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Bacterial pathogenesis > Lecture 6 - The intracellular bacterial pathogen > Flashcards

Lecture 6 - The intracellular bacterial pathogen Flashcards

1
Q

Evading the immune system by becoming intracellular is a strategy to…

A

Persist in the host

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2
Q

What is the process and markers of phagosomal mutation?

A
  • Early: forms quickly following invagination of the plasma membrane
  • Endosome defined by specific markers: EE - Rab5, LE: Rab7/9, Lysosome (63hydrolyses): vATPase, Lamp1
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3
Q

How do bacteria overcome their need for iron?

A

Iron necessary for: replication in most bacteria

Problem: very little free in the cell

Strategy to overcome: bacteria produce structures/compounds to steal iron

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4
Q

What does the cell produce to kill bacteria?

A

ROS to kill bacteria, proteases, peptidaes for degrading, change pH on membrane so bacateria can’t cross membrane

bacterial strategy to overcome: catalyses on the surface convert ROS

Bacteriostatic: Nutrient deprivation (Lactoferrin, NRAMP1)

Bectericidal: Membrane permeabilization (Defensins, Cathelicidins)

Hydrolysis: Carbohydrates (Lysozyme, beta-hexosaminidase, beta-glucuronidase); Lipids (Phospholipase A2); Proteins (Cysteine proteases, aspartate proteases, serine proteases, carboxypeptodases, aminopeptidases)

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5
Q

What strategies are used by professional intracellular bacterial pathogens to modulate phagosome maturation?

A
  1. Avoid delivery to the lysosome e.g. salmonella, mycobacterium tuberculosis, chlamydia trachomatis, coxiella
  2. Removal from the degradative comparatment e.g. legionella, turns itself into a piece of ER and is no longer sent to the lysosome to avoid degradation
  3. escape into the cytosol e.g. listeria
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6
Q

What bacteria use the cells as protection from antibiotics and a further resevoir of bacteria?

A

Chlamidya treat with antibiotics and person gets better. But then has a replapse.

TB lies dormant for many years, when old people have weakened immune system get TB symptoms

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7
Q

What does it mean in flurescence microscopy when the average fluorescence peak shifts?

A

If the peak shifts to the left it means the bacteria has divided. As get 1/2 the fluoresence when the bacteria divide. Uses thin liquid stream, laser excites the fluorophore and a detector measures the emission wavelength. Tagged with antibody that has a fluroesing molecule attached. Fluorescence dilution.

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8
Q

Where does non replicating salmonella persist and how can this be proved experiemtnally?

A

Non replicating salmonella persists in the immune organs by the rapid formation of nonreplicating persisters in macrophages

Tested by fluoresence diltions testing on multiple sites

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9
Q

How was it shown experimentally that salmonella persist by the rapid formation of nonreplicating persisters inmacrophages

A

Salmonella grown in LB medium and in the presence of antibiotic some bacteria are not killed.

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10
Q

What are required for the formation of macrophage induced nonreplicating persisters in response to vaculolar stimuli?

A

TA modules

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11
Q

What are the features of a pathogenic vacuole? (maintnance of vacuolar compartments)

A
  1. Cytoskeletal recuritment
    1. membrane stability e.g. salmonella chlamydia
    2. Organelle translocation e.g. T. gondii
    3. Membrane dynamics e.g. salmonella
  2. Cholesterol modulation
    1. Cholesterol accumulation to block lysosome fusion e.g. myco bacterium
    2. Cholesterol removal via acylation for membrane dynamics e.g. salmonella
  3. Selective fusion with host vesicular traffic
    1. Endocytic e.g. chlamydia, mycobacterium
    2. Exocytic e.g. chlamydia, salmonella
    3. ER-derived vesicles e.g. Brucella, legionella
  4. Secreted microbial effectors
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12
Q

What are the bacterial scretory systems?

A

Type I: ABC transporters through OMP

Type II: Sec machinary

Type III: Tat machinery

Type III

Type IV: Vir complex

Type VI: CIpB ATPase

Used by salmonella, chlamydia

Type VII: through the mycomembrane (mycobacterium)

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13
Q

What membranes are there in a host cell?

A

Plasma membrane

Internal membranes (nucleus, ER, golgi, vesicles)

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14
Q

When does the early endosome form?

A

Quickly following invagination of the plasma membrane

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15
Q

What is the pH pf the early endosome?

A

pH6-6.5

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16
Q

What is the pH of the late endosome?

A

pH5.5

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17
Q

What is the pH of the lysosome?

A

between 4.5 and 5.0

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18
Q

How can you define the status of an endosome/phagosome?

A

By the lpid/protein markers that are integral to the membrane

EE/EP: Rab5

LE: Rab7

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19
Q

What are the rab proteins?

A

small gtpases

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20
Q

What is another name for the multicesicular body?

A

Late endosome

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21
Q

What are the markers for the lysosome?

A

VATPase/Lamp1

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22
Q

What is the activty of the VATPase in lysosome?

A

pumps in protons to degrade :

  • DNA
  • RNA
  • proteins
  • lipids
  • carbohydrates
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23
Q

What are the professional dendritic cells?

A
  • Monocytes
  • Macrophage
  • Dendritic cell
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24
Q

What sized particule is taken up by phagocytosis?

A

>0.2µm (bacteria)

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25
Q

What is the process of phagocytosis?

A
  1. Professional phagocytic cells (macrophages, monocytes, dendritic cells) take up particles from the external environment (>0.2µm)
  2. This forms an ealy phagosome with the same markers as an early endosome
  3. Early phagosome sequentially fuses with the early endosome which undergoes the defaul pathway to deliver the particle to the lysosome - including an increasing acidification of the lumen and switch from Rab5 presenting to Rab7 presenting to VATPase/Lamp1 producing
  4. Degradation of the particle by 63 hydrolases
26
Q

What is the interplay of iron between the host and the intracellular bacterium?

A

Most bacteria need iron to replicate Fe2+

The cell transports iron that has been bought in by tranferrin out of the cell efficiently and produces lactoferrin which collate the free iron in the cell

Many intracellular bacteria produce siderophores which bind with high affinity to iron to remove the iron before the cell can transport it out

27
Q

Give exmaples of some of the pathogen and host defense and attack mechanisms

A

Host microbial factors against pathogen defensive mechanisms

Cell produces reactive oxygen species, and reactive nitrogen species by sox2 and nox2 - by macrophages (oxidative burst) kill bacteria by damaging the DNA/protein

VTPase - acid environment (cell), bacteria can pump out protons to prevent acidification

Cysteine proteases/aspartine/serine (typically in the lysosome) phospholipase A2 (degrades lipids) (cell)

Catalyases (bacterial surface) convert hydrogen peroxide (cell) to water and oxygen so it no longer produces ROS

Antimicrobial peptides (cell): change pH membrane so peptides can’t enter, proteases (bacteria)

NADH oxidase (cell) broken down by proteases (bacteria)

28
Q

What is the benefit of living inside the cells?

A
  • Evasion of the immune cells
  • No binding complement, antibodies
  • If can negate delivery to lysosome and survive in cell
  • Warm, nutritious environment (if remain on pathway of internalisation)
29
Q

Examples of strategies ued by professional intracellular bacterial pathogens to modulate phagosome maturation (Mycobacterium tuberculosis)

A

Mycrobacterium tuberculosis

  • arrests in early phagosomal stage
  • never loses rab5, change lipid content, stays with pi3phosphate
  • prevents deleivery to lysosome
30
Q

What are the general strategies used by professional intracellular bacterial pathogens to modulate phagosome maturation

A
  1. Avoid delivery to the lysosome e.g. salmonella, mycobacterium tuberculosis, chlamydia trachomatis, coxiella
  2. Remove yourself from the degradative compartment e.g. legionella
  3. escape into the cytosol e.g. listeria
31
Q

Why was it suspected that bacteria can form persisting resevoirs in the host?

A

e.g. people with chlamidya often have relapses (same serovar) evidence suggest can have reseviors of bacteria

TB can live for 70 years (slow growing) if a person is infected and reach old age where immune system not as efficent could develop TB, however only 10% ever get active TB infection

32
Q

How do you interpret a fluoresence dilution graph?And method. (paper)

A

Flow cytometry

  • Attach a fluorophore to an antibody
  • looking at fluorescence intensity as go to right increasing fluoresence
  • count: number of cells
  • Log scale
  • Must look at the mean fluoresence (peak) the shift along the x axis denoting dilution of emission aka replication
  • e.g. taken salmonella expressing GFP, then use fluroesence dilution (FD)
    • induced expresion of GFP, then stop induction
    • if bacteria then divides get half emission in the bacteria, seen in dilutions of a half
  • Thin stream of wavelenth laser with a wavelenth that excites fluorophore emission picked up by detector, takes average flurosence of each bacterium
33
Q

How quickly are the nonreplicating persisters generated? (paper)

A
  • Bacteria treated with antibiotic cefotaxime: black - indicates salmonella in LB, antibiotic kills bacteria (number of CFUs) ; blue - survival in BMM after treament with antibiotic ; red - LB media after 30 mins internalisation and treatment with antibiotic ; green - surivial in BMM without cef treatment
  • After 30 mins get persistent bacteria that are no longer killed by antibiotic
  • Showed also in a LB broth compared to BMM macrophages got high survival after just 15 mins - environment of the macrophage giving prosurvival characteristics to the bacterium
34
Q

What is the metabolic status of persisters? (paper)

A
  • Using spectrophotometry
  • Bacteria GFP positive, induce dsRed so if have arabinose get transcription and translation of dsRed protein (show metabolism)
  • If araibinose added to the salmonella are they still metabolically active? Increase in dsRed
  • Put salmonella into macrophages and extract and put into LB.
  • Viewed shift in GFP to the left showing that after being in the macrophages the bacteria are still alive and replicating - but only those that were metabolically active, if ALL were dividing all of the column would shift over but only the top does - If not metabolically active then there is no shift.
  • In the macrophage only those that are metaboliclly active can regrow
  • Also looked at regrowth in niave bmm
  • only a small proportion begin regrowing

Have dormant bacteria - not dead but not metabolically active

Replicating bacteria, not necessarily persisting but will go on to infect other cells

Non replicating metabolically active bacteria (even though some won’t regrow - some damage has occured) and are extracellular growth compenent and can regrow and re infect

35
Q

How was it shown that TA modules are required for formation of macrophage induced non replicating persisters in response to vacuolar stimuli? (paper)

A
  • Have toxin-antitoxin system cleave toxins to reduce replication
  • Found that the ability to form a persister (using chemicals that prevent pahocytosis - LatB or CytD) dependent on phagocytosis
  • This resulted in a drop in number of persistors, need acidic environment to form a persister
  • If don’t have genes and can’t form toxin-antitoxin systems cant form persisters
  • 14 loci in salmonella genome: if mutated get reduced persistance
36
Q

What are the mechanisms of intracellular survival employed by intracellular bacteria?

A
  • Modulation of the cytoskeletonn (salmonella, chlyamida)
    • salmonella produces salmonella induced filaments which it uses to cause membrane deformation - get greater curvature of a tubule making the tip of the tubule more fusagenic, improved accessibility of nutrients
  • Cholesterol modulation (accumulation/removal/inferenece with membrane stability) (TB)
    • Membrane fusion dependent on lipids in membrane - prosurvival phenotype if can control this
  • selectively fuse with vesicular traffic
    • need to remain fusagenic with vesicles coming inside the cell, (need rab, monk and snare proteins for fusion) - the same needed for fusion with vesicles coming inside the cell how is it selective? avoiding delivery to the lysosome?
  • secrete microbial effector proteins
37
Q

What was shown in the Internalisation of salmonella with macrophages induces formation of nonreplicating persisters paper?

A

Shows that salmonella (food poisoning, typhoid) can go inside cells and persist, be metabolically active

3 main points:

  • Need to be internalised in acidic environment
  • Happens quickly (within 15 mins)
  • Identified some of the genes to regulate the response of generating these persisters (14 loci involved in the toxin-antitoxin system)
38
Q

How was it shown that salmonella injected into mice persist in the lymph node?

A
  • e.g. taken salmonella expressing GFP, then use fluroesence dilution (FD)
  • induced expresion of GFP, then stop induction
  • if bacteria then divides get half emission in the bacteria, seen in dilutions of a half
  • Looked at lymph nodes after injection into a mouse, light doesn’t change - bacteria have not replicated
  • These recovered bacteria put into LB and can see there is regrowth (light divides, shift to the left)
  • Used cherry fluorophore to identify bacteria by consitiutive expression (red and green = yellow) if not divided collection of cells will be yellow, as the cells start to divide the green gets halved but red expands
  • If no antibiotic fed to the mouse - bacteria divide in the lymph
  • With antibitiotic, extract cells (which are not dividing and not being killed) and do regrowth in LB both get regrowth
  • shows bacteria persisting in the mice in the presence of antibiotics
39
Q

What is the main problem if you are an enveloped bacteria and you want to get out of the cell but not fuse with the vacuolar membrane?

A
  • Have to prevent fusion
  • (Monk rab snare) proteins neccessary for fusin found on the cytoplasmic side of the host cell
  • Have to interfere with machinery on the wrong side of the membrane
  • need a way of modifying them
  • endosomal membranes are a barrier (small solutes can go through) not necessarily got transporters for transport through the membrane
    • Outer membrane and inner (bacteium), plama membrane (cell)
  • T3SS: Needle-like complex and tip-like complex allows bacteria to secrete proteins through its membranes into the host cytoplasm through the small peptidoglycan layer (bacterium) T3SS - e.g. salmonella chlyamidya
  • Mycobacterium has T7 secretion system, can secrete effector proteins into the lumen of the membrane
  • Can have other secretory systems and mulitple
40
Q

How does pathogenic salmonella actively cause its own uptake?

A
  • Infective salmonella serovar often flagellated bind to epithelia and can transcytose - uses pathogenicity island
  • Assembles T3SS and injects host proteins into the mammalian cells, causeing its own uptake from the apical surface to the basal lateral surface
  • circulating macrophages & monocytes - salmonella uses T3SS from spi1 (pathogenicity island) to get into macrophage
  • then assemble second T3SS using spi2 then within the phagosome bacteria start replicating, rupture, release for more infection
41
Q

Why is T3SS thought to be related to flagellum?

A
  • have similar structures
  • basement complex, small tube, needlike protrustion, tip proteins/caps
  • three families of T3SS, yersinia, chlmydia, salmonella
42
Q

What is the structure of the T3SS?

A
  • basal body
  • needlelike complex
  • tip complex
  • translocon secretes effector proteins
  • ATPase at bottom (energy requiring process)
43
Q

What is the assembly of the T3SS complex?

A
  • form base in outer membrane and inner membrane
  • needlelike proteins are t3ss proteins
  • basement complex starts secreting needle proteins
  • needlike complex assembles
  • secretes tip proteins evolved for getting proteins into host cell cytoplasm
44
Q

Can we predict whether proteins encoded in the genome are a T3SS protein?

A
  • May have an RNA encoded sugnal
  • most proteins require chaperone to bring them to the end of the T3SS
  • chaperone and unknown sequence at n terminus for secretion
  • increase in camp shows = secreted
45
Q

What of the structure of the T3SS genetic material and the associated proteins?

A
  • chaperones are encoded next to the effector proteins
  • have the same loci in the genome
  • chaperones have similar structures - common mechanism of the chaperone (structure) for recruiting effector protein to base of complex
46
Q

Why does salmonella need a T3SS once it is in the cytoplasm?

A

over 60 effector proteins secreted from salmonella into host cytoplasm

47
Q

What is the genome structure of salmonella?

A

4.9 million base pair geome

90 000kb vir plasmid (spi 1-pathogenicity island) : several T3SS effector proteins

Several T3SS in spi2

chaperones nearby to the T3SS proteins and effectors

48
Q

How were the important proteins for salmonella intracellular survival identified?

A

STM

  1. synthesis a piece of random piece of DNA: Nk[20]: twenty repeating units of N (A C G T) K ( G, T) results in a forty-mer
  2. hind3 resutrction enzyme site: AAGCTT 6 base cutter and append to the 40mer
  3. (synthesised dna is variable region)
  4. use PCR to add primer p1 and primer p2 (same 5’ and 3’ ends) (invariant ends)
  5. Put DNA into a transposon and into bacteria
  6. DNA will be inserted randomly into the genome
  7. also have selectable marker to isolate DNA that has been transformed
  8. isolate individual bacteria and make a pool (mutant library) in a 96 well plate with an individual bacteria per well
  9. each individual bacteria will have an individual transpon
  10. pcr all indiviual mutants using primer 1 and 2 with radioactivity
  11. use resitrction enzyme site to cleave off primers
  12. spot one microliter from each into the 96 well grib format onto a surface that will bind DNA
  13. lyse bacteria and hybridise probes (heat up to denature then allow to anneal to dna on the membrane) to check the ability to pcr an individual clone is still valid (not c rich that would prevent pcr)
  14. put bacteria into mice and those bacteria that have been mutagensised may be killed (if it was pro survival)
  15. extract and homogenise the spleen, take out bacteria
  16. isolate individual bacteria and make an output pool (mutant library) in a 96 well plate with an individual bacteria per well
  17. each individual bacteria will have an individual transpon
  18. pcr all indiviual mutants using primer 1 and 2 with radioactivity
  19. use resitrction enzyme site to cleave off primers
  20. spot one microliter from each into the 96 well grib format onto a surface that will bind DNA
  21. compare between input pool to identify genes necessary for survival
  22. know exactly what bacteria used, extract genetic material and see what gene has been inactived

survival genes

49
Q

What was found in the orgininal signiture tagged mutagenesis in salmonella

A

T3SS effector and complex proteins are survival proteins

50
Q

What is the life cycle of chalmadyia trachomatis?

A
  • STI
  • can sterilise males, due to inflammatory response
  • obligate pathogen - cannot survive outside human host cell
    • small genome
    • lost most of its genes
    • dependent upon ATP
  • leading cause of trichoma blindness in the world, flies crawl on faecal matter and on peoples eyes - get infefction
  • different serovars, eye different to STI form
  • Infectious elementary bodies get phagocytosised, rapdily enter cell, and differentiate in endosome into reticulate bodies within 12 hours
  • then get inclusion membranes where the bacteria are in close appositon with the membrane, as have a T3SS need to be close to secrete things into the host
  • then get a regression to the EB, exocytosis and delivery of the EBs to infect neighboroughing cell
  • infected cells cause infalmmatory response - promotes a strong immmune response
  • can also get a persistant form - relapse
51
Q

What are the features of chlamydial T3SS

A
  • slightly different from salmonella
  • ATPase
  • chaperones
  • needleike complex
  • less info becuase obligate - cant genetically manipulate always inside eukaryotic host
52
Q

What are the features of chlamydial pathogen-host protein-interactions

A
  • secretion system secretes cytokines/microbial products which can can give protein response: release of IκB (need to be released to release NF-κB)
  • ct441 (chlamidya) secretion inhibits NF-κB so there is no immune response
  • deubiquitinases
  • interfere with SNARE proteins (ct813)
  • interfere with small gtpases (rab, phosphoronusitase, kinase, phosphotases)
  • downregulate the immune response to survive inside the cell
53
Q

How has genetic manipualation of chlamydia been achieved?

A
  • chlamydia has a vir plasmid
  • chlamydia can’t be transformed as usual with plasmids
  • took chlamydia plasmid and added e.coli plasmid to form a large plasmid and introduced penecillin resistant gene
  • normal chlamydia under penecillin treatment can’t grow
  • managed to transform ebs with calcium chloride
  • creating penecillin resistance chlamydia
  • forces chlamydia to become antibiotc resitant
  • can put gfp gene into view inclusion bodies (full of ebs)
  • route to introduce new genes in chlamydia
  • e.g. strain that can’t make starch (iodine staining)
54
Q

Why do most intracellular pathogens target the same pathways?

A

limited number of biochem reactions going on inside cell

  • salmonella secretes sop b
    • which manipulates phosphoinositides (sop b phosphotase)
    • changes maturation of the salmonella containing vacuole
    • can now start changing lipids of phagosomal membrane
  • TB uses a similar mechanism
55
Q

Aside from T3SS what other secretory systems are there?

A
  • yersinia secretary system
  • Epec system
  • system of plant pathogen pqudomonas syringe
56
Q

How are proteins secreted through the T3SS?

A
  • secreted proteins are folded in bacterium
  • bind to chaperone protein
  • taken to the base of secretion complex
  • energy from ATPase unfolds the protein
  • unfolded protein is passed through the complex
  • when exits it refolds (don’t require chaperones for refolding)
57
Q

Why is it difficult to identify the secreted effector proteins from salmonella?

A
  • look at primary amino acid sequence can’t see activity
  • have little homology to eukaryotic proteins
  • can’t identify functions
  • cifA - recruits scip andmodulates vesicular trafficking
  • ssej - unknown function, stops microtubules from collapsing
  • sscl - ubiquitin protease
    • lysine residue host ubiqutinates it -> degraded, above makes it survive in the cell long
58
Q

What is Salmonella SpvC and what is it’s mode of action?

A

spvC - phosphosthreonine lyase

All map kinases are phosphorylated on hydroxyl threonine or tyrosine (active site) = phosphothreonine

Kinase phosphorylates, phosphotase removes phosphorylate

SpvC a phosphothreonine lyase, removes phosphate and with a beta illumination reaction removes oxygen

completely destroys threonine no more phosphorylation

MAPKKK phosphorylates map-kinase-kinase which phosphorylates map-kinase, inactivates ERK

59
Q

What are the features of legionella pneumoniphillo for avoiding getting sent to the lysosome?

A

Ligenella pneumoniphilo

  • modifies membrane
  • recruits proteins to phagosomal membrane turns itself in ER membrane
  • Ribsomes bind to surface
  • No longer sent to lysosome
60
Q

What are the features of listeria monocyte genes for avoiding getting sent to the lysosome?

A

listeria monocytogeneis

  • unpastereurised dairy products
  • produce listeria lysinoma phospholipases - proteins permeabilise and degrade membrane
  • no longer on route to lysosome (cytoplasm)
61
Q

Give an example of the mechanisms used to avoid being sent to the lysosome by professional intracellular cell Coxiella species

A
62
Q
A

Bacterial pathogenesis (8 decks)

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