Studying bacterial pathogenesis: approaches and methods

Question 1

Why is it important to study bacterial pathogenesis?

Answer 1

To improve bacterial disease :
- prevention, eg. vaccines
- diagnosis, eg. toxin detection
- treatment, eg. Identify new therapy targets

Question 2

Bacterial pathogenesis involves bacterial and host factors.

List these factors

Answer 2

Bacterial:
*Virulence factors
*toxins
*immune evasion
*attachment
*motility
*gene regulation
*acute v chronic v dormant phases
*spread
*resistance

Host:
*Immune response
Innate
*skin
*phagocytes
*complement
*Fe restriction
adaptive
*antibodies
*lymphocytes
*macrophages

Question 3

How do we prove that a bacterium is responsible for a particular disease?

Answer 3

Koch’s postulates:
1. The bacterium is found in all people with disease
2. The bacterium can be isolated from patients and maintained in pure culture
3. The pure culture can be innoculated into a human volunteer or animal model and cause symptoms of disease
4. The bacterium can be reisolated from the volunteer or animal

eg. Helicobacter pylori

Question 4

Describe Hypothesis-driven research

Answer 4

Hypothesis -
What is the question? Is it worth asking?

Methods -
Can an experiment be designed that can answer the question? That is feasible?

Results -
Are the results clear? How reliable is the data?

Conclusions -
Can we answer the question? How does the interpretation impact on our understanding of bacterial pathogenesis? What still needs to be done?

Question 5

To investigate bacterial pathogenesis we need:

Answer 5

A. Clinical observation and epidemiology

B. Models of disease
in vitro and in vivo

C. Appropriate strains of bacteria to test
Including genetically modified bacteria

Question 6

Describe the Clinical observation and epidemiology of bacterial pathogenesis

Answer 6

Epidemiology - the study of spread and distribution of disease; In bacteriology it often includes the discrimination of different strains/variants and their spread.

Question 7

Describe the Clinical observation and epidemiology of toxic shock syndrome

Answer 7

What is the problem? -Rare fatal shock in young women

Common features of cases? Which tissues are targeted? -Associated with tampon use, systemic shock

Are all people equally susceptible? - Rare, so probably not

How does it spread (eg. oral-faecal, airborne, sexually)? - Not clear

Epidemiological links between cases? Sources of outbreaks? - No

What is the causative organism (Koch’s postulates)? - Staphylococcus aureus, common in the nose

Are all strains equally pathogenic? - No, only those carrying the tst gene cause disease

Does killing of the bacterium reduce symptoms? - No, as toxin acts too quickly

Question 8

Describe the Clinical observation and epidemiology of Helicobacter pylori

Answer 8

What is the problem? - Gastric ulcers

Common features of cases? Which tissues are targeted? - Helicobacter pylori in gastric samples (although previously thought to be due to acid reflux)

Are all people equally susceptible? - No, since many carriers without disease

How does it spread (eg. oral-faecal, airborne, sexually)? - Oral-faecal

Epidemiological links between cases? Sources of outbreaks? - No?

What is the causative organism (Koch’s postulates)? - Helicobacter pylori

Are all strains equally pathogenic? - Yes

Does killing the bacterium reduce symptoms? - Yes

Question 9

Describe the Clinical observation and epidemiology of tuberculosis

Answer 9

What is the problem? - Chronic lung infection, esp. HIV patients, developing countries

Common features of cases? Which tissues are targeted? - Lung infection, can be dormant

Are all people equally susceptible? - No, many carriers without disease (dormant phase)

How does it spread (eg. oral-faecal, airborne, sexually)? - Airborne

Epidemiological links between cases? Sources of outbreaks? - Yes

What is the causative organism (Koch’s postulates)?
Mycobacterium tuberculosis

Are all strains equally pathogenic? - Yes

Does killing the bacterium reduce symptoms? - Yes, but killing is difficult and strains becoming more drug resistant.

Question 10

How are good epidemiological studies designed?

Answer 10

-Clear definitions of patients “with” and “without” the disease
-Sufficient numbers of patients to investigate
(statistical significance)
-Collection of relevant information and samples
-Consider - ethics, patients are complex, logistics

Question 11

Describe the models of disease (laboratory/controlled conditions)

Answer 11

1. Bacterial behaviour in rich or specialised growth media (in vitro)
2. Bacterial behaviour in laboratory conditions (in vitro) that mimic in vivo
3. Animal models (in vivo)

Question 12

Describe the purpose behaviour on/in rich or specialised growth media

give an example of a specialised growth media

Answer 12

e.g. agar plates or tryptone soy broth medium (TSB)

Useful to identify:
- growth requirements - nutrients, O2, CO2, Fe
- motility
- ability to digest proteins, lipids, carbohydrate, DNA, etc
- adherence
- morphology

Question 13

What are the advantages and disadvantages of studying behaviour on rich or specialised growth media?

Answer 13

Advantages
Inexpensive
Large scale is possible
Assays are flexible
Reagents easily available

Disadvantages
Doesn’t mimic in vivo situation
- no host interaction
- bacterial gene regulation is dependent on external conditions,
but rich conditions are not representative of infection

Question 14

Describe the Importance of bacterial gene regulation

Answer 14

Bacteria are single-celled organisms that are highly
responsive to environmental triggers.

Triggers include :
nutrients
oxygen
iron
temperature
bacterial pheromones
mammalian cells, hormones
Etc. etc.

Many bacterial virulence factors are only expressed in vivo, or in conditions mimicking those found in vivo

eg. Vibrio cholera expresses cholera toxin and pilin necessary for colonisation only in the human intestinal tract

eg. Corynebacterium diphtheriae only produces diphtheria toxin in low iron conditions such as those found in vivo.

Question 15

What are the disadvantages and advantages of studying bacterial behaviour in laboratory conditions that mimic in vivo?

Give examples of laboratory conditions

Answer 15

Advantages
Mimic host interaction and growth conditions

Disadvantages
More expensive, access to reagents, hard to scale-up,
not as reproduceable

eg. Blood, plasma, serum, Peritoneal dialysis fluid, Fresh mammalial cells, Cell culture & Tissue culture

Question 16

Give examples of bacterial behaviour in laboratory conditions that mimic in vivo

Answer 16

• Interaction with freshly-derived mammalian cells
• haemolysis of red blood cells
• phagocytes (eg. neutrophils, macrophages) mixed with bacteria to mimic
  -phagocytosis
  -bacterial killing
  -bacterial survival and replication
  -cellular killing

Question 17

Describe Cell culture and Tissue culture

Answer 17

Lawns of immortalised mammalian cells grown in flasks, eg. endothelial or epithelial cells

Or samples of tissue kept artifically alive, eg. skin or intestinal tissue

Used to grow obligate intracellular bacteria or bacteria with important intracellular phases eg. TB, Shigella

Question 18

Describe Animal models (in vivo)

Answer 18

Very powerful method, the practical ‘gold standard’ e.g. - mice, rats, rabbits, larger animals, flies, worms

Purified bacteria innoculated intraperitoneally, intravenously, orally, etc. Inoculum size/number of bacteria is important.

Outcome can be measured by :
ID50, LD50, symptoms, cfu in tissue, migration of immune cells, antibody response, cytokine response

Question 19

What are the advantages and disadvantages of Animal models (in vivo)

Answer 19

Advantages
Best available mimic of human infection in vivo
Transgenic animals available
Essential for trialing therapies, safety. Required before
human studies

Disadvantages
Ethics - licensing, restricted numbers, animals must not suffer unnecessarily
How should the bacteria be inoculated?
What outcomes should be measured?
Variation between different models

Question 20

Give an example of a study on Animal models (in vivo)

Answer 20

Example - meliodosis (Burkholderia pseudomallei)
(tropical infection in rice farmers with high mortality)

Q. How important is flagella?
Method :Compare wild-type strain and its isogenic
mutant pair with mutated fliC gene in models
- in vitro, motility yes
- BALB/c mice intranasal infection yes
(less bacteria in spleen and lungs)
- BALB/c mice i.p. infection yes
- invasion & replication in cultured human lung cells no
- C. elegans (worm) slow kill assay no
- hamster model no
- diabetic rat model no

Question 21

Describe the exploitation of bacterial genetics to investigate pathogenesis

Answer 21

Bacteria have relatively small and simple genomes.
Possible to study the whole genome, and genetically
manipulate bacteria to discover which genes are important
in pathogenesis.
These genes then become targets for vaccines, diagnostics
and therapy

Question 22

What is the purpose of Sequencing projects?

Answer 22

Provides overview of the capabilities and limitations of a bacterium
eg. capsule in Campylobacter jejuni
However, many predicted genes have no known function

Identification of putative/predicted virulence factors
e.g. toxins, immune evasion pathways, resistance genes, regulators, metabolic pathways, etc.

Question 23

What is the purpose of Comparative genomics?

Answer 23

Q. If some isolates of a bacterial species are more virulent than others, how can the differences be identified?

Many virulence factor genes are carried on MGE such as bacteriophages, pathogenicity islands, plasmids,
transposons
They can be found in some strains and not others. MGEs and variant genes can make up to 20% of the bacterial genome.
eg. diphtheria toxin in C. diphtheria
cholera toxin in V. cholera
Panton Valentine leukocidin in S. aureus

Question 24

How are Comparative genomics undertaken?

Answer 24

Whole genome sequencing is now affordable (£50)
Eg. Next generation sequencing – Illumina, IonTorent, MinION

Compare groups of strains that vary in
- Virulence
- Resistance
- Host-specificity
- Transmissibility

Identify associated
- genes
- Mobile genetic elements
- Single nucleotide polymorphisms (SNPs)/mutations

Using SNPs to identify the evolutionary history of
a species and how is it spreading

Question 25

What is the purpose of cloning to study bacterial pathogenesis?

Answer 25

Is a gene responsible for a phenotype?

Add a foreign gene to a bacterium, compare the two bacteria in an infection model.
Most useful if - one gene is responsible for the phenotype
- the parent bacterium is not fully virulent

Question 26

Describe the process for cloning

Answer 26

Question 27

Q. Which regions of the cloned gene are important?

Q. In a bacterial chromosome, which gene is responsible for a particular phenotype?

Answer 27

A. Site directed mutagenesis - introduce mutations into the cloned gene and then determine if it is still functional.

A. Randomly clone thousands of chromosomal fragments into thousands of E. coli (library), and screen for the clone that has the appropriate phenotype.

Question 28

Describe the use of Isogenic mutant pairs / knockouts (gene deletion) iin studying bacterial pathogenesis

Answer 28

Q. Is a gene necessary or essential for a phenotype or for pathogenesis?

Very powerful tool, especially for complex virulence pathways involving more than one gene.

Question 29

Describe the Construction of a knockout

Answer 29

Question 30

Describe the use of Transposon libraries in studying bacterial pathogenesis

Answer 30

Q. Which genes are necessary or essential for a given phenotype?

Transposons are fragments of naturally occurring DNA with discrete ends that encode transposase genes

Transposases catalyse the “jumping” of the transposon from one DNA insertion site to another.

For constructing libraries, we use transposons that insert randomly and carry a selectable antibiotic resistance marker.

Question 31

Describe the construction of libraries using transposons

Answer 31

after image:
Screen library by testing 1000s of individual Tn mutants
e.g. for capsule
e.g. for survival in phagocytes.
Identify mutants with missing phenotype.
Using genome sequencing, identify transposon insertion site
in those mutants, and therefore the gene that is deleted.

Question 32

Q. How to identify which genes are necessary to cause disease in an animal model, but without sacrificing thousands of animals?

Question 33

Describe the use of transcriptional genomics in the study of bacterial pathogenesis

Answer 33

Q. Which genes are expressed in vivo?
Presumably, only genes important for survival and virulence are expressed, so it is a marker of their importance.
Also, gene regulation pathways that respond to in vivo signals are potential targets for therapeutics.