Bacterial colonisation of the host

What are two ways that bacteria can respond to the environment?

• Chemotaxis

• Quorum sensing

Briefly describe how chemotaxis works

• Chemoreceptors at the ‘nose’ of the cell sense and transmit information via phosphorelay to the flagellar motor switch

• Switch controls direction of the flagella: CCW for swim, CW for tumble and random direction change

Briefly describe how quorum sensing works

• Switching on of virulence genes when it is detected that the bacterial population of a biofilm has reached a specific density

• Secretion of small signal molecule and sensing its concentration in the environment as a proxy of bacterial population in local environment

What are virulence genes?

• Specialised genes for bacteria’s strategy of invasion and damage

• Not constitutively expressed, but induced in response to the host environment

• Can be co-regulated

• Can be carried on plasmids or pathogenicity islands of the chromosome

Why is carrying virulence genes on plasmids advantageous?

• Gene can be at presence of less than one copy per bacterium in a population

• When selected for, HGT to others

• Proliferative advantage, expands until whole surviving population has it

What is a pathogenicity island?

• Regions of the chromosome dedicated to genes required for infection and survival within the host

• Evolved by integration of transposons, plasmids and other transformed DNA - evidence in that typically have G/C content that differs from surrounding chromosomal DNA

Give an example of genes held on a pathogenicity island, and briefly what they do

• Salmonella SPI-1 and SPI-2

• Entry into non-phagocytic cells, survival in macrophages respectively

• Increases dissemination of virulence traits

Why are pathogenicity islands advantageous?

• Localisation enables coordination

• Enables HGT to transfer all required virulence genes at once, without transferring large segments of the genome, as transferring only one or two factors alone would not necessarily increase the virulence of the recipient, as sometimes only increases virulence if all factors are working together at once, e.g. Salmonella

Simply, what are mechanisms bacteria use to initially colonise the required cell type

• Motility

• Surface adhesions

Describe how bacterial cell motility is important for colonisation

• Increases chances of coming into contact with host surface if it can move directly towards it, rather than passively waiting for chance encounter

• Some e.g. helicobacter pylori move through mucus

• Swim through liquids using chemotaxis, attracted to nutrients

• Some small populations can even coordinate movement to swarm across surfaces e.g. Proteus

Describe how cell surface adhesions are important for colonisation

• Enable stable attachment to host cells or extracellular matrix via glycolipid or carbohydrate receptors using pili adhesion proteins

• Can then ‘pull’ closer

• Overcomes initial repulsion between negatively charged membranes

Why are pili advantageous for colonisation?

• Overcomes initial repulsion between negatively charged membranes

• Can then ‘pull’ closer

• Allows long distance plasmid transfer (Again, without the need to come together despite electrostatic repulsion)

• Can adhere from a distance but then this localises toxin secretion

Describe how V. Cholerae colonises the small intistine epithelia

• Pili adhesins bind epithelia

• Secrete A-B toxin CTX

• Colonisation is secure and extracellular, so toxin can reach many cells but still localised to the small intestine

Describe how UPEC colonises kidney epithelial cell

• P-pilus adhesin proteins on the end of pili

• Binds GbO4 on kidney epithelia

• Stabilizes

What does UPEC stand for? What does it cause?

• Uropathogenic E.Coli

• Many of the UTI types

How does pneumonia overcome host barriers to colonise the nasopharynx?

• Polysaccharide capsule promotes survival in dissemination droplets

• Resists removal by mucous and cilia by pneumolysin release that forms pores in cilia so less efficient

• Secretes IgA protease that prevents opsonization so not targeted for phagocytosis by macrophages

• Enables migration to lower respiratory tract

What is the envelope of mycobacterium tuberculosis made of and what are the consequences of this experimentally / in the lab?

• Impermeable mycolic acid

• Resistant to drying and disinfectants

• Anti-microbial and immune-evading layer

• Difficult to detstain with decolourizer during gram staining, appears ‘gram neutral’ or ‘gram ghost’ under light microscope

What are the four factors of bacterial colonisation, and what bacteria model each one?

• Adhesion - EHEC

• Biofilms - P. aeruginosa

• Host defense evasion - Pneumonia

• Internalisation - Listeria, salmonella

(not all have to be done)

Describe how an acute UTI can lead to reccurent UTIs

• During acute, bladder epithelia that are infected with replicating UPEC will be degraded by toxin haemolysin until the infected epithelial cell detaches from the rest of the epithelium

• Other nearby UPEC can invade sub-surface epithelial cells, form a reservoir

• Reservoir protected from AB treatment once top layer of epithelium repaired

• Eventually, reservoir replicates, infects upper epithelium and can be released back into the urinary tract, second round of infection which can also migrate to the kidney

What species is colonization via pedestal adhesion restricted to?

• E.Coli

What is colonisation via pedestal adhesion?

• Tighter adhesion to epithelia by E.Coli, especially EHEC

• Initial P-pilus adhesion pulls EHEC closer

• Specific translocated intimin receptor (Tir) needle penetrates host cell

• Intimin on bacterial surface phosphorylates Tir

• Enables recruitment of host proteins inside cell that are involved with actin reconstruction

• Actin reconstruction and polymerization for formation of tightly bound ‘pedestal’ that fixes bacteria to cell

• Gives time for further cytoskeletal rearrangement and continuous injection of effector proteins that promote internalization and invasion

What is the purpose of colonization via pedestal adhesion?

• Gives time for further cytoskeletal rearrangement

• Continuous injection of effector proteins that promote internalization and invasion

Draw a detailed diagram of colonisation via pedestal adhesion

https://ibb.co/hFrx1fbG

Can all bacteria form biofilms? Why?

• No

• Requires genes for quorum sensing and specialised adhesion molecules

• Energetically costly to maintain

Where do we see biofilms clinically and why are they difficult to tackle (clinically)?

• P. aeruginosa on contact lenses - Conjunctivitis

• P. aeruginosa in CF patients

• Staphylococci on catheters - UTI

• Difficult to eradicate by immune system cytotoxic response and by antibiotics due to decreased SA:V

Why does P. aurginosa form biofilms?

• Gens encoding virulence factors for direct and indirect damage are co-regulated by quorum sensing

• Increased virulence and proliferation in the presence of biofilms

• Synergistic effect of anti-microbial polysaccharide slime secreted, forms thicker layers for drug e.g. fosfomycin to penetrate before can reach bacteria

Describe P. aeurigonsa biofilm formation in CF patients

• CF airways constantly inflamed, producing thick sputum with high [DNA, actin, salts] from apoptosis of neutrophils

• Local anaerobic environment, but facultative, so not a barrier to growth

• Pili binds DNA in sputum, many more binding sites available for non-epithelial initial adhesion

• Closer proximity to CFTR glycoprotein and epithelia, easier to adhere

Contrast P. aeruginosa activity in healthy vs wt patients

• Rarely form biofilms in healthy as colonisation of epithelia is random and dispersed

• Forms biofilms in CF as DNA in sputum is extra binding sites for initial adhesion, and can then use biofilm formation for localisation and coordination of colonisation when properly adhering to respiratory epithelia

Simply, what are the two mechanisms of colonisation via invasion, and what bacteria models it?

• Zipper mechanism of receptor-mediated endocytosis - Listeria

• Trigger mechanism by injection - salmonella

There are two mechanisms of colonisation via invasion, but what do they both do?

• Remodel host cytoskeleton by binding actin directly and / or subversion of host signal transduction

• Remodeling enables internalisation

Describe the zipper mechanism of receptor-mediated endocytosis as a general model

• Invasin proteins can be associated with pili or expressed directly on the cell surface

• Mimic eukaryotic ligands on extracellular matrix

• Single invasin is sufficient to promote host cytoskeletal rearrangement and internalisation into an endosome, so require relatively low expression

• Listeria models

Describe the zipper mechanism of receptor-mediated endoytosis, in the context of Listeria

• Invasins InlA and InlB are anchored directly to listeria cell surface

• Adhere to junction protein Ecad, or RTK Met respectively on GI epithelia

• Promotes cytoskeletal rearrangement and internalisation in to an endosome

Describe the trigger mechanism by injection of colonisation by invasion as a general mechanism

• Effector proteins injected into host cell through bacterial needles

• Proteins mimic euk proteins involved in actin polymerisation and signaling pathways to promote cytoskeletal rearrangement and internalisation into endosome

• Salmonella models

Describe the trigger mechanism by injection of colonisation by invasion, in the context of Salmonella

• Adheres using P-pili adhesion proteins to GI epithelia

• Inject SipA and SipC, mimic euk proteins involved in actin polymerisation

• Inject SptP, mimic euk proteins involved in signaling pathways

• Promotes cytoskeletal rearrangement and internalisation in to an endosome

• SipA,C and SptP are all held on a pathogenicity island within the Salmonella genome

How does V. Cholerae colonise small intestine epithelia?

• Fimbral adhesin binds epithelia

• External colonisation means continuous secretion of CTX toxin can reach large numbers of nearby cells

Simply, how does helicobacter py,lori colonise the stomach epithelia?

• Surface adhesins BabA and BabB bind