Molecular Mechanisms of Microbial
Pathogenesis and Virulence
Controlling Infectious Diseases
1. The pre-antibiotic Era- Improving Sanitation & hygiene education
•
Typhoid (Salmonella),
•
Plague (Yersina pestis)
•
Syphilis (Treponema pallidum)
2. The antibiotic Era (1940-70)- Defeat of infectious bacteria?
3. Post-antibiotic Era - Emergence and reemergence of infectious
diseases.
•
Deaths due to TB (3 million) & Diarrhea (3-4 million)
•
Legionnaires disease (Legionella pneumoniae)
•
Lyme Disease (Borriella burgdoferi)
•
Gastric ulcers & cancer: Helicobacter pyori
•
Campylobacter jejuni
•
7 strains of E. coli
•
Viral agents:SARS, Morbilli, HIV, Hunta
We have lost control over infectious diseases? Why?
1. Role of Antibiotics:
Overuse of antibiotics / antimicrobials has increased drug resistant
microbes; no new antibiotics have been discovered in the past 20 yr
Some examples of antimicrobial ressistance:
•
•
Pseudomonas aeruginosa dominate hospital burn units
Emergence of multiple (methicillin) resistant staphylococcus
aureus (MRSA); Some strains are now resistant to vancomycin,
the only effective remaining antibiotic
Decade
2. Improved standard of living:
Air conditioners and Legionella
3. Removing and / or substituting environments:
SARS, HIV, Lyme disease
4. Aging population, compromised immunity
Microbes adapt to the stress and pressure placed on
them
1. Physiological Adaptation – Quorum sensing
2. Genetic Adaptation
Physiology
Trojan Horse:
quorum sensing
Acquiring new genes
- Genetic
Various stages of bacteria eucaryote cell interaction during infection
Quorum sensing - bacterial signaling:
• Low molecular weight compounds
• Inhibit macrophage & T lymphocyte functioning
• Able to determine cell density- switch on / off genes
• Increase density to an effective infectivity dose without
alerting host of impending attack; pathogenesis genes not
switched on.
• Legionella in cooling towers
Pathogenic microbes subvert functions of hosts in many ways:Host signaling,
host-parasite signaling; parasite signaling
Acquiring New Genes- Horizontal Gene Transfer
& Genetic Recombination
1. Horizontal Gene Transfer Mechanisms:
• Transformation
• Transduction
• Conjugation
2. Fate of the Transferred Genetic Material:
• Incorporated into the hosts genome - the mechanism
• Not incorporated into the hosts genome
3. Consequences for the Host Cell:
•New functions
Genetic Recombination is:
• An exchange between two homologous
DNA molecules - general / homologous
recombination. What is the mechanism
• Results in a new combination of genes
Mechanism of recombination - E.coli model:
•Complex, 25 genes: recA genes essential &
universal
• Homologous DNA pair up
• Donor DNA nicked (nuclease) & displaced
(helicase) eg E.coli: RecBCD - nuclease +helicase
• Reunion of paired segments by strand
invasion (Single Strand Binding protein +
RecA complex)
•Exchange of homologous DNA - DNA
polymerase & DNA ligase
Recombination results in a new combination of genes
•Natural process
•Occurs extensively in all cell types (domains Bacteria, Archaea & Eucarya)
•Diploid - Eucarya (sexual reproduction)
•Haploid - Bacteria-Bacteria; Bacteria - phage.
•Important for all life- gene transfer mechanisms are different in
Bacteria / Archaea and Eucarya but recombination results.
•New genotypes evolve
•Random across the genome; directly proportional to the distance
Evidence for Horizontal Gene Transfer
Mitochondria - Evidence for
lateral gene transfer
Mitochondria – Classic evidence for lateral gene transfer and
genome miniaturization?
•Mitochondria are symbiotic bacterium in a eucaryote: multiply by
cell division, posses 300-400 proteins in a complex membrane (IM
& OM) with electron transport chain (aerobic respiration).
• Miniaturized genome: Humans- 16-18kb (37 genes, 13 proteins,
22tRNA, 12S & 16S rRNA (bacteria-like transcription &
translation & antibiotic susceptibility)
•Mitochondrial DNA has 5-10 times higher mutation rates than
nuclear DNA: possibly due to mutagenic environment (free radicals
& noxious agents) & inefficient repair system; May be responsible
for maternal inherited disorders
•Mitochondria use an extreme form of “wobble” for translation
(22 tRNA rather than the normal 30 tRNA molecules); some
codons specify aa that differ from the accepted universal code
•The nucleus contains genes for mitochondrial DNA replication,
transcription and translation; imported into the mitochondria as
polypeptides (remember self assembly of cell membranes)
Mitochondria genesis and function is a product of both the
mitochondria and nuclear–encoded genes. Consequently, a
mechanism for the coordination of transcription must exist.
Is this under mitochondrial (prokaryotic) control
The Human Mitochondria genome database at the URL http://www.mitomap.org/
Parasitism also leads to genome miniaturization
•Host provides metabolic and physiological requirements
•Mycoplasma genitallium: 580 kb, 470 predicted ORF; Similar to
the number of genes which encode human mitochondria. No M.
genitallium nuclear encoded genes found in the eucaryotic
nucleus.
•There is a price to pay for the reduced genome and maintaining
parasitism: gene addition. Significant number of unique genes
code for adhesins, attachment organelles and a number of
different membrane-surface antigens directed towards evasion
of the immune system
M. peumoniae genome is 816kb (an additional 209 ORFs). How did
this difference occur in the same genus? http://www.zmbh.uniheidelberg.de/M_pneumoniae/genome/G_Comparison.html Gene
transfer, of course!!!
Microbial Gene Transfer: How are genes
transferred? What are the consequences?
Gene transfer in the microbial world
•Involves a donor & a
recipient cell
• DNA is transferred by
•Transformation
•Homologous
recombination
•Transductuction
•Heritable traits
•Conjugation
Chromosome
Plasmid
Transformation
Remember that transformation may have different meanings
Cell Biologists interpretation: Transformed cell lines means
immortalized cells. This can occur by DNA mutation or by gene
transfer mechanisms
Microbiologists interpretation: Transformation is one the 3
methods of gene transfer
Don’t learn transformation in isolation. Make connects
with the knowledge you already have on cell membranes
Transformation:
•Cell death & DNA fragments released into the
environment
•DNA uptake by related recipient species
•DNA integration with recipients chromosome
•Recipient & its progeny cells = genotypically distinct
•State of the cell, state of DNA, integration of the DNA
•Competence, DNA uptake, Integration of DNATransfection, Artficially induced competence, DNA
transfer by electroporation
•Recipients should be competent- only some strains naturally competent; artificially induced competency (E.coli)
•Genetic (regulatory) basis for competency: Membrane-associated DNA binding protein, autolysin, nucleases
•Bacillus subtilis- Quorum sensing cell number dependent competency
•Gram -ve: uptake of ds DNA but periplasmic space nucleases renders it ssDNA
•Gram +ve: dsDNA degraded to ss DNA & then taken up (S. pneumoniae- 10 molecules of 15-20kb dsDNA bind
to DNA binding proteins but only 8kb ss DNA enters the cytoplasm)
•Initial DNA binding is reversible but later becomes irreversible
•After uptake, the ss DNA binds to competency-specific proteins- nuclease attack proof
Transformation under natural conditions:
• E. coli is not naturally competent. Uptake artificially induced by
electric field or chemicals. ds linear or ds plasmid DNA. Maximum
of 20% population transformed.
•Most human / animal microbes not naturally competent (exception
S. pneumoniae & Neisseria)
•Soil & water microbes naturally competent; 5% of cultivable soil
microbes are competent. Possible reason- soil is a harsh extreme
environment lacking nutrients. DNA of lysed cells binds to soil, is
protected from nucleases and becomes food. In the intestinal
tract, DNA is rapidly degraded by nucleases and not available.
“Adaptive induced strategy”
Transduction
Transduction: Bacteriophage (phage) mediated gene transfer
•Bacterium (host) phage specific interaction: phage attaches to
host cell’s surface receptors & injects DNA leaving the capsid
outside.
•Inside the cell, DNA can either:
(a) reproduce to form phage and lyse the host to release the
phage progeny (process called lytic) OR
(b) integrate into the host genome (process called lysogeny).
Lysogenic phage may contain phage-encoded toxin genes
(botulinum,diptheria, cholera and E. coli O157:H7 bloody
diarrhea & kidney failure). Lysogenic phage can be induced into
a lytic cycle
• Some phage progeny released from the lytic may contain host
DNA (transducing phage) which is transfered into a new host in the
next infection cycle. The foreign host DNA can integrate by
homologous recombination or be lost – the process is called
generalised transduction (see next figure)
Generalised transduction
Conjugation
Conjugation
Cell to cell contact between a donor and a recipient cell in
which genes are transferred.
Conjugation process requires different proteins- one protein
forms a bridge between the two cells (pili) and another is
required for transfer of the gene. The genes are located on
plasmids.
Conjugation
• Understanding Plasmids
1. Physical nature of plasmids
2. Replication of plasmids
3. Cell to cell transfer of plasmids by conjugation
4. Types of plasmids & their biological significance
5. Resistance Plasmids
6. Toxins & other virulence characteristics
7. Bacteriocins
Understanding Plasmids
Plasmids
are genetic elements
replicate independently
• Plasmids do not have an extracellular form like a virus
•Thousands of types of plasmids are known; E. coli has over 300 different
types
1. Physical nature of plasmids
• DS DNA
• Circular (but linear forms are also known)
• 1 - 1000 kb in size
• Supercoiled
• Plasmids can be eliminated - cured; F plasmid by acridine orange
• Episomes - plasmids that integrate into the host chromosome
• Isolation and purification possible
Understanding Plasmids
2. Replication of plasmids
• Involves normal cell enzymes
• Synchronised event during host cell division
•1 to >100 copies per cell- controlled by genes on the plasmid
•Many different types of plasmids can reside in one cell- regulated by genes on the
plasmid eg Borriella burgdoferi- 17 types of circular & linear plasmids. Incompatible
(Inc) group plasmids unable to coexist as the replication mechanisms are in common
• Similar replication mechanism to that for chromosomes - initiated at the origin of
replication, unidirectional or bi-directional
•Small size of plasmids means rapid replication- one-tenth time of cell division
•Phage phiX174 replicates by rolling circle which has a ss intermediate- ss DNA
plasmids
•Linear plasmids replicate by a priming protein bound to the 5’ end
Understanding Plasmids
3. Cell to cell transfer of plasmids by conjugation
Conjugation plasmids encode (i) mating pair functions and (ii) DNA
transfer (transmissibility) and replication.
Two types of types of plasmids are known: (i) self –transmissible
and (ii) conjugative transposons
Self-transmissible Plasmids: Large >30kb plasmids.Can transfer
themselves (F+); mobilisable plasmids: Unable to transfer
themselves as they lack the ability to form a mating bridge (F-)
Conjugative Transposons: Integrate into host chromosome. It
can mobilise the transfer of the chromosomal DNA from one
cell to another. Strains that transfer high large amounts of
chromosomal DNA during conjugation are called high frequency
recombinants (Hfr)
Insertion sequences (IS) assist in the
integration of transposons (Tn) into
homologus sites of recipients genome.
Different Hfr strains are produced as a
result
Genes for conjugative transfer
Origin of transfer
Replication & segregation genes of F plasmid
99kb F (Fertility) Plasmid Genetic Map (E. coli)
Understanding Plasmids
4. Types of plasmids & their biological significance
Remember- essential host functions not part of plasmid
genes
Pseudomonas- entire metabolic degradation pathways of
unique compounds – camphor, napthalene.
Cryptic plasmids – we know little of the plasmid functions
Understanding Plasmids
5. Resistance Plasmids (R plasmids)
R100 carries resistance for sulfonamdes, tetracyline,
chloramphenicol, spectinomycin, mercury. Broad enteric host
range- Escherichia, Proteus, Klebsiella, Salmonella, Shigella,
Understanding Plasmids
6. Toxins & other virulence characteristics
• Ability to attach and colonise a host
o E. coli Colonisation Factor Antigen (CFA) assists in
attaching to intestinal epithelia
• production of toxins, enzymes that damage the host
o E coli hemolysin- lyse RBS & E. coli enterotoxininduce salt and water secretion into the bowel
Understanding Plasmids
7. Bacteriocins
Bacterocins inhibit or kill related species or different strains of
the same species (limited inhibitory spectrum to antibiotics)
E. coli plasmids- Col
Insertion Sequences, Transposons & Integrons
Mobilised via plasmids.
Promote changes to the host DNA- Rearrange and or delete
genes
Integrons are IS elements or transposons which create and
move large gene clusters as a single unit - PAI