bacterial infections
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Bacterial Infections
Amith ReddyEastern New Mexico University
Infections in mankindInfections 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 infectionMolecular 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 genesDNA from a strain is digested with several
different restriction enzymesFragments separated by gel elctrophoresisFragments then submitted to Southern Blot
testA 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 HybirdizationAllows multiple bacteria to be detected
and identified in one sampleProbes are applied in horizontal lines
across a hybridization membrane◦The probes correspond to different bacterial
species16S 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
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Checkerboard HybridizationProbes 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.
FIGURE 21.1
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Virulence segmentsVirulence 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
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Pathogenicity Islands of Escherichia coliDifferent 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.
FIGURE 21.2
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
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 entryAttachment is the first step in many
infectionsThere 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.
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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.
FIGURE 21.3
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
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Assembly of Bacterial PilusThe 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.
FIGURE 21.4
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
The second step : InvasinsNot 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 bacteriaWith 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 competitionThe 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 acquisitionAlmost 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
SiderophoresSiderophores 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
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Acquisition and Uptake of Iron by EnterochelinFepA 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.
FIGURE 21.5
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Pathogenic bacteriaPathogenic 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)
29
UNN 21.1
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Bacterial toxinsBacteria 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
EndotoxinsEndotoxins 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.
ExotoxinsMost pathogenic bacteria have
toxins that deliberately harm the host.
Secreted by living cells
Mostly exotoxins are proteins.
Type I ExotoxinDo 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 ExotoxinAct on the cell membrane of the
target cellSome degrade the membrane
lipids themselves or create holes in the membrane
Hemolysin A disrupts the membrane of many types of animal cells.
Type III ExotoxinEnter a target cellConsist of two factors
◦Toxic protein ◦Delivery protein◦Several interesting examples
ADP-Ribosylating toxinsLarge family of toxins that hydrolyzes the
cofactor NAD and ADP-riboseThe 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 ADP-ribosylation, 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
37
ADP-Ribosylating ToxinsNicotinamide 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.
FIGURE 21.6
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
BacteriophagesCertain other bacteriophages can use
enzymes that utilize NAD and ADP-ribosylate 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.
CholeraVibrio 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 choleraeVirulence 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
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Regulation of V. cholerae Virulence GenesToxR 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.
FIGURE 21.7
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Cholera toxinCholera toxin consists of two protein subunits
◦ Encoded by ctxAB genesThe original A protein is split into two pieces
by a protease and linked by a disulfide bondThe B protein forms a ring like sturctue of five
subunits which surrounds the A subunitThe 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
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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.
FIGURE 21.8
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Cholera toxinAfter enterin the cell, the toxin splits
NAD into nicotinamide and ADP-ribose◦The ADP-ribose is used for ADP-ribosylate
target moleculesThe 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 toxinNormally, a G-protein will be activatated, bind
to a GTP. And then bind to adenyl cyclaseGTP 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 waterGTP analogs that cannot by hydrolyzed show
similar effects.
Heat-labile enterotoxinsCholera 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)
47
Mechanism of Action of Cholera ToxinIn 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.
FIGURE 21.9
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Anthrax toxinAnthrax is caused by the gram positive
bacterium Bacillus anthracisIn 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 cellsThispathogen is very similar to other
Bacillus species
Edema factor and Lethal factorAnthrax 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
protein-protein signaling Lyses macrophages Excessive release of interluekines results
in shock leading to respiratory failure and/or cardiac failure
Antitoxin therapyMost therapies rely on antibodies against
toxinsBut now, more gene related approaches
are beginning to emergeThe 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
53
Activation of Protective AntigenThe 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.
FIGURE 21.10
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
Polyvalent InhibitorPhage display is used to isolate
nonnatural peptidesThese peptides bind weakly to single
proteinsIf 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
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Dominant-Negative Toxin MutationsThe 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.
FIGURE 21.11
Biotechnology by Clark and Pazdernik Copyright © 2012 by Academic Press. All rights reserved.
SummaryBacterial 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
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