L13 - how bacterial alteration of the host cellular processes leads to disease

Question 1

What are the selective pressures that drive evolution?

Answer 1

• Day-night and hot-cold cycle
• Variable nutrient availability
• Unstable – rain, wind, heat
• Competition (from other bacteria)

Question 2

Solutions to withstand selective pressures

Answer 2

Resist being washed away

Get away from toxic/dangerous agents - move to nutrients

Survive nutrient limitations, bad times & prevent dissection

Question 3

How to bacteria resist being washed away?

Answer 3

Adhesins are used to stick to surfaces so they don’t wash away

They generate pili which are longer and allow the bacteria to contact things that are further away

Can also release sticky materials like DNA and polysaccharides
• These help bacteria stay in a desirable location

Question 4

How do bacteria get away from toxic/dangerous agents & move to nutrients?

Answer 4

Flagella to move faster & swim towards food/nutrients or away from home

Chemotaxis is a sensor to be able to detect concentration gradients

Question 5

How do bacteria survive nutrient limitations, bad times & prevent dissection?

Answer 5

Produce highly resistant spores

Lock themselves away in a dormant state and they can remain viable for years until the conditions become better when they can break out of the spores and start again.

Release enzymes to sequester nutrients

Question 6

Bacterial evolution

Answer 6

Modification of “old” functions / development of new ones
– Allow modification of existing functions to optimize fitness in an existing niche or to adapt to a new niche
– Although, this is a slow process of alterations which selection will select against or for

The key mechanism by which many bacteria evolve is by acquisition of new bits of DNA

Bacteria have no sexual life to facilitate exchange of alleles within a population

Question 7

Result of bacteria having no sexual life to facilitate exchange of alleles within a population

Answer 7

This function is fulfilled by horizontal gene transfer

Bacteria can obtain found DNA from outside of up to 100 kB in size (DNA has the capacity to encode complex processes)

Can encode entire metabolic pathways or complex surface structures – go from not having function to having it in one step

Genes can be taken up as naked DNA or transferred in the form of plasmids, phages, or conjugative transposons

Question 8

Bacterial DNA acquisition mechanisms

Answer 8

Bacterial transformation

Conjugation

Transduction (bacteriophages)

Selective pressures - retention / loss

Question 9

Bacterial transformation

Answer 9

Process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment

Question 10

Bacterial conjugation

Answer 10

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus

‘Bacterial sex’

Question 11

Bacterial transduction

Answer 11

Process of genetic recombination in bacteria in which genes from a host cell (a bacterium) are incorporated into the genome of a bacterial virus (bacteriophage) and then carried to another host cell when the bacteriophage initiates another cycle of infection

Question 12

Importance of toxins

Answer 12

Toxins can play an important role in taking on competitors, killing them and then using them as a source of nutrients

Question 13

How do proteins get across the membrane?

Answer 13

Very easy for gram-positive as it only has single membrane

Hard for gram-negative as it has two membranes

Question 14

Sec pathway

Answer 14

Comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane

Sec pathway allows bacteria that express ribosomes to either directly be delivered to an export pathway or following a chaperone to guide it

They then interact with a transport system and they pass through a central pore via signal sequence (at the N-terminus - has characteristics such as hydrophobicity)

Once protein has been fed through the channel the signal sequence can be cleaved off and the protein can diffuse away from the bacteria.

Question 15

TAT pathway

Answer 15

Twin Arginine Translocation (TAT) pathway

A protein export, or secretion pathway found in plants, bacteria, and archaea. In contrast to the Sec pathway which transports proteins in an unfolded manner, the Tat pathway serves to actively translocate folded proteins across a lipid membrane bilayer

Once protein is through the pore, the signal sequence is cleaved and this allows the protein to diffuse away

Question 16

Why is there competition for bacteria?

Answer 16

As bacteria propagated more and more over the world, there are much more bacteria fighting over the same amounts of nutrients

This also put pressure on bacteria to come up with new strategies, so it promotes the bacteria to be mobile to be able to move to new environments

Some pilus can retract and can pull bacteria across a surface

Question 17

How has competition aided the evolution of gram-negative bacteria to have a second membrane system as a protective shield?

Answer 17

Evolution of anti-killing mechanisms – 2nd membrane/shield

Now problem of how get proteins secreted across the second membrane system?

Selection pressure for bacteria to come up with solutions

Question 18

Why do we need specific secretion systems in gram-negative bacteria?

Answer 18

It has 2 impermeable membranes

The way they get the protein across the second membrane is achieved via different systems
• Autotransporter / type 5 secretion system
• Type 2 secretion system
• Efflux pump

Question 19

What are the systems used to get proteins across the second membrane in gram-negative bacteria?

Answer 19

• Autotransporter / type 5 secretion system
• Type 2 secretion system
• Efflux pump

Question 20

Autotransporter / type 5 secretion system

Answer 20

The pathway signals to get it into the periplasm and then the C-terminal part of the protein forms a pore in the membrane and then the vascular part of the protein is fed through the pore and can be then cleaved off so it can be secreted or released

If it is not released, it can act as adhesins on the cell surface of the bacteria

This system has evolved from the mechanism that allows the bacteria to stick proteins into the outer membrane.

Question 21

Type 2 secretion system

Answer 21

Has evolved from pili

The pili system was generated to produce adhesins and proteins are delivered TAT or Sec pathway to produce a pilus

The pilus is generated by adding proteins that extend through the hole

The pilus has been duplicated and modified to invent the secretion system

Now, there is a pseudo pilus and substrates go into the cytoplasm where they can fold (or make oligomers) and interact with the pseudo pilus that then extends and pushes the protein through the porin where it is released

The pilus then retracts so that it can load another substrate.

Question 22

Efflux pump secretion system

Answer 22

Efflux pumps are usually associated with the removal of toxins so they don’t get passed the inner membrane and kill the bacteria

This system has been duplicated and modified to allow the bacteria to secrete proteins in a single step

These proteins have a signal sequences that gets them to the membrane protein

This forms a continuous conduit and the protein is then secreted across both membranes in a single step –> it goes directly from the cytoplasm to the outside the cell where it can diffuse away

Question 23

How can bacteria overcome competition?

Answer 23

Obtain/modify functions to grow faster or better on available resources – outcompete

Work together – symbiosis (mutualistic relationship)

Murder – ‘stab to death’ – Type 6 Secretion System (T6SS)

Question 24

Type 6 Secretion System (T6SS)

Answer 24

A molecular machine used by a wide range of Gram-negative bacterial species to transport proteins from the interior (cytoplasm or cytosol) of a bacterial cell across the cellular envelope into an adjacent target cell

A protein tube surrounded by a contractile sheath

Similar to the tail of T4‐bacteriophages

Injects antibacterial ‘effector’ molecules into (periplasm/cytoplasm) other bacteria to kill (if no antidote)

Question 25

What provided a lot more competition for bacteria?

Answer 25

Unicellular eukaryotes & protozoa

Question 26

What are protozoa?

Answer 26

• Protozoa ("unicellular animals"), Historically, single‐celled organisms with animal‐like behaviours, such as motility and predation Include: • Cilliates • Flagellates • Amoebae

Question 27

How do ciliates move?

Answer 27

Move using hair‐like structures called cilia

Question 28

How do flagellates move?

Answer 28

Move with help of whip‐like structures called flagella

Question 29

How do amoebae move?

Answer 29

Move by the use of foot‐like structures called pseudopodia

Question 30

What is the key food source for amoebae?

Answer 30

Bacteria Bacteria need to find ways of preventing them from being killed by the phagocytic cells amoebae and this drives evolution

Question 31

How do protozoa take in food?

Answer 31

By osmotrophy or phagocytosis

Question 32

Osmotrophy

Answer 32

Absorbing nutrients through cell membrane

Question 33

Feeding by phagocytosis

Answer 33

Engulfing particles via pseudopodia As amoebae do

Question 34

Where do protozoa digest their food?

Answer 34

In stomach‐like compartments called vacuoles

Question 35

What happens when amoebae interact with bacteria?

Answer 35

They internalise them which allows the cell to encompass and pull the bacteria into a food vacuole Once in the vacuole, a lot of antimicrobial enzymes are targeted to the vacuole to kill the bacteria, break it up and provide food for the eukaryotic cell This also drives selection

Question 36

What is symbiosis?

Answer 36

This comes from a Greek word simply meaning 'living together' and can be used to describe any association between two organisms

Question 37

What is mutualism?

Answer 37

This can be used to describe an association in which both organisms apparently benefit

Question 38

What is commensalism?

Answer 38

In this association one organism [the commensal] benefits, and the other [the host] is apparently unaffected.

Question 39

What is parasitism?

Answer 39

In this association one organism [the parasite] benefits, and the other [the host] is adversely affected [weakened, sickened, damaged etc]

Question 40

The phagocytic process of engulfing bacteria

Answer 40

1. The bacteria engulfed by eukaryotic cells enter into a phagocytic vacuole 2. The vacuole fuses with early & late endosomes, forming a phagolysosome 3. Contains anti-microbial products within to kill/destroy nutrient source This drives the evolutionary race between the host cell & the bacteria

Question 41

How do bacteria try to win the evolutionary race between the host cell and itself?

Answer 41

They are trying to find ways to inhibit this process & to deploy mechanisms of secreting factors to kill eukaryotes, or disable them, or live within them It is thought that this is where bacteria learnt host cell physiology – understand by trial & error what bits of DNA were needed to not die Bacteria have evolved lots of mechanisms to interfere with all the different activities that take place within the organelles of a eukaryotic cell

Question 42

How do secreted proteins get into eukaryotic cells?

Answer 42

Some function at the surface Some toxins are delivered that are not enough to lyse the cells but are just enough to trigger signalling cascades which alter the biology of the eukaryotic cells which perhaps will inhibit things like phagocytosis. Other proteins have adapted to understand what receptors are on the cells Proteins also have to evolve strategies to get into the cells

Question 43

How do some bacteria function at the surface of eukaryotic cells?

Answer 43

Diffuse across from the bacteria, interact with the eukaryotic cell and some insert spontaneously into the lipid bilayer of cells, and these can cause lysis Ions and water can flow through so that the cell bursts

Question 44

How have some bacteria adapted to understand what receptors are on the cell?

Answer 44

Some secreted proteins can interact with the receptor and trigger signalling cascade which alter the biology of the cell

Question 45

How have bacteria evolved strategies to get into the cells?

Answer 45

Many proteins have two structures composed of three domains One of these is a catalytic domain that does the modification of the target host protein. The binding domain helps get the protein into the cell, or to interact with the cell. The translocation domain helps get the bacteria into the host cytoplasm so it can interact with the target

Question 46

How does the diphtheria toxin act?

Answer 46

Diphtheria toxin is secreted, it interacts with a specific receptor, this triggers endocytosis As the vesicles mature, they become more acidic (the acid is there to help break down the components of the vesicles) This increase in pH alters the structure of these proteins to allow the catalytic domain to be translocated across the membrane The protein inserts into the host membrane in a pH dependent manner, forms a pore to allow the catalytic subunit to enter the cytoplasm where it can diffuse to its target, for example the ribosomes to prevent protein synthesis If a cell cannot generate new proteins, it dies

Question 47

How do exotoxins get into the cell?

Answer 47

Interacts with the receptor to trigger its uptake by a vesicle, but it has a different strategy to get into the cytoplasm It exploits retrograde transport which involves a vesicle being trafficked to the Golgi and then components are added to the Golgi and then transferred across to the ER From the ER they are passed into the cytoplasm Usually, this cytoplasmic step in host cells is for proteins that don’t fold correctly and are targeted into the cytoplasm where they are degraded But this bacterial protein avoids the degradation process so it can diffuse away to its target, modify it to alter its biology, causing alternations to cellular processes

Question 48

Bacterial mechanism to deliver proteins directly into the cytoplasm

Answer 48

One of these is the type 3 secretion system which is considered to be evolutionarily related to the flagella. Instead of secreting proteins to make flagella, it secretes proteins to form a needle-like structure and this allows the bacteria to deliver proteins directly from the bacterial cytoplasm into the host cell cytoplasm. Similarly, there is a type 4 secretion system which is related to conjugation – instead of inserting plasmids into recipient bacteria, this allows the bacteria to deliver proteins into target eukaryotic cells. The type 6 secretion system (T6SS) has also been shown to deliver proteins directly into eukaryotic cells

Question 49

Bacteria in the GI tract

Answer 49

The GI tract is the key environment for humans to get nutrients and fluids However, it is a very competitive environment (due to sheer amount of bacteria) Dysfunction leads to diarrhoea The host has evolved ways to prevent nutrients being stolen by bacteria

Question 50

How has the GI tract evolved ways to prevent nutrients being stolen by bacteria?

Answer 50

* Stomach acid * Peristalsis * Mucus * Glycocalyx * Secretory IgA * Antimicrobials

Question 51

Role of the small intestine

Answer 51

Take up all retrievable nutrients & all but ~1.5 litres of water The key evolution in mammals is the generation of a large enough surface area to be able to take up most of the nutrients and fluids quick enough to outcompete the bacteria This is achieved through mucosal folds, villi and microvilli

Question 52

Why are most bacteria kept out of the small intestine?

Answer 52

Allows to accumulate in the large intestine because it is the site of all the nutrients that the host has not been able to exploit and allows the bacteria to replicate there and break down the nutrients The host gets about 10% of its energy from the bacteria breaking down these components

Question 53

What is the gut lining composed of?

Answer 53

Enterocytes Final stages of enzymatic digestion liberates small molecules for absorption Sole site in the digestive tube for absorption of amino acids and monosaccharides

Question 54

What are goblet cells?

Answer 54

Produce/maintain a protective mucus blanket by synthesizing/secreting high‐molecular‐weight glycoproteins known as mucins

Question 55

What are enteroendocrine cells?

Answer 55

Produce gastrointestinal hormones/peptides in response to stimuli Initiate digestive actions and detecting harmful substances triggering protective responses eg vomit

Question 56

What are paneth cells?

Answer 56

Secrete anti‐microbial compounds into the lumen of the intestine

Question 57

Key features of enterocytes?

Answer 57

* Tight junctions * Adherent junctions * Desmosomes * Gap junctions

Question 58

Tight junctions in enterocytes

Answer 58

Impermeable junctions that prevent molecules from passing through intercellular space They are found at the apical (top) region of most epithelial tissue types. Some proteins in the plasma membrane of adjacent cells are fused/interlocked ‐ they prevent certain molecules from passing between cells of epithelial tissue.

Question 59

Adherent junctions in enterocytes

Answer 59

An adhesive belt anchors adjacent cells A transmembrane linker protein attaches to actin microfilaments of the cytoskeleton and binds adjacent cells

Question 60

Desmosomes in enterocytes

Answer 60

Main junctions for binding cells together They are scattered along abutting sides The cytoplasmic side of each plasma membrane has a plaque which are joined between cells by the linker proteins. Intermediate filaments go across the cytoplasm and anchor these desmosomes together at opposite sides of cells

Question 61

Gap junctions in enterocytes

Answer 61

Are passageways between two adjacent cells They let small molecules move directly between neighbouring cells The cells are connected by hollow cylinders of protein and function in inter‐cellular communication

Question 62

Barrier function of tight junctions

Answer 62

Forms a continuous seal Serves as a selective barrier to small molecules and total barrier to large molecules

Question 63

Fence function of tight junctions

Answer 63

Prevents diffusion of membrane components between Apical and Basolateral surfaces enabling delivery of components (lipids/proteins) to specific locations to produce specialised features (eg microvilli) or functions (immune surveillance by enriching antigen‐detecting receptors on host‐privledged surfaces)

Question 64

What happens when theres disruption of tight junctions?

Answer 64

Is linked to a diarrhoeal, usually inflammatory, outcome Fluids and ions can then diffuse in between and antigens would come in and cause an inflammatory response

Question 65

Specialised properties of intestinal epithelia

Answer 65

* Microvilli are finger like extensions of the PM – they are abundant in epithelial tissue of the small intestine & the kidneys * They maximise SA, acting like a filter, & resist abrasion thanks to actin microfilaments at their bases * Number & shape correlate with absorptive capacity * Contain transporters to uptake ions, nutrients & fluids * Most pathogens cannot get through this impenetrable barrier

Question 66

What happens if theres a loss of microvilli on intestinal epithelia?

Answer 66

Is diarrhoeagenic – if you cannot take in ions and nutrients then the water won’t follow the ions, it will stay in the gut and lead to diarrhoea

Question 67

When did multicellular eukaryotes and mammals evolve?

Answer 67

Multicellular eukaryotes evolved only during the past 1‐2 billion years Mammals proliferated massively only during the past 65 million years

Question 68

Why was the evolution of mammals good and bad for bacteria?

Answer 68

* Lovely ‘New’ Niche for bacteria – constant temperature with continuous supply of nutrients inside mammals * BUT Highly hostile environment (anti‐infective host factors and competition) – many bacteria in host = competition * Host has evolved lots of mechanisms to prevent bacteria accumulating within the small intestine but allows the bacteria to accumulate in the large intestine * Bacteria do undertake very important role in host physiology (mutualistic relationship) – they break down nutrients which the host then benefits from and they help train the immune system * Very few bacteria cause disease in a healthy human (issue in young, old, immuno-compromised)

Question 69

Is E. coli gram negative or gram positive?

Answer 69

Negative

Question 70

What E. coli is a safe strain?

Answer 70

E. coli K12 Work‐horse of molecular biology (cloning genes onto plasmids) Earliest model for study of bacterial biology (fundamental understanding of cellular processes & biotechnology implications)

Question 71

What E. coli causes diarrhoeal disease?

Answer 71

Small Intestine: • Enteropathogenic E. coli (EPEC) • Enterotoxigenic E. coli (ETEC) • Diffusely adherent E. coli (DAEC) Large Intestine: • Enterohaemorrhagic E. coli (EHEC) • Enteroinvasive E. coli (EIEC)

Question 72

How did E. coli evolve to cause diarrhoeal disease?

Answer 72

Evolved through acquisition of new DNA – provides new capacities so the bacteria an survive in the large intestine or to colonise the small intestine and alter the biology which leads to disease