GTP. The G protein then activates adenylate cyclase. Normally, the GTP is
hydrolyzed and the G protein returns to its inactive state. Cholera toxin cleaves NAD and attaches the
ADP-ribose group to an arginine in the G protein. This prevents the G protein from splitting GTP.
Consequently adenylate cyclase does not get turned off and continues to produce cyclic AMP.

47

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


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

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

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

FIGURE 21.10
Activation of Protective Antigen
The pag gene of the pOX1 plasmid encodes the protective antigen (PA) of B. anthracis. PA is
synthesized as an inactive precursor that is cleaved and assembled into a ring structure.

53

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


FIGURE 21.11
Dominant-Negative Toxin Mutations
The PA63 protein (protective antigen) binds the lethal factor (LF) and edema factor (EF) and
transports them into the target cell cytoplasm via an endocytotic vesicle. The dominant-negative
inhibitory (DNI) mutant of the PA63 protein (purple) assembles together with normal PA63
monomers (pink) to give an inactive complex that cannot release the LF and EF toxins from the
vesicle into the cytoplasm.

55

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


Bacterial infections

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (20)

En vedette

En vedette (16)

Similaire à Bacterial infections

Similaire à Bacterial infections (20)

Plus de Amith Reddy

Plus de Amith Reddy (6)

Dernier

Dernier (20)

Bacterial infections