Lecture 3 - Exopolysaccharides and Membrane Proteins Flashcards
The structure of the cell wall is important to the integrity of the cell. One way to kill bacteria is to:
attack the cell wall, since without it bacteria CANNOT survive
Gram Negative Summary
2 membranes, inner and outer
Peptidoglycan layer is in between the 2
Space between the 2 membranes (IM & OM) is the periplasmic space
Gram Negative bacteria stain PINK/RED in gram stain
Gram Positive Summary
Have a thick layer of peptidoglycan and no outer membrane.
Will stain purple in a gram stain
Most useful antibiotics target the _______ specifically ___________ of the _______
Most useful antibiotics target the MEMBRANE specifically PEPTIDOGLYCAN of the CELL WALL.
Some antibiotics are useful against gram positive bacteria but not gram negative bacteria. Why?
Because the gram negative bacteria have an outer membrane that the drugs cannot penetrate. Other drugs have to be designed to go through the outer membrane of gram negative bacteria.
The CAPSULE is
also called exopolysaccharide capsule or glycocalyx (not good names since not always composed of sugar, sometimes amino acids) USE CAPSULE!
Outside the cell wall,
secreted by the cell,
very defined
the EPS
EPS = extracellular polymeric substance
also called the slime layer or exopolysaccharide (not good names because it is sometimes composed of sugar but NOT always - also proteins or extracellular DNA.)
EPS is not well defined and you cannot see it very well.
It is a matrix that bacterial cells produce that forms a cocoon that shelters and allows bacteria to form a biofilm.
How is capsule stained such that it is visible under microscopy?
The bacteria have a ghostly shade around them which is the capsule. It lies outside of the cell wall and is secreted by the cell, very define, unlike the EPS.
The stains used generally bind to charged particles, but the capsule is NOT charged therefore we cannot stain the capsule specifically. Instead, we use a counter stain to stain everything but the capsule which is why it appears as a ghostly white outline.
What is the main difference between capsule and EPS?
The capsule does NOT detach
What is a biofilm
Biofilms allow cells to attach to a surface. Plaque is a biofilm. Cells attach to a surface (like enamel, epithelial cells, tongue) with a slime layer that allows them to stick, otherwise they would be washed away. In the oral cavity it is ALWAYS a biofilm.
Are tge CAPSULE and EPS necessary for bacterial cell survival?
NOT all Bacteria have capsules or EPS
Both the capsule and EPS are NOT required for the bacteria to grow.
If you mutate bacteria so they cannot produce the capsule, it will grow just as well in a laboratory setting. This is NOT the case with the CELL WALL - bacteria cannot grow without a cell wall!
ALTHOUGH NOT REQUIRED FOR GROWTH, THE CAPSULE IS VITAL IN SURVIVAL. CAPSULES AND SLIME LAYERS ARE HELPFUL IN THAT THEY ARE:
- physical barriers
- chemical barriers
- protect against dessication
- protect from phagocytosis
What are the Functions of the EPS and Capule?
- PHYSICAL BARRIERS
compounds from the environment can’t penetrate capsules and slime layers well - CHEMICAL BARRIERS
some chemicals can’t penetrate the EPS or just get stuck in it - PROTECTION AGAINST DESICCATION
the cell can survive much longer because the capsule and slime layer create a moist microenvironment - PROTECTION FROM PHAGOCYTOSIS
if protected with capsule, cells of immune system can’t really see the bacteria.
The Capsule can be seen as a __________
VIRULENCE FACTOR
What is a virulence factor?
Virulence factors are properties that help the bacteria to cause infection, specifically helping in one or more of the following:
1. colonization
getting into the body through the skin, respiratory tract, etc
- survival
needs to be able to survive in new environment - establishment/multiplication
avoid being detected by the immune system or grow faster than it can be killed - nutrient obtainment
bacteria needs toxins or enzymes that allow it to harm tissues to get nutrients (Ex: breaking down RBC’s)
Why can cells of the immune system not detect bacteria if capsule is present?
Immune cells usually look for the LPS, which will be concealed by a capsule. the bacterial cells outside of the body will grow well without a capsule, but inside the body the capsule is needed or the immune system will see it and attack it.
What is immunoevasion and what aids with it?
Avoiding of the host’s immune response
The capsule aids with immunoevasion.
Viruses are the most abundant organisms on the planet. Viruses gain entry into the body via a surface receptor on the bacteria. What happens if bacteria is covered by a capsule?
The virus won’t see the receptor and can’t penetrate the cell. If bacteria were covered by EPS, virus would have to go through it which would be difficult.
What can disrupt the cell membrane?
Toxins and detergents
What can protect disruption of the cell membrane from toxins and detergents?
Matrix - EPS - endopolymeric substance OR
Capsule provide some protection against both!
Functions of the EPS
true for just EPS not usually for capsule.
- Nutrient source: the matrix can be a food source since it is composed of proteins and sugars.
Example: S. mutans which causes caries, produces a sugar matrix.
Biolfilm is a gel that is very sticky/slimy and it can attach to a surface of other bacteria, creating an aggregation of cells which is more difficult for the immune system to handle.
What bacteria is said to cause caries?
Streptococcus mutans
What is the structure of biofilm?
The EPS forms a biofilm:
Biolfilm is a gel that is very sticky/slimy and it can attach to a surface of other bacteria, creating an aggregation of cells which is more difficult for the immune system to handle.
What is Streptococcus pneumoniae?
Gram Positive Bacteria
Causes upper respirator infections (pneumonia)
Form a slime layer
If colonies from fresh clinical isolates are smooth colonies, what does this indicate?
Presence of a capsule around the bacteria
If you continue reproducing same bacteria with smooth colonies, from plate to plate, they will eventually form rough colonies. What does this mean and why does it happen?
This means that bacteria have lost their capsules.
Smooth - with capsule
Rough - without capsule
In a lab setting, the bacteria no longer need the capsule anymore so they wont make it because it costs them a lot of energy to do so.
Why is it important to use fresh strains in the laboratory w/ regard to virulence factors?
Fresh clinical isolates or fresh strains are critical for studying virulence factors because bacteria tend to modify their need for certain virulence factors when in a lab setting - since they don’t require them and they require much energy to keep them around.
Ex: loss of the capsule
How many capsules can Streptococcus pneumoniae make?
S. pneumonia can produce 90 different polysaccharides to make different capsules.
Cells that are capsulated are __________ if they do not produce a capsule they are ______ because the immune system will easily kill it
VIRULENT
NON VIRULENT
What is the Classic Griffith experiment?
In the 1920’s people were trying to determine if genetic material was protein, DNA/RNA.
Experiment done with Mickey, a mouse.
- If injected w/ rough strain - Mickey lives
(no capsule, non-virulent, detected by immune system and bacteria killed) - If injected w/ smooth strain - Mickey dies
(capsulated, virulent, not detected by immune system) - If injected w/ heat killed smooth strain - Mickey lives
(though capsulated, bacteria was killed with heat so no effect) - If injected with MIX of Rough strain and heat killed smooth strain - Mickey dies
(this means that something was passed from the heat-killed cells into the rough non-capsulated live cells, which killed Mickey)
What was passed from dead to live cells - protein or DNA?
Determine this by adding protease or DNAse to injections
add PROTEASE to this mixture - Mickey still dies
add DNASE to this mixture - Mickey lives
THIS PROVES THAT THE GENETIC ELEMENT IS DNA - NOBEL PRIZE.
What is the thing that allow bacteria to establish itself in the host and cause disease?
Give an example
VIRULENCE FACTOR
Ex: Capsule it helps the bacteria be bale to survive in organisms without being detected by the immune system.
What is specific to EPS?
- nutrient trap (matrix of sugars can be broken down and used for food in times of need)
- attachment to surfaces
- cell aggregation (helps associate multiple bacterial cells)
When you grow bacterial colonies, you swab a sample and streak it on a plate, single cells grown into _____
COLONIES - CFU = colony forming unit
Each colony is millions of cells that started from one cell.
What did the Griffith Experiment Determine?
This classic experiment determined that DNA is the genetic element which is the particle that contains genetic material.
What is Streptococcus pyogenes?
Gram Positive
Causes strep throat, rheumatic fever, puerperal fever.
Produces a capsule (mostly sugar) -
NAG-glucuronic acid - this has the same structure as hyaluronic acid, which is part of the human extracellular matrix. If the human body recognizes S. pyogenes, this means it will attack not just the bacteria but also cells in the body, leading to rheumatic fever.
Graph: Non-capsulated S pyogenes will not kill mice only wild type bacteria protected by capsule do!
Why bacterium contains NAG-glycuronic acid and why is this dangerous?
Streptococcus pyogenes
NAG-glycuronic acid has the same structure as hyaluronic acid, which is part of the human extracellular matrix. If the human body recognizes S. pyogenes, this means it will attack not just the bacteria but also cells in the body, leading to rheumatic fever.
What Is Streptococcus pyogenes Gram and What does it Cause?
GRAM POSITIVE
Causes strep throat, rheumatic fever, puerperal fever.
Does Streptococcus pyogenes produce capsule?
Yes - NAG-glycuronic acid (same structure as hyaluronic acid in human extracellular matrix - dangerous because if visible to immune system - then body will attack invader cells as well as its own - leading to rheumatic fever)
What Is Bacillus anthracis Gram and What does it Cause?
GRAM POSITIVE
Causes severe upper respiratory infection before migrating throughout the body.
Has 2 plasmids: pXO1+ (produces toxin) and pXO2+ (produces capsule)
Capsule is made of amino acid poly-D-glutamic acid (NOT SUGAR)
Does Bacillus anthracis produce capsule?
Yes capsule is made of poly-D-glutamic acid (not a sugar - AMINO ACID)
Which bacteria has 2 plasmids and what is distinct about their functions?
Bacillus anthracis has 2 plasmids
pXO1+ and pXO2 +
pXO1+ produces the toxin
pXO2 + produces the capsule
Who was Ignaz Philipp Semmelweis?
Noticed that rate of childbed fever was 3x higher in dorctor’s wards than nurse’s wards. Women after birth would get childbed fever because in doctor’s wards (student doctors) did not have hand-washing while the nurses used bleach/vinegar solutions. Semmelweis was palced in an asylum for his theory, which made the hospitals look bad.
Nevertheless, HAND-WASHING REDUCED THE MORTALITY RATE TO BELOW 1% AND HELPED KILL BACTERIA STREPTOCOCCUS PYOGENES
In graph of Bacillus anthracis (2 plasmids pXO1+ and pXO2 +) displaying viral load in the lung or spleen.
- Outcome if wildtype has both toxin and capsule and it infects ________________
- What happens if you knock out pXO2+ __________________
- If wildtype has both toxin and capsule and it infects BOTH THE LUNG AND MOVES TO SPLEEN (has BOTH the TOXIN (pXO1+) and the CAPSULE (pXO2+) so it can move to the spleen and not be detected by the immune system.
- If you knock out pXO2+ then the CAPSULE IS GONE however pXO1+ (TOXIN) is still present, therefore you just have establishment in the lung itself but no migration to the spleen (since without capsule, immune system detects it.)
Streptococcus mutans
Gram Positive
ACIDOGENIC - produces acid that causes caries in enamel (@ pH 5.5 and lower).
To stay attached to teeth, it produces EPS made from glucan. EPS makes it harder for saliva to reach bacteria and buffer acid produced. Sucrose (glucose+fructose) is very important for S mutans to produce glucan. When sucrose breaks down to form glucose + fructose, the bond breaks and high energy is produced which allows EPS to be made. Eating sucrose causes greater occurence of dental caries.
What Is Streptococcus mutans Gram and What does it Cause?
Gram Positive
Produces acid that causes caries in enamel (pH 5.5 and <)
Produces EPS made of GLUCANS. (SUCROSE IS CRITICAL FOR THIS!)
Does Streptococcus mutans produce EPS?
Yes - Produces EPS made of GLUCANS. (SUCROSE IS CRITICAL FOR THIS!)
High energy from breakdown of sucrose into fructose and glucose gives the energy necessary for S mutans to produce EPS. Thus eating sucrose causes greater occurrence of dental caries.
Eating sucrose causes greater occurence of dental caries. Why?
Because sucrose is very important for the S. mutans to make its EPS. Sucrose is Glucose + Fructose, breaking the high energy bond of sucrose produces the energy required for S. mutans to produce the EPS.
Which bacteria requires glucans?
On the graph, what happened in rats, if you know out ability to produce glucans.
Streptococcus mutans requires glucans to produce its EPS.
If ability to make glucans is knocked out, the bacteria find it hard to survive. There is still a small bit of caries occurring in no bacteria mutant rats because other bacteria present that cause caries but not to such a level as those in abundance of glucans.
Staphylococcus aureus
Gram Positive
We know it as MRSA - it is Methicillin-resistant Staphylococcus aureus.
It likes high salt concentration, lives in nose and on skin, A lot of strains are drug-resistant. One of the number one causes of non-viral eye infections.
Good at avoiding immune system because it has a polysaccharide on the capsule (poly-N-acetylglucosamine PNAG) which helps it attach to tissues and cells, form biofilm and protect it from our body’s defense.
What Is Staphylococcus aureus Gram and What does it Cause?
Gram Positive
MRSA - it is Methicillin-resistant Staphylococcus aureus. LIKES high salt concentration - lives in nose and on skin.
Has poly-N-acetylglucosamine PNAG polysaccharide on the capsule (helps protect it from immune system)
What is PNAG?
poly-N-glucosamine - a polysaccharide on the capsule which helps attach STAPHYLOCOCCUS aUREUS to tissues and cells, form biofilm, protect it from our body’s defense systems.
On a graph if you knock out the ability of S. aureus to produce PNAG, what happens with regard to microphages in the body?
Microphages are much more effective at attacking S. aureus and killing it. This explains the much lower survival rate of the PNAG mutant
Capsules are helpful in protecting bacteria, but downside is that capsule components can also be:
weakly immunogenic - this means our bodies can recognize parts of the capsule to initiate an IMMUNE RESPONSE
What is a vaccine?
A dead or inactive for of virus, which does not make you sick, but does cause your body to produce antibodies (active immunization)
What is active immunization?
When you get a vaccine which does not make you sick but causes your BODY TO PRODUCE ANTIBODIES.
If you are bitten by a snake, what sort of immunization will help?
PASSIVE IMMUNIZATION
you will be given the antibodies. (
How do you test in the lab if something is a virulence factor (with mice)
Knock it out and compare to wild type. If mutant no longer causes infection, then knocked out factor is virulence factor. You can further try to reintroduce the factor into the mutant and see if it will cause the infection again.
The tree of life is shown to represent what?
how distantly related one organism is from another. They all originated from a common ancestor and evolved from there.
_____ look like bacteria and sometimes grow in colonies like yeast, but in metabolism they are much closer to eukarya than bacteria.
FUNGI
____ are called ancient microbes and are usually found in _____ environments
ARCHAEA are called ancient microbes and are usually found in EXTREME ENVIRONMENTS LIKE HOT SPRINGS - there are No human pathogens within archaea.
Bacterial cell walls have:
psuedopeptidoglycan
Lysoszyme will do what?
break beta 1,4 cross links in peptidoglycan of mainly gram positive bacteria. it will not degrade cell walls of archaea because it has different structure
What is the S layer?
Paracrystalline protein layer Some bacteria (gram positive and negative) and some Arhcaea have a paracrystalline protein layer (s layer)
This s layer is a layer of proteins the cell secretes located outside the cell wall and is attached to the cell wall and functions similarly to the capsule.
It is not well defined among bacteria so it is not good for vaccines.
The S. layer is resistant to lysozymes.
It only allows partial permeability for toxins, and helps stabilize the membrane.
The bacteria may be able to survive without it. It also helps to hide bacteria from the immune system.
The good thing about the membrane is that it is impenetrable - compounds wont leak trough it but the bad thing is —–
it is difficult to get needed compounds through the barrier.
The good thing about the membrane is that it is impenetrable - compounds wont leak trough it but the bad thing is it is difficult to get needed compounds through the barrier. Therefore, you need ……..
MEMBRANE PROTEINS
For a gram negative bacteria, proteins on the inner membrane are _______ and proteins on the outer membrane are _______
inner membrane proteins
outer membrane proteins
Are there outer membrane proteins in gram positive bacteria?
NO but there could be an S layer
What is the S layer specific to?
Some Gram positive and gram negative bacteria.
What are the functions of the MEMBRANE PROTEINS?
- transport of materials from outside into the cell
- some proteins serve as secretion systems - take materials from inside and bring them outside
- Sensing proteins bind molecules from the environment which leads to up regulation or down regulation of certain genes
- proteins can have a role in attachment (to other cells, to teeth, etc..) These proteins are a virulence factor
- Some proteins can anchor the capsule to the cell
- Toxins
- Enzymes
What is a porin?
Found in outer membranes of gram negative bacteria only, they cross the outer membrane a few times, usually produce a 3 barreled channel. The channels (small openings) lead from outside of the cell into the periplasmic space.
Allows compounds to diffuse from high concentration to low concentration.
Not everything can pass through a porin - channels are usually selective by size since porins are small - average sugar can go through it and amino acids. Lysozyme us too large and cant passthrough so this is why gram negative bacteria are resistant to lysozyme. the lysozyme can/t get to the peptidoglycan.
Chloroplasts and mitochondria have their own DNA and probably originated as gram negative bacteria.
Where are porins found?
In the outer membrane of gram NEGATIVE BACTERIA ONLY
Mitochondria and chloroplasts have their own DNA and probably originated as
gram negative bacteria
Where are porins found?
on the outer membrane of gram negative bacteria only
Which bacteria are resistant to lysozyme? Why?
Gram Negative because lysozyme is too large and cannot pass through porins (which are located on the outer membrane of gram negative bacteria)
