L8/9- Salmonella Flashcards
What type of bacteria are they
Gram -ve dacultative intracellular (don’t need to be)
What 2 types are there
Salmonella enterica serovar typhimurium or typhi
Typhi causes typhoid fever and can go systemic and lethal
Tm causes salmonellosis and is local
How have some adapted to not be killed in acidic stomach
Acid shock proteins activating at sensitive ph
What are pathogenicity islands and spi1/2
Gene clusters needed for virulent factors
T3ss1 is for invasion inside affecting family of rho gptases
2 is for survival inside a scv
Both have needle complex, basal body and translocon
By systemic, where can typhi disseminate
Spleen,ln,liver,gall bladder
Why are m cells preferred route of salmonella
No mucus
Plus it can be take n up by mac or dc where it can migrate to mln and escape out to disseminate
What else can sample lumen and take them up
Crocs epi dc
What in mucus helps regulate inflammation
Trefoil peptides
Which types of phagocytes cna carry salmonella to the circulation for dissemination
Cd18+
Can use this indep of its t3ss
What happens when they eventually want to get released from macrophages
Cause inflammation eg via Il1b release from mac
Then either get killed ef by neutrophils or enter epi cells absolaterally
Which protein family regulates tight junctions because they have effect on the cytoskeleton
Rho gtpases
Disrupting their function would cause what
Change in junction organisation and cell membrane actin to promote entry
What are gaps
Gtp activating proteins
Hydrolyse gtp inactivating the gtpases
Give 3 members of the family and what they do
Rho - formation of focal adhesions (ecm to actin) and stress fibres
Rac- forming extrusions/lamellipodia from plasma membrane
Cdc42- forming finger extrusions (filopodia)
What do the extrusions from rac and cdc42 need
Actin polymerisation
What effects can tm have that doesn’t affect these rhos for their entry (by their effectors)
Down reg of zo-1 and dephos of occludins
What are the effector proteins which induce the ruffling of the membrane/ structural changes
Sipa,sipc, sopb, sope, sope2
What does sopb do
It’s a phosphatase needed for macropinosome formation
It indirectly stimulates cdc42 activity allowing extensions filopodia of membranes needed for macropinosome/pinocytosis
What do sope and e2 do
Act as gef activating rac1 and cdc42 to induce cutokskeletal extrusions for bac uptake
What do sipa and c do
Bind with actin and cause bundling to help with macropinocytosis
Which protein is recruited by cdc42 and rac1 for actin polymerisation for extrusions
Actin related protein arp 2/3
What late acting effector proteins (released after entry) help reverse cytoskeetal arrangements
Spt P
What does late acting avra do
Stabilise tj again
and inhibits inflammation response quickly by ikb blocking degradation
Which spi2 protein blocks mhc2 presentation on dc
SteD
How do scv acquire nutrients when inside cell
SifA induces tubules which provide nutrients to them
Which immune receptors key in early stages of salmonella and how do they induce proinflam cytokines
Prr
Myd88
Activate mapk signalling pathway and activate ap1 nkfb tf
Proinflam cytokines exp like Il1b,23,18,tnf,12,ifny
How can stm also induce compelsmtn
C3 recognises o-antigen on its lps
Which tlr shown v important in esrly process to stop invasion of mln
Flagellin tlr5
What does tlr1/2 recognise
Curli ligand in the ecm of salmonella biofilms
What does lps bind to on cell surface
Lps binding protein which then interacts with
Cd14 gpi anchored which interacts with tlr4
What do nod1 and nod2 also recognise if intracellular
Dap acid (nod1)
Muramyl dipeptide (nod2)
How do they activate nkfb also
Through rip2 adapter protein
When we’re nod 1/2 crucial for in mice salmonella
Crucial in infection when salmonella expressed their spi2 t3ss - ie intracellular
Which t3ss1 proteins can be delivered and activate nlrc4 receptor
Prgj from needle complex
And flagellin can be injected
What does this do
Associate with caspase1 and asc protein and allows cleavage Il1b and il18 to active forms by caspases 1
= drives neutrophil response and il18 drives ifny = mac activation
= inflammation
Which bacteriophage derived effector protein can some strains have that can activate caspase 1 by unknown mechanism
Sope
Which chemoattractant from salmonella induced macrophages causes basolateral influx of neutrophils from circulation
Il8
What does hepoxilin hxa3 do instead
Allows migration to the apical/lumen of neutrophils through tj
Which effector causes production of hxa3
It is an arachidonic metabolite
SipA induced lipid signal cascade , activation of pkc, and release of arachidonic acid from plasma membrane
Converted into hxa3 by 12 lipooxygenase
Why would sipA induce it’s own inflammation against salmonella
Thrives in it
How can neutrophils bind basolateral through rolling and adhesion
Their B2 integrins
What is the hypothesis of watery diarrhoea
Spi1 effector induced inflammation and rapid neutrophil influx (via sipA) causes impaired barrier and leakage of extra vascular fluid
Also neutrophils migration can lead to Cl sexretion by epi cells which causes influx of water for osmolarity
What is extrusion
Cell mechanism to signal they are dying and other cells induce actomyosin ring releasing dying cell into the lumen
Why is this bad
Can cause salmonella escape to lumen, and either infect other cells or exit host in transmission cycle
Which amp forms nano nets around salmonella and stops adhesion instead of killing them
Hd6
Which illness is there a deficit of this
Ibd crohns
Activation of caspases can cause what type of killing of macrophages (inducing the release of more inflammation or killed by neutrophils)
Pyroptosis
Which chemokines produced in the nkfb activation pathways eg nod or tlr cause neutrophil recruitment
Il23 inducing th17 inducing il17= chemokine
Il1b
TNFa
Which 2 induce ifny macrophage activation pathway
Il12 and 18
What from th17 by il23 from nkfb can cause release of amps and lipocallin2
Il22
What pathogenicity island important for effector blocks lysosomal trafficking when salmonella inside
SpiC
How do typhi cause gallstones
Associated with biofilm formation
Other than general gut barrier. Give 2 chemicals form Microbiota needed for colonisation resistance
Bacteriocins - gram-ve rna,dna and protein disruption
Indole - works with scfa to downregulated spi1 gene cluster exp
What other things physically stop colonisation
Carbon source limitation
Iron limitation eg via nissle 1917 (largest siderophore acquisition)
Adhesion competition
and physical adhesion since there’s around 10^11 in colon
Which genes found in salmonella crucial for early colonisation in mice- through mice studies with 500 mutants and screened which ones did not survive after mutants made
Chemotaxis, lps genes,
niFe-hydrogenase subunit genes hybA and hybF
FrdA fumarate reductases complex encoding
Why would niFe dehydrogenase be needed for early colonisation to overcome cr
Allows to use dihydrogen released from Microbiota femenrtariln as an alternative electron source in anaerobic conditions = promotes growth
Is this only relevant before inflammation induced fitness
Yes! Early colonisation before inflammatory help
What is the evidence for inflammation being the fitness advantage using spi1/2 mutants
Mutants in spi1/2 who cannot induce inflammation themselves werent able to outcompete / grow Microbiota in a dss-colitis mouse model
Less than 1% of colonisers if mutant
90 fold increase in colonisation when compared with wt salmonella
What from fermentation by Microbiota can be converted to tetrathionate and give the steps
H2s
Converted to thiosulfate by colonic epi cells bc it is toxic
This can react with the migrated chemotaxtic neutrophils via hxa3 ros they release
Causes oxidation of thiosulfate into tetrathionate
Why does tetrathionate matter in our competition
Used as an alternate electron acceptor in anaerobic conditions , allowing them to grow on poor carbon sources
Tetrathionate more efficient for energy than normal fermentation is = our growth
How does il22 from inflammation also cause outcompetition
Induces release of amp lipocallin2 which will block iron acquisition of commensals like ecoli but salmonella is immune because they have a modified siderophore (salmochellin)
How is host nitrate produced by inflammation
Effectors recognised like sopE (nlrp3) can induce release of proinflam ifny (from il18 by caspases) = induces production of NO by inos
Also induced dual oxidase 2 activity forming hydrogen peroxide (ros)
Ros will react with NO to form peroxynitrite which isomerises to nitrate
Why is this important
Can be also used as alternative electron acceptor (same as tetrathionate but nitrate is higher in hierarchy as has more redox potential) = more energy generated
Why are the aea important
Because they are not used by obligate commensals anaerobes
Give a potent poir carbon source only respired during production of alternative resp electron acceptors
Ethanolamine part of lipid membrane shed during epi cell turnover
What enhances salmonella fitness through chemotaxis
Flagella motility
How does this work
Sense ligands inducing methylation of a receptor
in inflamed gut like sense new nitrate or tetrathionate and undergo growth blooms through chemotaxis movement by flagella
Which proteins were crucial in this process when tested mutants in them on mice and nutrient agar plates
Methyl accepting chemotaxis proteins (mcp proteins)
Eg tsr and aer
Mutants had reduced fitness survival and lack of swarming to nitrate/tt
How is it suggested HGT can occur during inflammation/Microbiota dysbiosis between commensal enterobacteraciae and pathogenic strains eg ones that are antibiotic resistant or stx
Because they are facultative anaerobes meaning they can use nitrate for respiration and out grow in inflammation (seen in conditions like Ibd)
There is therefore more chance they come into close contact with virulent strains
Give some modes of hgt with close contact
Conjugation (exchange plasmids)
Transformation (uptake of naked dna)
Which other fermentation product can be used as an alternative carbon source in both aerobic and anaerobic s.tm respiration
1,2 propanediol
Fermentation of what produces this
Rhamnose or fucose
What gene cluster do they have for degradation of 1,2 propanediol
PduA-X
Which aea during inflammation allowed to use this as carbon source under anaerobic conditions
Tetrathionate
Which enzyme mutation involved in aerobic respiration alongside mutation in moa (for synthesis of molybdopterin required for enzymes which allow use of tt/nitrate as electron acceptors) proved that it is a combo of aerobic and anaerobic respiration salmonella uses for 1,2 propanediol use
Cytochrome bd oxidase II
Why did they find Microbiota needed to be present to generate this for growth advantage
Loss in gf mice showed no advantage
B fragilis and theta did though generate it
Explain how epithelial oxidation through antibiotic treatment occurs
Reduced clostridia butyrate production and therefore reduced b-oxidation and need to use glycosylis releasing oxygen
How can this provide environment where salmonella grows rapidly just after antibiotic treatment
Have bd cytochrome oxidase II for the use of oxygen as a final electron acceptor (the most efficient way)
After 4/5 days of antibiotics clostridia became present again but salmonella still grew , how? Also what 2 enzymes required for the most efficient transmission/shedding
T3ss induced inflammation was used instead to drive further dysbiosis where eventually butyrate will fall again (depletion of clostridia) - shown via mutants of spi
and grow on both nitrate and bd oxidase II electron acceptors
Nr and cytochrome bd oxidase were required for best transmission (shown through mutants)
What we’re conclusions of this mice model experiment using mutants
Early antibiotic treatment = need oxygenation/low butyrate
Later = need inflammation, dysbiosis and can use both nitrate and bd oxidase for efficient transmission/shedding
What was found to bloom in dysbiosis of colitis induced mice (3rd experiment example)
Enterobacteraciae / ecoli
Why is this
Formate is found enriched in dysbiotic and inflamed gut
Can use formate oxidation as a growth fitness advantage
Where does formate come from which isn’t present in the gf mice model of dss
Microbiota fermentation eg by b theta
METAGENOMICS ANALYSIS EXAMPLE- Which 2 enzymes were necessary (upreg in inflamed gut) for the dysbiotic microbiome in dss-mice (majorly ecoli) to use formate to grow (formate oxidation) and why
formate dehydrogenase - allows coupling of electron acceptors (oxygen in this inflammatory environment) to the electron donor(formate)
and cytochrome bd oxidase for aerobic resp
What was found the electron acceptor in formate oxidation
Oxygen as aerobic resp was found necessary (cytochrome bd oxidase needed)
(Inflammation is an oxidative state)
What marker of inflammation (produced from nos reaction with ros) was shown to induce formate dehydrogenase so that ecoli can use formate as an electron donor in inflamed gut
Nitrate
What does the spi-7 capsule vi on s typhi prevent
Complement fixation
At which point according to colitis mouse models with s.tm ko of nod1/2 show it’s importance in dampening the survival of s.tm in cells
When they are expressing spi-2 effectors forming the scv
Which d-alanine importer is important for salmonella survival against neutrophil D amino acid oxidase (DAO) which kills it via oxidative damage
DalS
Which ecoli strain been proven probiotic for salmonella infections eg via siderophore and Curli ligand
Nissle 1917
Which bacterial species important for toxin delivery to kill salmonella via t6ss
Bacteroides
What is the evidence from 2 mouse studies that Microbiota are important for cr and reduced inflammation
Study 1-
Low shedder mice were treated with antibiotics and converged to super shedders showing increased colonisation/invasion and increased shedding of salmonella
Study 2-
Antibiotic treatment induced worse salmonella colonisation and inflammation. Even after 3 weeks from the antibiotic treatment this still was the case showing the long lasting effect a dysbiosis can have
When spi1/2 mutants we’re introduced into il10- mice models of colitis what happened
We’re able to outcompete Microbiota again and overcome cr
Shows inflammation required
What is ttrSR
Gene cluster of tetrathionate sensor and response regulator encoded by salmonella and some ecoli strains which permit use of tetrathionate as an aea via ttrBCA
What is the ttr BCA cluster for
Encode tetrathionate reductase
