L13 - how bacterial alteration of the host cellular processes leads to disease Flashcards
What are the selective pressures that drive evolution?
- Day-night and hot-cold cycle
- Variable nutrient availability
- Unstable – rain, wind, heat
- Competition (from other bacteria)
Solutions to withstand selective pressures
Resist being washed away
Get away from toxic/dangerous agents - move to nutrients
Survive nutrient limitations, bad times & prevent dissection
How to bacteria resist being washed away?
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
How do bacteria get away from toxic/dangerous agents & move to nutrients?
Flagella to move faster & swim towards food/nutrients or away from home
Chemotaxis is a sensor to be able to detect concentration gradients
How do bacteria survive nutrient limitations, bad times & prevent dissection?
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
Bacterial evolution
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
Result of bacteria having no sexual life to facilitate exchange of alleles within a population
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
Bacterial DNA acquisition mechanisms
Bacterial transformation
Conjugation
Transduction (bacteriophages)
Selective pressures - retention / loss
Bacterial transformation
Process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment
Bacterial conjugation
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’
Bacterial transduction
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
Importance of toxins
Toxins can play an important role in taking on competitors, killing them and then using them as a source of nutrients
How do proteins get across the membrane?
Very easy for gram-positive as it only has single membrane
Hard for gram-negative as it has two membranes
Sec pathway
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.
TAT pathway
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
Why is there competition for bacteria?
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
How has competition aided the evolution of gram-negative bacteria to have a second membrane system as a protective shield?
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
Why do we need specific secretion systems in gram-negative bacteria?
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
What are the systems used to get proteins across the second membrane in gram-negative bacteria?
- Autotransporter / type 5 secretion system
- Type 2 secretion system
- Efflux pump
Autotransporter / type 5 secretion system
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.
Type 2 secretion system
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.
Efflux pump secretion system
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
How can bacteria overcome competition?
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)
Type 6 Secretion System (T6SS)
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)
What provided a lot more competition for bacteria?
Unicellular eukaryotes & protozoa
What are protozoa?
• Protozoa (“unicellular animals”), Historically, single‐celled organisms with animal‐like behaviours, such as motility and predation
Include:
• Cilliates
• Flagellates
• Amoebae
How do ciliates move?
Move using hair‐like structures called cilia
How do flagellates move?
Move with help of whip‐like structures called flagella
How do amoebae move?
Move by the use of foot‐like structures called pseudopodia
What is the key food source for amoebae?
Bacteria
Bacteria need to find ways of preventing them from being killed by the phagocytic cells amoebae and this drives evolution
How do protozoa take in food?
By osmotrophy or phagocytosis
Osmotrophy
Absorbing nutrients through cell membrane
Feeding by phagocytosis
Engulfing particles via pseudopodia
As amoebae do
Where do protozoa digest their food?
In stomach‐like compartments called vacuoles
What happens when amoebae interact with bacteria?
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
What is symbiosis?
This comes from a Greek word simply meaning ‘living together’ and can be used to describe any association between two organisms
What is mutualism?
This can be used to describe an association in which both organisms apparently benefit
What is commensalism?
In this association one organism [the commensal] benefits, and the other [the host] is apparently unaffected.
What is parasitism?
In this association one organism [the parasite] benefits, and the other [the host] is adversely affected [weakened, sickened, damaged etc]
The phagocytic process of engulfing bacteria
- The bacteria engulfed by eukaryotic cells enter into a phagocytic vacuole
- The vacuole fuses with early & late endosomes, forming a phagolysosome
- Contains anti-microbial products within to kill/destroy nutrient source
This drives the evolutionary race between the host cell & the bacteria
How do bacteria try to win the evolutionary race between the host cell and itself?
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
How do secreted proteins get into eukaryotic cells?
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
How do some bacteria function at the surface of eukaryotic cells?
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
How have some bacteria adapted to understand what receptors are on the cell?
Some secreted proteins can interact with the receptor and trigger signalling cascade which alter the biology of the cell
How have bacteria evolved strategies to get into the cells?
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
How does the diphtheria toxin act?
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
How do exotoxins get into the cell?
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
Bacterial mechanism to deliver proteins directly into the cytoplasm
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
Bacteria in the GI tract
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
How has the GI tract evolved ways to prevent nutrients being stolen by bacteria?
- Stomach acid
- Peristalsis
- Mucus
- Glycocalyx
- Secretory IgA
- Antimicrobials
Role of the small intestine
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
Why are most bacteria kept out of the small intestine?
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
What is the gut lining composed of?
Enterocytes
Final stages of enzymatic digestion liberates small molecules for absorption
Sole site in the digestive tube for absorption of amino acids and monosaccharides
What are goblet cells?
Produce/maintain a protective mucus blanket by synthesizing/secreting high‐molecular‐weight glycoproteins known as mucins
What are enteroendocrine cells?
Produce gastrointestinal hormones/peptides in response to stimuli
Initiate digestive actions and detecting harmful substances triggering protective responses eg vomit
What are paneth cells?
Secrete anti‐microbial compounds into the lumen of the intestine
Key features of enterocytes?
- Tight junctions
- Adherent junctions
- Desmosomes
- Gap junctions
Tight junctions in enterocytes
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.
Adherent junctions in enterocytes
An adhesive belt anchors adjacent cells
A transmembrane linker protein attaches to actin microfilaments of the cytoskeleton and binds adjacent cells
Desmosomes in enterocytes
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
Gap junctions in enterocytes
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
Barrier function of tight junctions
Forms a continuous seal
Serves as a selective barrier to small molecules and total barrier to large molecules
Fence function of tight junctions
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)
What happens when theres disruption of tight junctions?
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
Specialised properties of intestinal epithelia
- 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
What happens if theres a loss of microvilli on intestinal epithelia?
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
When did multicellular eukaryotes and mammals evolve?
Multicellular eukaryotes evolved only during the past 1‐2 billion years
Mammals proliferated massively only during the past 65 million years
Why was the evolution of mammals good and bad for bacteria?
- 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)
Is E. coli gram negative or gram positive?
Negative
What E. coli is a safe strain?
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)
What E. coli causes diarrhoeal disease?
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)
How did E. coli evolve to cause diarrhoeal disease?
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
