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

Various stages of bacteria eucaryote cell interaction during infection

Trojan Horse: quorum sensing

Physiology

Acquiring new genes - Genetic

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

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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

Chromosome Plasmid

•Involves a donor & a recipient cell• DNA is transferred by

•Homologous recombination

•Heritable traits

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

ConjugationCell 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

Plasmidsare 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)

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

Insertion sequences (IS) assist in the integration of transposons (Tn) into homologus sites of recipients genome. Different Hfr strains are produced as a result

Replication & segregation genes of F plasmid

Genes for conjugative transfer

Origin of transfer

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 enterotoxin- induce 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