Immunity - Mechanism of bacterial infection

What is Gram stain?

What is Gram stain?

Developed by the Danish bacteriologist Hans Christian Gra,

Heat fixed slide of tissue is stained with crystal violet with purple color

The stain is attached to peptidoglycan in the bacterial cell will, which then iodine is added to bind to the crystal violet and trap the crystal violet in the cell so it doesn’t float away

Then alcohol or acetone is added

Lastly, stain with safranin or carbol fuchsin (pink)

How are gram positive and gram negative bacteria identified by Gram stain?

Gram positive retain colour of the crystal violet dye

Gram negative do NOT retain the crystal violet dye because they have an outer membrane which the dye cannot get to them

What are Acid Fast bacilli….

Organisms that are tend to be called gram positive

They have a different structurewhere they have a lipid outer layer and do not stain properly under the Gram stain

Example: mycobacterium tuberculosis

Carbol fuchsin (red) in phenol driven into bacilli with heat= Ziel Neelsen stain

Motility of bacteria

Some bacteria exhibits motility property . They contain outward motor structure which help them with

Swarming - Swimming

Even bacteria without outboard motor can also be motile through a couple of mechanism

Twitching (plus retraction) - Gliding (focal adhesion) - Sliding (spreading though an organism that divide)

Aerotolerance

And what are

Obligated aerobe

Micro arophile

Facultative Anaerobe

Aerotolerant Anaerobe

Obligate Anaerobe

The bacteria that require aerobic respiration would be found in the top of the tube

Micro aerophile can grow in small amount of oxygen

Facultative anaerobe can either use areobic respiration or fermentation whcih does not require any oxygen. can find in any oxygen concentration

Biofilms

Microbes creating a sticky matrix which protect them from the immune syste,m , resistant from the antibiotic around them.

What is the cycle of biofilm?

• Initial attachment

• Irreversible attachment

• Biofilmmaturation

• Buidling up into high rise building but in bacterial form

• Dispersal and aggregation

• Planktonic cells release

Cycle repeats

Genetic material of a bacteria

They can have most of their DNA inside the nucleoid but they can have the ability to have extra bits of the DNA in the form of plasmid

Pan genome =

core + accessory gene

The same species can be quite different if they have got different accessory genes

they can express genetic ability, turning on and off genes lead to different phenotypes

Transduction, transformation and conjugation

Transduction : virus injecting the genes to the cell, cell hence picking up new traits from the virus

Transformation: bacteria just picking up plasmid which are the extra bits of material from the environment, for example, when a bacteria die, its genetic information is busted out to the environment so a cell can suck it of

Conjugation: two different species of bacteria comes together, form a bridge and transfer genetic material

Pathogen

microbes capable of causing damage to the host

opportunistic pathogen

take advantage of an opportunity to cause disease

Intra vs extracellular pathogen

Extracellular pathogen: can be anywhere, on the skin or in the soil

Obligative intracellular: required to be inside a cell

Facultive intracellular: can be outside or inside the cell

Host range

Influenza A: very broad host range, in human and a lot of animals

Influenze B C: host restricted

How to cause an infection : steps

Step 1: Establish a foot hold

Step 2: Evade host defences

Step 3: Proliferate

Step 4: Cause damage

What are the ways that bacteria stick in Step1: Establish a foot hold

• Non specific adhesion molecules which are often reversible ( Lipotechoic acids of Staphylococcus aureus and Alginate capsule of Pseudomonas aeruginosa)

• Specific adhesins from grame negative bacteria: Pili and fimbriae or in Gram positive bacteria: cell wall protein .there is Microbial suface components recognising adhesive matrix molecule

Step 1: Establish a foot hold EPEC example

Adhere to something, preventing being washed away by finding and sticking and achoring to the right niche. Taking advantage of the sticky ness

Example: Ecoli . (EPEC) which has initial contact with intestinal cells mediated through bundle-forming pilus (BFP), changing the appearance of the cell which then leads to distinctive lesion characterised by effacement of brush border microvilli and intimate attachment of bacteria to cell. T

This is relate to the LEE: locus of enterocyte effacement. They do not come inside the cell but inject own proteins into the cell and change what the cell does

Firstly through the translocation pore EspD/B (T3SS) it punches EspA subunit through into the cell membrane, and like a spring punch its way through the host cell and injects a bunch of proteins. Eventually pump in their own proteins that express as receptors on the outside of the cell which is called the Translocated intimin receptors (Tir) which binds to Intimin on the bacteria’s membrane. Which creates an intimate connection betwen the host and the bacterial cell.

Step 1: Establish a foot hold , example of Neisseria meningitidis (Gram negative bacteria)

Step 1: Pili attach to receptor on nasopharyngeal cell surface

Step 2: Pilli retract

Step 3: Bacterial outer membrane adhesin attaches to receptor on cell surface

Step 1: Establish a foot hold , example of Staphylococcus aureus (Gram positive bacteria)

Step 1: lipoteichoic acids mediate initial attachment to cell surface

Step 2: MSCRAMMS (Microbial Surface Components Recognizing Adhesive Matrix Molecules) mediate stronger interactions with host matrix proteins, depending on the type of cell, can bind to fibrogen, collagen, fibronectin.

Step 2: Evade host defence

Example of host defence:

• skin, mucus barrier. secretes antibacterial compounds (cationic antimicrobial peptides, lysoyme, secretory IgA, lactoferrin)

• Iron restriction: because iron plays as an important cofactor in many bacterial enzyme, it is restricted as a line of defence, we do not havemuch free iron running around in our body

Evading the host defence: complement factor proteolysis

Complement factor proteolysis:

Influencing complements ability to bind to the bacteria

example is Streptococcus pyogenes which release the C5a peptidase which is a protease that break down 5a complement which prevents MAC

Evading the host defence: Phagocytotic killing

Phagocytotic killing:

• Usually the bacteria get phagocytosed after opsonisation but in some cases the engulfed bacteria can produce toxin which leads to phagocyte death but bacterial survival

• eg. bacillus anthracis

• Or in mycobacterium tuberculosis: they do not release toxic but detoxify ROI or prevent fusion with lysosome therefore the bacteria still survive after being engulfed

Evading the host defence: mimicking and pretending to be from human

Mimicking and pretend to be coming from the human:

• Usually the antibodies would recognise the foreign antigens on the surface bacterium by binding the Fab region to the antigen receptor and then promote the destruction and the disposal of the pathogen, but

for example:, like Staphylococcus aureus Protein A, they actually binds the antibody the other way around. The Protein A of Staphy binds the to antibody Fc region (not Fab), thus inhibiting the complement activation, phagocytosis and ADCC (antibody- dependent cellular cytotoxicity).

Staphylococcus aureus bacteria also exhibit MSCRAMMS which binds to the extracellular matrix protein, which means that it cover itself in a layer of host proteins, as well as being involved in attachment, so it gets recognise as self.

Evading the host defence: resisting antimicrobial fluid

Resisting antimicrobial fluid:

• Altering surface charge to repel cationic peptides

• Producing proteases eg to cleave sIgA

• Producing a physical barrier eg capsule and outer membrane

Invading by mask and hide so the immune defences do not see the invader as NON SELF, for example, haivng a poorly immunogenic capsule so the immune system cannot recognise

Evade host defence: Example from invasion by Listeria

Entry into the cell by the protein Internalin that it has, being taken into a cell vacuole, but then it destroy the vacuole so it could be free living inside the cell

Actin builds up and form comet tail and pushes the Listeria from one cell to another, an essential spasm to keep Listeria intracellularly.

Masking and hiding by being inside the cell.

Evade host defence: Masking and hiding: capsule - Example by Streptococcus pneumoniae.

Contains poorly immunogenic polysaccharide

They use the capsule to hide proteins on their surface that are normally what are recognised.

Surface exposed protein antigens are recognised by antibodies.

Complement deposited on surface of bacterium

Thus easily phagocytosed (opsonised) complement mediated lysis

Many vaccines composed of capsular polysaccharide so pathogen easily recognised

Evading host defence: Masking and hiding example - Mycobacterium tuberculosis

Mycobacterium tuberculosis stay inside macrophages and abuse the cell to get nutrient

Evade the host defence: another mimicking type - binding factor H - Neisseria meningitidis type b & M protein of streptococcus pyogenes

If C3b forms on a cell it can target it for complement-mediated damage

Serum factor H binds to our cells and degrades any C3b forming on the surface to protect us from damage

But sialic acid capsule of Neisseria meningitidis type b & M protein of streptococcus pyogenes bind fH and evade complement killing, so they do not get degraded by complement cascade

Evading the host defence: complement disruption from stapylococcus aureus

Stapylococcus aureus blocks 5a receptors by secreting CHEMotaxis Inhibitory Proteins of Staphylococci (CHIPS)

Feeding CHIPS to the complement which has higher binding affinity to 5a, therefore blocked! Cannot bind to bacteria

Evading the host defence: antigenic variation

Usually, the antigen on the bacteria are recognised by antibodies so they can be attached. And the amount of specific antibodies for that antigen would be increased so next time when the antigen is introduced the adaptive immune response is quicker and faster.

However, the bacteria can change its antibodies so it can no longer be recognised

Example: Neisseria meningitidis pilin, outer membrane proteins

Evading the host defence: releasing decoys

Decoys are the outer membrane blebs containing lipopolysaccharide and the bacteria release those outer membrane blebs as immune decoy so it would overwhelm the immune system by doing that and the immune system would be busy phagocytosing the blab while the bacteria itself commence invasion.

Evading the host defence: staying out of reach

To get far away and protect themselves from the membrane attack complex and complement C3b, E coli would produce really long O-antigens on its surface

How does our body get hold of iron

Lactoferrin in secretions

Transferrin in blood

Stored as ferritin in cells

Functional in enzymes

How do bacteria gets iron from us? four ways

• By the secretion of Siderophores(small iron chelators) which are specific binding proteins eg, for transferrin

• Increasing the available iron by destroying cells which eventually release irons by haemolysin, inducing protease activities

• Adapt to parts of the cell where there is a lot of iron, for example to high iron niche of phagosome

• Evolving enzymes that use other co-factors eg, Mn