Mechanisms and principles of microbial pathogenesis

Question 1

Define mutualism. Where is this seen?

Answer 1

Where both the microbe and the host benefits from the co-existence and neither suffers, e.g. normal flora, gut flora.

Question 2

What is a commensal microbe? Where is this seen?

Answer 2

Similar to mutualism, but the microbe benefits and the host doesn’t, e.g. normal flora, gut flora.

Question 3

Define a parasite.

Answer 3

A microbe that benefits from the host, but the host suffers. This is when disease arises.

Question 4

List Koch’s postulates [4]. What are the exceptions?

Answer 4

1. Microbe is in diseased tissue but not in normal tissue (ex: pathogen can be a coloniser and only sometimes cause disease, e.g. S. aureus, H. pylori)
2. Microbe can be isolated from diseased tissue as a pure culture (ex: non-culturable organism, e.g. T. pallidum requires PCR identification; disease may require multiple organisms)
3. Microbe can cause disease when inoculated into an animal or man (ex: N. gonorrhoae has no animal model)
4. Microbe must then be reisolated in pure culture from animal or man (ex: disease requires multiple microbes, e.g. peridontal disease such as tooth decay)

Question 5

List the molecular Koch’s postulates [4]. What are the exceptions?

Answer 5

1. A virulence gene is associated with a microbe that cause disease, but is absent or inactive in strains that fail to cause disease (ex: rendundant virulence factors, e.g. PVL (cytotoxin) positive CA-MRSA cause more severe disease, but not essential)
2. Disruption of a gene in a virulent strain causes avirulence (ex: PVL positive MRSA)
3. Introducing a cloned gene into an avirulent strain causes virulence (ex: PVL positive MRSA)
4. The gene is expressed during infection

Question 6

What are the benefits of studying virulence factors?

Answer 6

It allows us to fight disease more effectively:

• New treatments (drug targets, other therapeutics)
• Design vaccines
• New diagnostic markers (understand how disease takes place, or how an individual responds to treatment)
• Epidemiological markers (genotype pathogens, track them through populations)

Gives us an insight into host biology:

• Toxins: signalling (e.g. the neuromuscular junction and Clostridium botulinum and perfingens which interfere with cell signalling at the neuromuscular junction, these have been important in understanding neurotransmission)

Can give insights into molecular evolution

Question 7

What makes a successful pathogen? [5]

Answer 7

1. Colonise the host or tissues
2. Persist in the presence of a variety of host defences, e.g. immune response. Many pathogens can avoid, subvert, or circumvent these host and cellular defenses.
3. Replicate
4. Spread
5. Cause disease

Question 8

What are the three stages of pathogenesis?

Answer 8

1. Colonisation
2. Invasion
3. Proliferation

Question 9

What are the two branches of immunity protecting against infection?

Answer 9

• Innate immune response: non-specific constitutive host response
• Adaptive immunity: specific

Question 10

Outline adherence and entry of a pathogen. Give examples.

Answer 10

The pathogen needs to adhere and enter through mucosal surfaces, which is mediated by a specific ligand-receptor interaction.

• For example, in HIV the ligand is gp120 which interacts with CD4 receptors in T-helper cells.

These interactions determines the tissue tropism, the type of cells that a pathogen targets as well as the organism.

• e.g. feline leukaemia virus causes leukaemia in cats, but not humans.

Some pathogens enter into non-phagocytic cells and utilise cellular attributes to suit the needs of the pathogen.

• e.g. Yersinia spp. can exist inside cells, taking advantage of preexisting pathways: Inv ligand on surface interacts with B1 integrins on host cell surfaces; Listeria has an InlB/E ligand that interacts with E-cadherin or C-met on host cells.

Question 11

What are the two host environments a pathogen can exist in? Give examples of each.

Answer 11

1. Extracellular
   
   • ​​Vibrio cholera: on the surface of the small intestine, produce toxins and causes disease.
   • Diptheria.
2. Intracellular (obligate/facultative)
   
   • Can survive in unique compartments, e.g. Tuberculosis invades macrophages and survives in the phagolysosome)
   • Some can persist within the cytosol, e.g. Listeria
   • Some can survive in lysosomes, e.g. Coxiella leishmania

Question 12

Outline dissemination.

Answer 12

Dissemination can mean the spreading of a pathogen from one organ to another, or from host to host within a population. There are various methods of transmission, including oral-fecal, aerosols, and sexual transmission.

Question 13

What are the two types of pathogens? Give examples.

Answer 13

1. Primary pathogens: regularly cause disease in at least a portion of immunocompetent individuals.
   
   • Always cause disease: Chlamydia, Neisseria gonorrhoea, influenza
   • Sometimes cause disease: Staph. aureus, Strep. pyogenes, H. pylori.
2. Opportunistic pathogens: only cause disease in immunocompromised hosts.
   
   • Pseudomonas, enterococcus.

Question 14

How are different virulence factors identified? Give examples.

Answer 14

• Purify proteins: secreted proteins or abundant surface molecules, easily identified in cultured media, e.g. cholera toxin, diptheria toxin.
• Serum antibody probes: highly antigenic proteins.
• Microarrays, proteomics and promoter traps: stage-specific genes/genes only expressed in the host, as opposed to those needed to survive in other environments.
• Signature tagged mutagenesis: gene products required for survival of the pathogen in cells/hosts, can knockout genes to see if they’re essential pathogenesis.
• Metagenomics: other gene products that are unique to pathogens, e.g. Candida vs saccharomyces are very similar in terms of their genome, one is pathogenic and the other is not, looking at the differences in their genomes can help identify virulence factors.
• Animal models: used routinely as a tool to extablish the pathophysiology of disease, used because it is not ethical to infect humans, raises questions of validity (different physiologies, susceptibilities), expensive, difficult to screen, e.g. Tuberculosis in mice is good to study active disease, but is poor in understanding latent disease as granulomas do not form.

Question 15

How are virulence factors quantified using animal models?

Answer 15

Animal models are used routinely as a tool to extablish the pathophysiology of disease, and are used because it is not ethical to infect humans. However, it raises questions of validity (different physiologies, susceptibilities), is expensive, and sometimes difficult to screen, e.g. Tuberculosis in mice is good to study active disease, but is poor in understanding latent disease as granulomas do not form.

The animal model can reflect the human disease:

• End points (LD50) can be measured in animals based on dose of innoculant, which is used to quantify virulence of a specific strain.
• Determine which organs are colonised.

Question 16

What are the advantages and disadvantages of using a tissue model to study virulence?

Answer 16

Advantages:

• Cheaper
• Simpler
• Controlled environment
• High-throughput screens (automated)

Disadvantages:

• Immortalised cells lines are used which are essentially cancerous cells, e.g. THP1 macrophages, and they constantly proliferate, unlike normal cells. They do not give a completely accurate picture: altered behaviours compared to normal cell types, but act as a good model.
• Does not give the whole picture, i.e. in vivo there are multiple cell types.
• Does not accout for the different environments in vivo.

Question 17

What are the ways in which microbial genetics is studied?

Answer 17

• Introduce DNA (transformation, transduction, conjugation, electroporation).
• Genetic markers (drug resistance, enzymes such as b-gal, GFP (green fluorescent protein) to track expression).
• Random gene knockouts to see their effects.
• Episomal expression (plasmid expresses the virulence factor in a knockout, molecular Koch’s postulates).
• Targeted mutations to the chromosome, if the gene is known.

Question 18

List some techniques used to manipulate bacterial genomes to study virulence factors.

Answer 18

• Transposons in insertional mutagenesis.
  
  • Sequences are targeted in a chromosome for disruption, to establish virulence of a gene. Insertion sequences (transposases) are found naturally in the bacterial genome, and their ability to move in and out of the genome is used to target specific genes. This is done with a selectable marker (tetracycline resistance) alongside the knockout gene. Bacterial genes transcribed as operons, so individual genes can be knocked out.
• Episomal expression using a shuttle vector, which is grown in E. coli and the target pathogen. Allows for complementation of a virulence factor knockout.
• Targeted mutants (secreted proteins/toxins/antigenic proteins) and genetic screens/selections (adhesion, cytotoxicity, invasion, survival genes, growth genes).
  
  • Transposon mutant screen: loss of function, however doesn’t work on virulence factors with redundant phenotype, or essential for survival.
  • Complementation screen: gain of function.
• Microarrays for gene expression detection, present in virulent strains but absent in avirulent strains.
• Proteomics, differences in virulent/avirulent strains.
• Metagenomics, comparing genes between healthy and diseased states.
• Signature tagged mutagenesis (STM).

Question 19

Outline signature tagged mutagenesis (STM).

Answer 19

Signature-tagged mutagenesis (STM) is a genetic technique used to study gene function.

• The gene in question is inactivated by insertional mutation; a transposon is used which inserts itself into the gene sequence.
• When that gene is transcribed and translated into a protein, the insertion of the transposon affects the protein structure and (in theory) prevents it from functioning. In STM, mutants are created by random transposon insertion and each transposon contains a different ‘tag’ sequence that uniquely identifies it.
• If an insertional mutant bacterium exhibits a phenotype of interest, such as susceptibility to an antibiotic it was previously resistant to, its genome can be sequenced and searched (using a computer) for any of the tags used in the experiment. When a tag is located, the gene that it disrupts is also thus located (it will reside somewhere between a start and stop codon which mark the boundaries of the gene).

STM can be used to discover which genes are critical to a pathogen’s virulence by injecting a ‘pool’ of different random mutants into an animal model (e.g. a mouse infection model) and observing which of the mutants survive and proliferate in the host. Those mutant pathogens that don’t survive in the host must have an inactivated gene, required for virulence. Hence, this is an example of a negative selection method.

Caveat: genes may not necessarily be involved in virulence, they could equally likely be a housekeeping gene.

Question 20

P. aeruginosa / C. elegans

Outline a case study in which a virulence factor was identified in a pathogen. What methods were used?

Answer 20

Pseudomonas aeruginosa (PA14 strain) is known to kill Caenorhabditis elegans. Using systematic mutagenesis of PA14 to identify mutants that do not kill C. elegans, phenazines were identified as a mediator of killing:

• The ability for worms to survive on a PA14 lawn was tested, all die within 3-4 days. Fast killing is observed on higher osmolarity medium → what is the mechanism behind this?
• The rapid kinetics of fast killing suggest diffusible toxins: growing worms indirectly from the lawn (filters) still killed the worms → proven!
• To identify the bacterial factors, transposon mutants for defective process were created. Fast killing mutants contained a single TnphoA insertion.
  
  • Flanking DNA was amplified using inverse PCR, sequenced, and the translation products were identified using GenBank and BlastX.
  • The DNA sequence tags in all mutants had homologies to pseudomonads, their function was affirmed using a complementation screen.
• Two mutants had insertions in the operon regulating phenazine production, strongly suggesting it mediated killing.
  
  • Deletions of the regulatory genes phnAphnB confirmed this, allowing survival of worms.
• Pyocyanin, the most extensively characterised phenazine, has been shown to be cytotoxic to eukaryotic and prokaryotic cells by forming ROS.
  
  • Worms with altered responses to oxidative stress were tested for resistance to fast killing: resistance/susceptibility of nematodes to oxidative stress correlated with resistance/susceptibility to fast killing → pyocyanin kills using oxidative stress, we have the mechanism!
• The bacterial factors involved in fast killing were tested in other models for their relevance in pathogenesis (Arabidopsis and mouse): some mutants caused lower mortality, and some none → these are relevant virulent genes!!