Studying bacterial pathogenesis: approaches and methods - unfinished Flashcards
what is bacterial pathogenicity?
the ability of a bacterium to inflict damage on the host
- involves bacterial and host factors
why is it important to study bacterial pathogenesis?
To improve bacterial disease :
- prevention, eg. vaccines
- diagnosis, eg. toxin detection
- treatment, eg. identify new therapy targets
Bacterial factors - name some
Virulence factors
-toxins
-immune evasion
-attachment
-motility
-gene regulation
(sense and adapt)
acute v chronic v
dormant phases
spread
resistance
Host factors - name some
Immune response
innate skin phagocytes complement Fe restriction adaptive antibodies lymphocytes macrophages
How do we prove that a bacterium is responsible for a particular disease?
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)
Hypothesis-driven research
- what does it consist of?
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?
To investigate bacterial pathogenesis what do we need?
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.
what is epidemiology?
the study of spread and distribution of disease - in bacteriology often includes the discrimination of different strains.
what questions come up when looking at epidemiology?
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?
Examples to consider - toxic shock syndrome
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.
Examples to consider - Helicobacter pylori
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
Examples to consider - tuberculosis
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
How are good epidemiological studies designed?
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
different models of disease?
- Bacterial behaviour in rich or specialised growth media (in vitro)
- Bacterial behaviour in laboratory conditions (in vitro) that mimic in vivo (i.e. mimic what happens in the host)
- 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.
- Bacterial behaviour in rich or specialised growth media (in vitro)
- explain this further
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.
- Bacterial behaviour in rich or specialised growth media (in vitro)
- advantages and disadvantages
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
bacteria are singled celled organisms that are highly responsive to….
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.
Many bacterial virulence factors are only expressed where?
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.
- Behaviour in laboratory conditions that mimic in vivo
- adv and disadv
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
Cell culture and Tissue culture
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
- Animal models (in vivo) - the practical ‘gold standard’
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
ID50
(infectious dose – that causes symptoms of infection in 50% of animals)
LD50
(lethal dose – that kills 50% of animals)
- Animal models (in vivo) - the practical ‘gold standard’
- adv and disadv
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…
- Animal models (in vivo) - the practical ‘gold standard’
- an example?
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
C. Exploiting bacterial genetics to investigate pathogenesis
-explain this further
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.
Phenotype
- a bacterial behaviour that can be measured
Genotype
- a variation in the genome that can be detected
Sequencing projects
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
- Comparative genomics
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
- Cloning
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
qualities of a good vector?
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.
