Pathogenesis and the Microbiota

Question 1

Non-pathogenic members of normal flora are “commensals”
What is commensalism?

Answer 1

Commensalism – An association between 2 organisms in which one benefits and the other derives neither benefit nor harm

Question 2

What is Mutualism?

Answer 2

Mutualism – Symbiotic relationship where both organisms benefit

Question 3

What is an Opportunistic pathogen?

Answer 3

When a commensal organism causes disease given the right circumstances

Question 4

What is the first step in disease progression?
What are some associated colonisation virulence factors?

Answer 4

Pathogen introduced into host and infecting

Adhesins
Invasins
Nutrient acquisition
Motility
Chemotaxis
Etc.

Question 5

What follows colonisation in disease progression? (hint - 2 paths)
What causes either one to occur?

Answer 5

Diseased host - Virulence factors causing damage
Asymptomatic carrier - Host defences

Question 6

What follows diseased state in disease progression? (hint - 3 paths)
What causes each to occur?

Answer 6

Death
Resolve to asymptomatic carriage - Host defences
Resolve infection and recover - Host defences

Question 7

What is possible from the asymptomatic state?
Likelihood?

Answer 7

Can revert back to diseased state
Rare

Question 8

What are some host defences involved in some of these disease progression steps?

Answer 8

Physical barriers
Complement
Macrophages
Antimicrobial peptides
Adaptive immunity
Etc.

Question 9

What are some damage virulence factors involved in progression from infection to diseased state?

Answer 9

Exotoxins
Endotoxins
Proteases
DNase
Lipase
Haemolysin
Etc.

Question 10

Why is death by disease much less common in the modern day?

Answer 10

Modern healthcare and medicine like antibiotics and vaccines

Question 11

What are Koch’s 4 postulates?

Answer 11

1) Microorganism must be found in abundance in all organisms suffering from the disease but not in healthy organisms
2) Microorganism must be isolated from a diseased organism and grown in pure culture
3) Cultured microorganism should cause disease when introduced into a healthy organism
4) Microorganism must be re-isolated from the diseased experimental host and identified as being identical to the original causative agent

Question 12

What are the Koch’s postulates for the molecular era designed for? (hint - genes)

Answer 12

Examining the potential role of genes and their products in the pathogenesis of infection and disease

Question 13

What are the 3 Koch’s postulates of the molecular era? (hint - measurable)

Answer 13

1) Phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species
2) Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence
3) Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity

Question 14

What are the 2 general methods of studying pathogenesis?

Answer 14

Genetic manipulation of organism - Some readout of virulence and look for a change in how they cause disease
Reductionist biology - Identifying virulence factors (e.g. toxins degradative enzymes, invasins, adhesins etc.)

Question 15

Many virulence factors are produced by both pathogenic and non-pathogenic organisms. What question does this raise?

Answer 15

If a protein we think is a virulence factor is produced in both then is it a virulence factor or not?

Question 16

What is tetanus toxin?
What does it cause?
Potency?
Virulence factor and how this benefits bacteria?

Answer 16

Neurotoxin that induces rigid paralysis –> Death

2nd most potent toxin known; LD50 of 2.5ng/kg

This toxin is definitely a virulence factor; Evolved to kill host
Beneficial to bacteria as Clostridium tetani is anaerobic; Dead host is a large anaerobic source of nutrients for bacterial growth

Question 17

What is OmpA?
What is it essential for?
What does its loss cause?
Why is this confusing?

Answer 17

Dominant E. coli outer membrane protein

Essential for evasion of macrophage killing and invasion of the blood-brain barrier

All E. coli strains have this, but most E. coli are harmless
Its loss in pathogenic E. coli reduces pathogenicity

Don’t know if its a virulence factor

Question 18

What does reverse genetics aim to do?

Answer 18

Seeks to assign a function to a particular gene/sequence
Identify gene for virulence factor and mutate the gene to see how it affects function

Question 19

What is the process of reverse genetics?

Answer 19

Starts with hypothesis
Uses directed mutagenesis; Targeting specific gene
Hypothesis directs experimental analysis e.g. Targeting what we think is a gene encoding a toxin, so we test for lack of toxicity in mutant

Question 20

What does forward genetics aim to do?

Answer 20

Seeks to identify the genetic basis of phenotype
Know bacteria X phenotype, but don’t know how they do it/the gene encoding phenotype

Question 21

What is the process of forward genetics

Answer 21

Does not require prior knowledge of genetic basis of phenotype
Uses random mutagenesis
Experimental approach designed to screen for phenotype e.g. Know bacteria produce a toxin, randomly mutate bacteria and look for mutant that has lost ability to make toxin

Question 22

In reverse genetics. if we have geneX, what can we do to find phenotype? (hint - complement)

Answer 22

Knockout geneX and test phenotype
Complement the knockout and test phenotype
Can also do a more subtle mutation (e.g. loss of function, gain of function, dominant negative) and test how this affects phenotype

Question 23

What is reverse genetics backed up by normally?
Drawback of reverse genetics?

Answer 23

Backed up by another sort of data e.g. biochemical assay to demonstrate an enzyme activity, protein-protein interaction etc.

Can edit genomes at will but can’t use all tools in all organisms

Question 24

What are the 2 ways of making a knockout?
- Which is better?
Examples of techniques?

Answer 24

Insertional (into middle of gene to disrupt)
Deletion
- Deletion is generally better

Lambda red, group II introns (TargeTron), Homologous recombination, Phage transduction, CRISPR

Question 25

What do the different knockout techniques boil down to?

Answer 25

Making a version of a gene that we want and then convincing the bacteria to swap their version of the gene for the one we've made

Question 26

How do we complement a gene? (hint - 2 ways) What is needed for each?

Answer 26

Plasmid - Selectable markers, origins, transformation/conjugation, promoters etc. Insertion at a distal locus - Suitable insertion site, homologous recombination, unstable/conditional plasmid, counter selection etc.

Question 27

Why is insertional mutagenesis complementation generally better than plasmid complementation

Answer 27

Gene is put back in as a single copy and is expressed similarly to how it would've been originally Plasmids tend to be present at high copy numbers e.g. could introduce 400 copies of a gene, altering expression greatly relative to WT

Question 28

What does a forward genetics screen for mutants that have lost the phenotype require? (hint - scalable)

Answer 28

Requires a relatively straightforward and scalable phenotypic screen Relies on random mutagenesis so can be challenging to identify the responsible mutation Screen must be straightforward and scalable as it can be extremely labour intensive

Question 29

How is random mutagenesis now done? How does this work?

Answer 29

Transposon mutagenesis Transposon randomly inserts into genome Screen library of transposon insertions Screen pools of many transposon mutants using TraDIS/Tn-Seq

Question 30

How does TraDIS work? (hint - casette)

Answer 30

Transposons with antibiotic resistance cassette randomly inserts in genome of millions of bacteria in a single pool Sequence each bacterial genome from transposon out into the genome Map these sequence reads onto bacterial genome to see where each transposon is inserted Gaps in insertions show essential genes need to survive

Question 31

How does TraDIS work in the context of conditional essentiality?

Answer 31

Apply different stresses to bacteria Map the genomic DNA of surviving bacteria Compare libraries before and after stress to see which genes are essential for survival in that certain condition Can the do targeted mutagenesis of essential genes and study them

Question 32

How can we use TraDIS to see which genes are essential for sporulation in C. difficile?

Answer 32

Sporulate library of of C. difficile transposon mutants Kill all the vegetative cells and sequence again Only mutants capable of sporulating will be present - With no insertions in essential sporulation genes

Question 33

Pros and Cons of using rodents as animal models?

Answer 33

Pros: Inbred lines - Reduces genetic variation Small, cheap, reproduce quickly Lots of available tools for rodents Cons: License restrictions and highly regulated Ethical issues Mice aren't humans; Not fully accurate so needs to be taken with a pinch of salt

Question 34

Pros and Cons of using fish and non-vertebrae organisms as alternative models?

Answer 34

Pros: Less ethical concerns Lots of tool in development; Particularly for zebrafish Light touch regulation; Up until a week old, you can do lots with these embryos without legal restrictions Can use large group sizes for statistical robustness due to small size Cons: Some organisms are even further away from humans; Less accurate representation More primitive immune systems

Question 35

Pros and Cons of using cultured cell lines and developed organoids (multiple cell types mixed together) as alternative models?

Answer 35

Pros: Can use human cells Normally immortalised so easy to culture and scalable No ethical issues Cons: Usually cancer cells so have many genetic changes (e.g. alterations in surface protein expression); Can behave differently to normal cell No disease; So only model specific aspects of virulence (e.g. adhesion, invasion, intoxication) Often behave quite differently to the parental cell type/tissue

Question 36

Define Microbiota

Answer 36

Complex microbial community that inhabits a particular environmental niche (bacteria, viruses and microeukaryotes)

Question 37

Define Microbiome Why have microbiota and microbiome become interchangeable?

Answer 37

Combined genetic material/potential of the microbiota We can only really see the genetics; Don’t tend to culture these bacteria

Question 38

The human microbiome consists of at least 150x more genes than the human genome; What does this mean?

Answer 38

Enormous metabolic potential in the microbiome

Question 39

Where is the largest biomass and diversity of microbiome in the human body? What can be said about the different ecosystems that make up humans? (hint - essential)

Answer 39

GI tract We are made up from many distinct ecosystems, each with their own environmental conditions that influence the species that live in us; These species are essential

Question 40

Why don't we use culturing to see what bacteria are present in a sample? (hint - conditions)

Answer 40

Culturing is typically quite difficult; Complex environments and many species for which we don’t have reproducible growth conditions

Question 41

What is the main way of determining what bacteria are present in a sample? - Main sequencing tech for short-reads?

Answer 41

Sequencing; Typically illumina (short-reads) but long-read tech starting to be adopted (Nanopore and PacBio)

Question 42

What are the 2 long-read sequencing methods that are starting to be used with Nanopore and PacBio? - Any cons and why?

Answer 42

Amplify mix of 16S rRNA - Gives you composition at genus and occasionally species level and their proportions, but doesn’t tell you anything else about the bacteria; 16S rRNA is highly conserved but drifts slightly from species to species - Metagenomics – Shotgun sequence everything; With good data you can even start to piece individual genomes back together to uncover certain features

Question 43

What areas of the body have larger and smaller populations of bacteria present?

Answer 43

Least populated areas are the eyes and dry areas of skin Wetter parts of the skin have higher bacterial abundance GI tract has very high abundance of bacteria - Mouth - Stomach - Small intestine - Colon

Question 44

What is the Kitome?

Answer 44

Microbiome of the kits and reagents used to study microbial DNA samples A lot of the DNA sequenced and observed was present in the kits and reagents

Question 45

How was the kitome uncovered? How can you see the unique contaminating kitome of each lab? - Avoidable?

Answer 45

3 labs with their own methods took a sample of Salmonella and performed serial dilutions They found Salmonella, but got increasing numbers of reads associated with other bacteria with each dilution This microbial DNA was not included in any samples, so it must be present in the kits By removing the Salmonella you can see the unique contaminating kitome of each lab - This can't be avoided

Question 46

When does the kitome affect results? (hint - density)

Answer 46

Doesn’t affect high density microbiomes (e.g. faeces); High density sample will completely overshadow the small contamination Not the case for low density samples

Question 47

The gut microbiota is incredibly diverse. What is observed through WGS of samples instead of just species identification?

Answer 47

Despite large species diversity between samples, metabolic potential is conserved - Many ways to achieve the same biochemical processes and equilibrium --> Health of microbiome and host

Question 48

What factors can affect microbiota composition?

Answer 48

Age; Babies have different microbiota to older age groups Diet Health status Gender Geography and Ethnicity? ANTIBIOTICS

Question 49

What do broad spectrum antibiotics do to mouse microbiota? Recovery?

Answer 49

Mouse microbiota is altered Microbiota doesn’t recover even after weeks; Despite returning to normal diet

Question 50

What is the microbiota important for?

Answer 50

Training the immune system; Immune tolerance and modulation Colonisation protection Metabolic functions - Amino acid and vitamin synthesis - Digestion of some complex carbohydrates

Question 51

What are some consequences of microbiota disruption?

Answer 51

Disruption of microbiota has been linked to obesity and type II diabetes Disruption can also cause loss of colonisation resistance, leading to infectious disease - Microbiome protects itself from invading bacteria, which protects us

Question 52

What is the link between Bifidobacterium Species and C-Section babies? Increasing disease?

Answer 52

Babies delivered via C-section have a different microbiome with a particular reduction in Bifidobacterium Increase in C sections correlates with increase in cases of asthma, allergies, eczema, childhood obesity etc.

Question 53

What can be done to correct the lack of colonisation in C-section babies? - Risky?

Answer 53

Vaginal swabbing seems to be able to correct this - Potentially risky and needs more data

Question 54

What is different about preterm infants microbiome? More likely to develop and susceptible to what? (hint - perfringens) - Traits of this

Answer 54

Weird microbiome and far more likely to develop asthma Highly susceptible to infection with Clostridium perfringens - Fast moving/growing bacteria - Necrotising enterocolitis; Digestion of large intestine - Low survival rate in infants

Question 55

What is dysbiosis and what is it a prerequisite for? What happens as this is cleared

Answer 55

Disruption of healthy microbiota Prerequisite for C. difficile Infection (CDI) - People with healthy microbiota are immune to this infection Microbiota recovers as CDI is cleared

Question 56

What is a new and effective treatment for CDI? Risks? What needs to be done to become mainstream? (hint - defined)

Answer 56

Transfusion of donor faeces is very good at treating CDI Has been linked to infections from the faeces and some deaths Need defined microbiota transplantation; Figuring out species in faeces that have therapeutic effect and growing them into artificial community of good bacteria to have same positive effect as full faecal transplants - Works in mice

Question 57

What could a potential Koch's postulates for microbiota studies look like? (4 postulates) (hint - microbiota signature, ameliorate) Feasibility? (hint - unculturable)

Answer 57

Identify all species present in both healthy and diseased individuals; Allows for identification of a microbiota signature for a disease state (what is different in diseased person) Isolate, purify and culture relevant bacterial species (particularly the different ones) Intervene and ameliorate the symptoms of disease by introducing the missing species or correcting the microbiota composition Detect and re-isolate the species in the microbiota to show it is ‘fixed’ Questionable feasibility, especially due to unculturable organisms in the gut Sequenced and cultured all species in mice microbiota; Starting this for humans too