Bacterial survival and damage

How are neutrophils important in the anti-bacterial response

• When activated, apoptose and release their DNA

• DNA is very sticky due to charge density

• Clumps and forms big complex that bacteria can get stuck in

• NET - neutrophil extracellular traps

How can Staphylococcus and Streptococcus survive in an activated-neutrophil environment?

• Make puss filled with enzymes

• Collagenase breaks down connective tissue

• Coagulases induces fibrin clot formation

• DNase breaks down DNA in pus from activated neutrophils

What are two different mechanisms of bacterial spread between cells, stating an example bacteria for each

• Lyse vacuole and spread by forming new vacuoles during exit and entry - Listeria

• Replicate inside entry vacuoles, rely on movement of host cell throughout body - salmonella typhi

How does Listeria spread from cell to cell?

• Lyse entry vacuole, released into cytosol

• Further cytoskeletal rearrangement induced alongside replication packages in new vacuole

• Released, lyses this for free movement

• Enters new cell in new vacuole

How does Salmonella typhi spread form cell to cell?

• Replicates in entry vacuoles inside macrophages

• Spread by macrophage movement through lymphatic and blood vessels

• Increased movement during immune responses, symptoms worsen

What are the three main categories of the host defence?

• Physical / chemical barriers

• Early immune

• Induced immune

State the physical / chemical barriers used as host defences

• Skin

• Mucous membranes

• Lysozyme

• Limited free iron availability

Explain the physical / chemical barriers used as host defences

• Skin: acidic, high salt limits growth

• Mucous membranes: traps bacteria, tight junctions limit invasion, includes cilia in airway

• Lysozyme: in tears and saliva, splits peptidoglycan, osmotic rupture

• Limited free iron: sequestered and compartmentalised, lactoferrin (breast milk, WBC, tears) and transferrin (RBC, semen)

State the early immune host defences against bacteria

• Complement

• Macrophages

Explain the early immune host defence against bacteria

• Complement: alternative pathway activated by bacteria

• Macrophages: bind via lectins, engluf, destroy bacteria in phagolysosomes

State the induced immune host defence to bacterial infection

• Antibody production

Explain the induced immune host defence to bacterial infection

• IgG opsonizes bacteria

• Fc-mediated phagocytic killing - antibody dependent cellular cytotoxicity

• Other Ig can also be used in neutralisation to prevent antigen binding receptors and adhering / internalization

How do bacteria overcome the hosts barrier of limited free ion availability?

• Bind iron at much higher affinity than host by secreting siderophores that capture iron and bring back to bacterium

How do bacteria overcome the host barrier of stomach acid?

• Stomach acid designed to kill by disrupting metabolic enzyme activity

• Helicobacter pump H+ back out off their cell, or cleave urea into NH3 to increase concentration of basic compounds and neutralise the acid

Simply, what are four ways bacteria can overcome the early immune defence of macrophages - give an example bacteria for each

• Macrophage paralysis - Yersinia

• Resist phagocytosis - P. aeruginosa

• Kill - E.coli

• Survival within - Salmonella

Describe how Yersinia can cause macrophage paralysis to overcome the early immune host defence

• Inject protease YopT into macrophage using needles

• Targets small GTPases to disrupt the cytoskeletal rearrangements needed for engulfing the bacteria

• Inject YopP

• Acetyl transferase activity inhibits signaling and triggers apoptosis of the macrophage (more of a kill mech)

Describe how P. aeruginosa can resist phagocytosis to overcome the early immune defence

• Surrounded by ‘slime’ capsule of non-PAMP glycoprotein polymers

• Physical block to phagocytosis (Steric hinderance)

• Most of these glycoproteins do not have affinity for complement factor B, so C3b cannot accumulate to direct macrophage engulfing and ADCC

Describe how E.Coli can kill macrophages to overcome the early immune host defence

• Secrete pore-forming toxins: the cytolysins

• Destroy macrophage membrane integrity

• Triggers apoptosis

Describe how Salmonella can survive within the macrophage to overcome the early immune host defence

• Inhibits fusion of phagosome with lysosome

• If this happens anyway, can resist oxidative pressure exerted by phagolysozymes to maintain bacterial integrity

How does Salmonella overcome the host defence of the induced immune response?

• Switches expression of surface antigens in phases

• Flips invertible promoter region that is flanked by recombination sequences that alter which flagellin protein is produced

How does Staphylococcus aureus overcome the host defence of the induced immune response?

• Expresses protein A on surface

• Binds Fc regions of Abs

• Prevents opsonization as C1q needs to recognise Fc region and active the classical pathway

What are two ways of overcoming the host defence of the induced immune response - give an example bacteria for each

• Evade recognition by switching expression of surface antigens - salmonella

• Binding Fc - Staphylococcus aureus

What are the two main types of toxin that cause direct damage - include two bacteria and their respective toxins for each

• Cytolysins e.g. E.Coli haemolysin / Helicobacter VacA

• A-B toxins e.g. C. Botulinum BoNT / Cholera CTX

Describe the action of E.Coli’s haemolysin toxin

• Targeted cell lysis, at lower concentrations can disrupt target cell’s signal transduction

• Can disable immune cells and assist tissue damage to promote spread

• UPEC cytolysin causes destruction of upper epithelium so can establish reservoir deeper in epithelium for recurrent infection

• Also allows break out of reservoir when replication is significant again

Describe the action of Helicobacter’s VacA toxin

• Disturbs anion balance

• Disrupts junctions between cells so can penetrate deeper in the GI epithelia

Describe the general action of A-B toxins:

• Specific catalytic activity dependent on A and B subunits used

• Receptor-mediated endocytosis by B binding receptors

• Connected by single disulphide bridge that is reduced in the lumen, mechanism unclear but could be breaking DSB between A and B and then reforming it in A in a way that both activates but also releases it from B

• Intracellularly active component (A)

Describe the action of V. Cholerae’s CTX toxin

• B binds GM1-ganglioside on host GI epithelia

• Receptor mediated endocytosis

• Endosome transports to ER

• Exact mechanism unclear but could be break of DSB and then reform within A in a way that both activates it and releases it from B

• Active A translocates to cytosol

• Binds Arg on Galphas, preventing intrinsic GTPase activity

• Remains active, maintains high cAMP

• Disturbs Na+ and Cl- membrane pumps

• Ion imbalance leads to mass water and electrolyte loss into gut lumen

Describe the action of C. Botulinum’s BoNT toxin

• A-B toxin absorbed from stomach

• B domain binds motor neurone receptor

• Receptor mediated endocytosis

• Acidification in endosome, reduces DSB

• A released into cytoplasm of motor neurone

• Cleaves V-SNARE synaptobravin

• Zipper complex now unable to form

• Can no longer release ACh to stimulate muscle contraciton

• Flaccid paralysis

Simply, what is meant by indirect damage

• Damage due to host immune response

• Acute inflammation too much / too long / in the wrong place

• Chronic inflammation

What triggers there to be too much / too long of an acute inflammation response?

• Gram neg LPS released from dead bacteria

• Binds TLR4 on macrophages

• Triggers signaling pathways that activate NFkB and MAPK cascades to rapidly increase cytokine synthesis and secretion

• Basis of septic shock

What causes septic shock?

• Too much inflammation

• Lipid A (a LPS) very strong PAMP for strong immune response

• Killing large numbers of bacteria simultaneously leads to large influxes of lipid A

• Too much NFkB and MAPK activation of cytokine gene expression

• High fever, low BP. increased clotting

What triggers acute inflammation to happen in the wrong place?

• Bacteria cross endothelium into blood, and can cross further past BBB

• Does not aid bacterial survival, bad all round

• Inflammation in brain due to LPS Lipid A

• Meningitis

What are two examples of chronic inflammation-causing bacteria that cause indirect damage?

• Helicobacter pylori - chronic inflammation of GI lining predisposes gastric cancer

• Myco TB - granuloma formation, inherently necrotic

To protect themselves from immune identification, bacteria can…

• Prevent their destruction by macrophages

• Reduce immune activation at the surface

• Form a reservoir using toxins

Why does antibiotic treatment for helicobacter often increase chance of persistance?

• Elimination of sensitive population so that only the stationary phase (non-dividing, less susceptible) cells remain

• When they leave stationary phase and replicate, they establish the resistance gene within the population

Describe when and how Mycobacteria become chronic

• Most infections resolved by strong active response, but 10% do not completely clear, with AB and immune-response resistant persistant bacteria held within granulomas

• Represses indirect damage by an acute immune response going on too long by surrounding infected macrophages with phagocytes that can fuse into giant, polynuclear cells

• This is then surrounded with T / B / DC/ neutrophils by the chemokine storm

• Begin recruiting fibroblasts or accumulate calcium salts to from an ECM which completely walls off the infected cells from the rests of the immune system

• Centre is caseous necrotic

• Short term benefit but risks reactivation long term

Describe how mycobacterium infects and begins colonisaiton

• Inhaled by droplets

• Phagocytosed by alveolar macrophages

• Replicates inside macrophages

• Small number disseminate with macrophage movement to other regions of the lungs and even lymph nodes

Stat about HIV co-infection with TB

• 6x more likely to fall ill with TB than HIV negative counterparts

• Leading cause of death among AIDS patients

Describe how chemokine storms result in granuloma formation

• Infected macrophage releases TNF-alpha to recruit neutrophils

• Neutrophil also release TNF-alpha for further neutrophil recruitment and to activate the infected macrophage to upregulate oxidative pressure - positive feedback

• TNF-alpha spike causes recruitment of NK, T, B and uninfected macrophages around the infected core

• Eventually, B and T release IL-10 and TGF-beta to promote Treg downregulation of the inflammatory response (prevent sustained indirect damage)

• Any bacteria still residing within centre macrophages remain in the centre, cause necrosis

What main host factor contributes to whether Mycobacterium will form tubercles (granulomas)?

• Weak initial immune response e.g. already immunocompromised

• IFN-gamma and TNF-alpha produced by CD4 and CD8 activate macrophages to strongly control mycobacterial infection

How is a mycobacterial infection conntrolled by a strong immune response?

• IFN-gamma and TNF-alpha produced by CD4/8

• Activate macrophages to increase ROS production and increase phagolysosome and vacuole acidification

• TNF-alpha also activates further macrophages and neutrophils

How do mycobacterium interfere with the adaptive immune pathway?

• Inhibit IL-12, preventing Th1 polarisation

How does helicobacter pyolori cause pathology?

• Neutralises gastric acid by secreting urease which splits urea into ammonia, raising pH and decreasing acidity

• Gastric mucous less viscous, easier to swim towards epithelia

• VacA disrupts anion balance (apoptotic) and cell junctions so penetration is easier

Describe how helicobacter establishes chronic infection

• Induces IL-8 secretion by epithelia, recruits neutrophils

• VacA disturbs anion balance (apoptotic) and cell junctions, forming pores

• Penetrate lower layers to establish reservoirs

• Cells lose structure and flatten, also gain resistance to apoptosis (increase gastric cancer risk)

• Macrophage recruitment of CD4 and B cells further disrupts chemical homeostasis

• B cells secrete IgA/G that clear external bacteria, but cannot access those that have established reservoir below surface epithelium

What is the treatment for helicobacter?

• Amoxicillin and clarithromycin 2x a day for at least a week

• PPI

• Premature termination of bacterial protein synthesis by action at bacterial 50S