Molecular Mechanisms of Microbial Pathogenesis and Virulence - PowerPoint PPT Presentation

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Molecular Mechanisms of Microbial Pathogenesis and Virulence

Controlling Infectious Diseases

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?

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 aeruginosadominate 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

Acquiring new genes - Genetic

Trojan Horse: quorum sensing

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.uni-heidelberg.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

Transformation
Transductuction
Conjugation

Homologous
recombination
Heritable traits

Involves a donor & a
recipient cell
DNA is transferred by

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 DNA-Transfection, 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

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

UnderstandingPlasmids
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

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

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

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

Replication & segregation genes of F plasmid

99kb F (Fertility) Plasmid Genetic Map (E. coli)

Origin of transfer

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

5. Resistance Plasmids (R plasmids)

R100 carries resistance for sulfonamdes, tetracyline, chloramphenicol, spectinomycin, mercury. Broad enteric host range- Escherichia, Proteus, Klebsiella, Salmonella, Shigella,