Studying bacterial pathogenesis: approaches and methods - unfinished

Question 1

what is bacterial pathogenicity?

Answer 1

the ability of a bacterium to inflict damage on the host

- involves bacterial and host factors

Question 2

why is it important to study bacterial pathogenesis?

Answer 2

To improve bacterial disease :

• prevention, eg. vaccines
• diagnosis, eg. toxin detection
• treatment, eg. identify new therapy targets

Question 3

Bacterial factors - name some

Answer 3

Virulence factors

-toxins
-immune evasion
-attachment
-motility
-gene regulation
(sense and adapt)
acute v chronic v
dormant phases
spread
resistance

Question 4

Host factors - name some

Answer 4

Immune response

innate	
  skin 
  phagocytes
  complement
  Fe restriction
adaptive
  antibodies
  lymphocytes
  macrophages

Question 5

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

Answer 5

Koch’s postulates (19th century)

The bacterium is found in all people with disease
The bacterium can be isolated from patients and maintained in pure culture
The pure culture can be innoculated into a human volunteer or animal model and cause symptoms of disease
The bacterium can be reisolated from the volunteer or animal

eg. Helicobacter pylori (causes gastric ulcers, potentially cancer)

Question 6

Hypothesis-driven research

- what does it consist of?

Answer 6

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

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

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 7

To investigate bacterial pathogenesis what do we need?

Answer 7

A. Good clinical observation and epidemiology

B. Good models of disease
in vitro and in vivo

C. Appropriate strains of bacteria to test
this can be enhanced by genetic engineering

genetic engineering -bacteria are small and have small genomes, to which we can knockout genes or add genes.

we can then study whats happening with bacteria and host more efficiently, compared to other organisms.

Question 8

what is epidemiology?

Answer 8

the study of spread and distribution of disease - in bacteriology often includes the discrimination of different strains.

Question 9

what questions come up when looking at epidemiology?

Answer 9

What is the problem?
What are the common features of cases? Which tissues
are targeted?
Are all people equally susceptible?
How does it spread (eg. oral-faecal, airborne, sexually)?
Are there epidemiological links between cases? Sources of
outbreaks?
What is the causative organism (Koch’s postulates)?
Are all strains equally pathogenic?

Question 10

Examples to consider - toxic shock syndrome

Answer 10

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 11

Examples to consider - Helicobacter pylori

Answer 11

What is the problem?
Gastric ulcers

Common features of cases? Which tissues are targeted?
Helicobacter pylori in gastric samples

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 12

Examples to consider - tuberculosis

Answer 12

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.

Dormant bacterium, common, infection activated in immuno-compromised hosts

Question 13

How are good epidemiological studies designed?

Answer 13

Clear definitions of patients “with” and “without” the disease

Sufficient numbers of patients to investigate (statistical significance)

Collection of relevant information and samples (may requires ethical approval, logistics may get in the way (below points essentially))

Consider - ethics

• patients are complex
• logistics

Question 14

different models of disease?

Answer 14

1. Bacterial behaviour in rich or specialised growth media (in vitro)
2. Bacterial behaviour in laboratory conditions (in vitro) that mimic in vivo (i.e. mimic what happens in the host)
3. Animal models (in vivo)

Laboratory models, control conditions – control where bacteria go. Reproducible manner. Very diff to patients. Unsure of when infected / what happened before. Can control parameters this way.

Question 15

1. Bacterial behaviour in rich or specialised growth media (in vitro)
   - explain this further

Answer 15

Can manipulate conditions.
can see if the bacteria have any growth requirement or particular nutrients they need to survive.

eg. aerobic / anaerobic. / require CO2 to grow, some need iron for growth and signalling.

flagella – motility, virulence factor, to go towards food.
Many bacteria may be able to adhere to particular host tissues.

Morphology – can see capsule (protect from immune response)

These are easy to study in laboratory.

Question 16

1. Bacterial behaviour in rich or specialised growth media (in vitro)
   - advantages and disadvantages

Answer 16

Advantages

Inexpensive
Large scale is possible
Assays are flexible
Reagents easily available

Disadvantages

Doesn’t mimic in vivo situation - usually would be stressed + fighting for survival
No host interaction
Bacterial gene regulation in response to wrong signals

Question 17

bacteria are singled celled organisms that are highly responsive to….

Answer 17

environmental triggers

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

Bacteria will produce receptor on cell surface, and binding will lead to a change in conformation = change in phophorylation of membrane protein. It can then interact with response regulator and change conformation of this gene. This gene is something that can bind to DNA at the promoter region.

Alters way RNA polymerase makes mRNA. More mRNA = increase protein (visa versa)

So outside enviroment can dictate which genes are expressed, which proteins produced, therefore function of bacteria. E.g. e.coli responding to glucose. High conc, will use it to make energy. Low = turn off.

Question 18

Many bacterial virulence factors are only expressed where?

Answer 18

in vivo (host) , or in conditions mimicking those found in vivo (innnnn hooooost)

eg. Vibrio cholera expresses
When in gut; 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 19

2. Behaviour in laboratory conditions that mimic in vivo

- adv and disadv

Answer 19

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

Advantages
Mimic host interaction and growth conditions somewhat

Disadvantages
Expensive, difficult to get reagents, hard to scale-up,
not as reproducible - samples will be diff, as different hosts

example: . Interaction with freshly-derived mammalian cells eg. haemolysis of red blood cells

eg. phagocytes (eg. neutrophils, macrophages) mixed with bacteria to mimic 
	phagocytosis
	bacterial killing
	bacterial survival and 
		replication
	cellular killing

Question 20

Cell culture and Tissue culture

Answer 20

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 21

3. Animal models (in vivo) - the practical ‘gold standard’

Answer 21

eg. mice, rats, rabbits, larger animals
flies, worms

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

Outcome measured by any or all of:
ID50, LD50, symptoms, cfu in tissue (count no of bacteria on plate), migration of immune cells, antibody response, cytokine response

Question 22

ID50

Answer 22

(infectious dose – that causes symptoms of infection in 50% of animals)

Question 23

LD50

Answer 23

(lethal dose – that kills 50% of animals)

Question 24

3. Animal models (in vivo) - the practical ‘gold standard’

- adv and disadv

Answer 24

Advantages

Best available mimic of human infection in vivo
Transgenic animals available
Can trial therapies

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 25

3. Animal models (in vivo) - the practical ‘gold standard’

- an example?

Answer 25

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

How important is flagella (fliC)?
(Compare wild-type strain and its isogenic
mutant pair with mutated fliC)

 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 26

C. Exploiting bacterial genetics to investigate pathogenesis

-explain this further

Answer 26

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 27

Phenotype

Answer 27

• a bacterial behaviour that can be measured

Question 28

Genotype

Answer 28

• a variation in the genome that can be detected

Question 29

Sequencing projects

Answer 29

Q. What is the bacteria capable of?

Thousands of bacteria now sequenced, including multiple strains of important pathogens

Provides overview of the capabilities and limitations of a bacterium
eg. capsule in Campylobacter jejuni (common cause of food poisioning, contaminated chicken). Prior to genome sequencing, didn’t know campylobacter had a capsule. Didn’t detect it before.

eg. Staphylococcus aureus, 100s strains sequenced
Identification of putative virulence factors eg. toxins, immune evasion pathways, resistance genes, regulators,
metabolic pathways, etc.

However, many predicted genes have no known function

Question 30

2. Comparative genomics

Answer 30

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. (Forms a fair amount)

eg. diphtheria toxin in C. diphtheria
cholera toxin in V. cholera
Panton Valentine leucocidin in S. aureus (causes sever skin and soft tissue infection)

Whole genome sequencing is now affordable -
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

Question 31

3. Cloning

Answer 31

Q. 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 32

qualities of a good vector?

Answer 32

A good vector will have restriction enzyme sites. And wont cut anywhere else, cloning region.

Good vector should also be able to replicate in bacteria (origin of replication region).

Will have something that will allow selection for those with plasmid. Have antibody resistance marker.
This case ampicillin. Only those which has vector, will be able to grow.