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MICR223 > MODULE 3 - Microbial Virulence Mechanisms > Flashcards

MODULE 3 - Microbial Virulence Mechanisms Flashcards

1
Q

how is adhesion important to an extracellular pathogen and give examples of these pathogens?

A

colonisation of host mucosal surfaces or artificial surfaces to allow for efficient delivery of exotoxins into the host cell

contact to surface of cell allows most effective toxin action

first step of biofilm formation is adherence to surface

e.g. Neisseria spp, S. aureus, uropathogenic E. coli, enteropathogenic E. coli

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2
Q

how is adhesion important to intracellular pathogens and what microbes do this?

A

critical step that precedes internalisation - adhesion first step to get the microbe inside cell where its safe

bacteria - listeria
protozoa - toxoplasma, plasmodium

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3
Q

what are the three things adhesion can be mediated by?

A

multi-component structures (pili)

single protein adhesins

a receptor derived from bacterium

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4
Q

what is a pili?

A

multicomponent structures made of lots of proteins, with the one at the tip mediating adhesion

they have lots of roles including in conjugation and adhesion

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5
Q

what is a P-pili expressed by and allow adherence to?

A

expressed by uropathogenic E. coli (UPEC) which colonise the urinary tract and infect the kidney

mediate bacterial adherence to epithelial cells of the bladder and kidney

responsible for 80% UTIs

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6
Q

what is the structure of the P-pilus?

A

PapG: adhesion that binds to a receptor on the surface of the human cell

PapA: the major structural component of the pilus

PapC: outer membrane protein that forms a channel that other P-pilus components are transported through (important for assembly of pilus)

PapD: chaperone that binds P-pilus components in the periplasm; prevents these proteins from being degraded and presents them to the PapC channel, helps with assembly of pilus

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7
Q

how does assembly of the p-pilus occur?

A

occurs in ordered fashion starting with tip protein PapG and ending with PapA and PapH

different components of pilus synthesised in periplasm, when in periplasm PapD chaperon keeps them folded correctly

no chaperon they get misfolded and degraded, PapG first protein to go through channel

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8
Q

what are pilicides and why could they be effective for treating UTIs?

A

chemicals that bind and inhibit the function of chaperones like PapD. Pilicides block P-pilus assembly and could eventually be developed into effective drugs to treat UTIs

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9
Q

what is the human surface receptor for the P-pilus?

A

globoside (a galactose containing glycolipid)

it contains polysaccharides and lipids and PapG binds to the polysaccharide component of the receptor

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10
Q

why might someone with UPEC not be responding well to antibiotics?

A

UPEC makes intracellular biofilms

after time this biofilm matures and some bacteria can disperse and go infect other cell

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11
Q

what is a Type IV pili mediate binding to?

A

mediate binding of bacteria to protein or glycolipid receptors on the surface of host cells (e.g. Neisseria, enteropathogenic E. coli (EPEC))

in the case of some bacteria e.g. Neisseria, type IV pili also promote internalisation of bacteria into human cells

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12
Q

what is Neisseria species?

A

gram negative diplococcus which commonly colonises nasopharynx (N. meningitidis) or urethra/cervix (N. gonorrheae)

causes meningitis and septic shock

causes gonorrhoea

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13
Q

outline the pathogenesis of N. meningitidis?

A

type IV pilis plays a critical role in bacterial internalisation into epithelial cells in nasopharynx ultimately resulting in spread to blood and then maybe even spread across blood-brain barrier

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14
Q

describe the structure of the Neisseria type IV pilus?

A

pilus extends from inner membrane through periplasm then outward

the pilus is dynamic in that it can grow and retract (this is ATP dependent)

PilE (pilin subunit) composes most of it

PilC binds host CD46 receptor

growth involves new pilin subunits being added onto base and retraction involves piling subunits being removed and this is done by ATPase

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15
Q

what is twitching motility mediated by type IV pili?

A

pili are dynamic and extend or retract due to addition or removal of new PilE subunits at the base (polymerisation and depolymerisation)

this results in twitching motility, which may bring adherent bacteria into close contact with the human cell

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16
Q

what are adhesins?

A

single proteins which will bind tightly to allow adhesion

binding often leads to bacterial internalisation in the case of many adhesins

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17
Q

what are two examples of single protein adhesins?

A

invasin protein of yersinia enterocolitica

InlA protein of listeria monocytogenes

these adhesins are bacterial surface proteins that bind to host surface receptors (usually proteins)

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18
Q

how does yersinia enterocolitica get internalised?

A

there is a receptor on the surface of intestinal M cells (NOT epithelial) which the invasin can target

once inside binds to macrophages which spread them around the blood and then they can go infect other shit

internalisation into M cells contributes to localised inflammation and bacterial dissemination

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19
Q

what is intimin?

A

single protein adhesion from enteropathogenic E. coli (EPEC)

in this case both the adhesion and the receptor for the adhesion come from the bacterium (it injects receptor into cell)

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20
Q

what is Tir?

A

the receptor for intimin which EPEC injects into host cell

interaction between intimin and Tir leads to actin filaments forming below Tir creating a pedestal which EPEC sits on on the cell

Tir is in the plasma membrane of human cell

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21
Q

what do EPEC pedestals do?

A

mediate tight and persistent adhesion to the intestinal epithelium

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22
Q

outline the two-step process that is EPEC adhesion to human intestinal epithelial cells?

A

step 1: binding of a type IV ‘bundle forming pilus’ to host cell

step 2: bacterial injection of Tir into the host cell plasma membrane. EPEC then binds to Tir through it’s bacterial outer membrane protein intimin. Tir/intimin interaction elicits the formation of a ‘pedestal’

degradation of microvilli is one thing which allows it to get closer to the cell for internalisation

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23
Q

is Tir essential for bacterial colonisation of intestine?

A

yeah

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24
Q

what are the three general mechanisms of invasion?

A

interaction with host surface receptors (active participation of both bacterium and host cell e.g. listeria)

injection of bacterial effectors into host cytosol (active participation of both both bacterium and host cell e.g. salmonella)

active penetration into the host cell (active participation of parasite only; host cell is passive e.g. toxoplasma, plasmodium (protozoans))

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25
Q

what is listeria monocytogenes?

A

gram-pos food borne pathogen

mainly affects immunocompromised, elderly or pregnant women

consequences of infection can include meningitis and abortion

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26
Q

why do microbial pathogens invade host cells?

A

access to an environment suitable for growth

protection from host antibody response

traversal of anatomical barriers

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27
Q

why does listeria monocytogenes invade host cells?

A

access to an environment suitable for growth (and bacterial spread)

protection from host antibody response

traversal of anatomical barriers (blood/brain barrier, fatal/placental barrier, intestinal cell barrier) they reach these barriers by travelling through the blood stream

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28
Q

what is internalisation of listeria mediated by?

A

mediated by a protein adhesin called InlA

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29
Q

what is E-cadherin?

A

surface receptor plays a critical role in cell-cell adhesion

listeria exploits it with InlA which interacts with

so many InlA proteins bind so many E-cadherin receptors that you get the host cell plasma membrane zipping up around the bacterium and eventually the two edges meet and fuse and you end up with bacterium in the cell

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30
Q

what type of cell does listeria target?

A

goblet cells

enterocytes are the main part of the intestinal epithelium for absorption, but goblet secrete mucus

InlA binds to e-cadherin receptor on the goblet cells which isn’t expressed or exposed on other cells

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31
Q

what is internalisation of Listeria driven by?

A

highly localised changes in the host F-actin cytoskeleton

actin polymerisation is very localised right around the bacterial cell which leads to localised change in the plasma membrane

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32
Q

what are microfilaments?

A

comprised of the protein actin

play critical roles in cell movement and cell division

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33
Q

what are microtubules?

A

made of alpha and beta tubulin

involved in cell structure, chromosome segregation and movement of cells (cilia or flagella)

larger width than microfilaments

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34
Q

how do we get cell motility from actin filaments?

A

polymerisation of actin monomers allow the filaments to grow, the addition of actin monomers at the plasma membrane interface generate protrusive force that drives eukaryotic cell motility

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35
Q

what host mechanism may be exploited to allow internalisation of listeria?

A

subversion of the actin polymerisation host mechanism normally used to drive eukaryotic cell motility

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36
Q

what is salmonella enterica?

A

gram neg species that includes serotypes typhimurium and typhi

causes gastroenteritis (typhimurium) or typhoid fever (typhi)

food borne usually from contaminated poultry or eggs

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37
Q

what are the benefits of invasion (internalisation) for salmonella?

A

provides access to a niche suitable for microbial replication

provides protection from the host antibody-mediated immune response

allows traversal of the intestinal barrier (salmonella enterica serovar typhi causes systemic disease)

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38
Q

what is the difference in infection between salmonella typhi and salmonella typhimurium?

A

infect M cells and are internalised (typhimurium stops here) and then get internalised into macrophages which are below the M cells which then spread them around body to stuff like spleen, bone, liver

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39
Q

what does internalisation of salmonella involve?

A

large scale re-modelling (ruffling) of the host cell surface

salmonella injects SopE protein which has ability to activate human proteins to induce actin filamemntation of larger structures

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40
Q

what regulates the actin cytoskeleton in mammalian cells?

A

small G proteins or the Rho family

eukaryotic rho family G proteins are active when bound to GTP and inactive when bound to GDP

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41
Q

what is toxoplasma gondii?

A

protozoan which causes encephalitis in immunocompromised or abortions in pregnant women

main host is cat as only animal it can reproduce in

humans infected by consuming tissue cysts in badly cooked meat or oocysts from cat faeces

asymptomatic in lots of people

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42
Q

what is the first site of infection for toxoplasma?

A

epithelial cells lining the intestinal lumen

toxoplasma actively induces its internalisation into these epithelial cells

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43
Q

what does internalisation of toxoplasma require from the pathogen and the host?

A

pathogen - active participation

host - nothing, he has no say

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44
Q

how does toxoplasma cause behavioural changes in rodents and why?

A

cats acquire toxoplasma from rodents

rodents infected with toxoplasma lose fear of cat odour

this is a mechanism to optimise parasite reproduction

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45
Q

what is gliding motility?

A

powers toxoplasma invasion

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46
Q

what are the stages of toxoplasma invasion?

A

contact (initial contact)

attached (gliding, apical attachment, MJ formation)

penetrating (penetration)

invaded (shedding, closure)

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47
Q

gliding motility and internalisation of toxoplasma into human epithelial cells is driven by what…

A

the actin and microtubule cytoskeletons of the parasite (NOT the human)

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48
Q

describe the process of toxoplasma internalisation?

A

parasite has proteins which bind to receptors on human cell

bacterial microtubule cytoskeleton is anchored but actin cytoskeleton can move and interact with microneme proteins. If actin cytoskeleton moves a certain way the microneme proteins push into plasma membrane and movement occurs

myocin (myoA) is what triggers actin to move in the first place

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49
Q

what occurs when a bacteria is internalised by a phagocyte?

A

when in phagocyte bacteria in vesicle called phagosome

phagosome comes from plasma membrane when bacteria is internalised

fusion of this with lysosome makes it acidic (phagolysosome)

well this happening reactive oxygen species and other shit fucking the bacteria up

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50
Q

what are the three main antimicrobial properties of phagocytes?

A

low phagosomal pH

production of reactive oxygen species (ROS)

cationic antimicrobial peptides (CAMPS)

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51
Q

what is phagosome maturation?

A

if non-pathogenic microbe internalised by phagocyte, phagosome matures

this involves pH dropping due to about 4.5 due to more and more V-ATPase molecules forming in phagosome membrane

these pump protons into it using energy from ATP hydrolysis

also some antimicrobial enzymes in phagosome which become most active at low pH e.g. cepthepsins

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52
Q

what are the stages of phagosome maturation and the pH at that stage?

A

early phagosome pH = 6.5

late phagosome pH = 5.5

phagolysosome pH = 4.5

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53
Q

how do more V-ATPases form on the membrane during phagosome maturation?

A

phagosome has a little compartment in it called an endosomal compartment

its like a small phagosome where endocytosis is always happening

early endoscope interacts with phagosome and you get a bit of V-ATPase on phagosome

this interaction involves fusion with phagosome and delivering of some of its contents including V-ATPases, cepthepsins and other enzymes

54
Q

how do phagocytes products reactive oxygen species (ROS)?

A

enzyme complex on phagosome called NADPH oxidase complex takes O2 and converts it to superoxide which is unstable so rips electrons off (oxidation) stuff like lipids and proteins in bacteria

can also be converted to hydroxyl radical which is another reactive oxygen species which does the same shit

55
Q

how are bacteria killed by cationic antimicrobial peptides (CAMPS)?

A

hydrophobic part interacts with fatty acid part of phospholipid bilayer on bacterial membranes

when this happens the positive charge is facing another direction so another molecule can come in and interact in same way so that the two positive parts are facing each other creating a pore

56
Q

what are cationic antimicrobial peptides (CAMPS)?

A

molecules in phagosome which are amphipathic (hydrophobic and hydrophilic on different sides of the molecule) and so can insert into bacterial membranes and create pores leading to lysis of bacteria

57
Q

what are some microbial methods of intracellular survival?

A

survival in acid compartments (salmonella)

avoidance of fusion with lysosome (legionella)

escape from vacuole (listeria)

58
Q

how does salmonella survive in an acidic compartment?

A

salmonella can survive in pH as low as 4 so in acidifying phagosome salmonella is allg

salmonella actually replicates best in late phagosome (called an SCV or salmonella containing vacuole)

also has two type III secretion systems (TTSS1 which is important for entry to cell and also TTSS2)

bacterial effector proteins injected into human cell through these secretion systems and some important for replication

also can cope with ROS and CAMPS

59
Q

how does salmonella deal with reactive oxygen species?

A

with enzymes superoxide dismutase (SOD) and catalase

NADPH oxidase complex converts molecular oxygen to superoxide

SOD made by salmonella can then convert superoxide to H2O2 however H2O2 converts itself to hydroxyl radical and so catalase made by salmonella converts this to H2O and O2

60
Q

how does salmonella protect against killing by CAMPs?

A

the two-component regulatory systems PhoP/PhoQ and PmrA/PmrB which covalently modify LPS

61
Q

how do the two-component regulatory systems PhoP/PhoQ and PmrA/PmrB work?

A

the two-component regulatory systems change gene express allowing LPS charge in outer membrane to be modified so that it is neutral and CAMP can’t bind

two-component reg systems are ways that organisms sense environment and then change their gene expression

PhoQ is in membrane and has kinase activity and PhoP stimulates gene expression

so PhoQ senses low pH or low magnesium to tell the bacteria its in phagosome

PhoQ then autophosphorylates a his residue and PhoP comes and takes that phosphate off PhoQ making PhoP undergo conformational change activating it to bind different promoters to turn on some genes e.g. those that covalently modify LPS

neutral charge of LPS repels CAMP from surface

62
Q

how is the LPS in the salmonella membrane able to be covalently modified for protection from CAMPs?

A

LPS composed of lipids and on lipid A part (core part) there are lots of phosphate groups which give it neg charge and enzymes expressed by PhoP covalently modify phosphates to neutralise charge thus repelling CAMP from surface

63
Q

what is the difference between the two-component regulatory systems PmrA/PmrB and PhoP/PhoQ?

A

work the same way

both sense low pH and are activated in phagosome

PmrA also turns on expression of genes and enzymes which modify LPS so it no longer binds CAMPs

main difference is that PmrA/PmrB seems to be a redundant system in salmonella but it is still there

64
Q

what is legionella pneumophila?

A

gram-neg rod which causes pneumonia (legionnaires disease)

infects protists (amoeba) and humans

facultative intracellular pathogen which replicates inside host cell by interfering with host trafficking pathways that would otherwise kill bacteria

65
Q

what kind of phagocytes does legionella live in?

A

lung macrophages

66
Q

how does legionella survive in phagocytes by altering host intracellular trafficking?

A

it has type IV secretion system which makes needle-like structure and injects proteins into human cell cytoplasm which do lots of shit but main one is prevent maturation of phagosome

prevents this by stimulating stimulating membrane recruitment from ER of host cell which then decorates phagosome membrane

this prevents interaction between phagosome and endoscope thus no maturation thus no antimicrobial properties so legionella can just replicate like crazy in phag/vacuole

67
Q

did type IV secretion systems and type III secretion systems co-evolve?

A

no! I don’t even think that is a word or makes sense

basically type IV ss just evolved independently of type III ss

68
Q

what are some features of legionella-containing vacuoles (LCVs)?

A

allow efficient growth of bacteria

lack antimicrobial characteristics (acidification, proteases, lysozyme, ROS)

contains membrane derived from ER (membrane flow from ER necessary for LCV formation)

69
Q

what is the purpose of forming a legionella-containing vacuole (LCV)?

A

legionella protects itself from phagocytic killing and also creates a niche productive for replication

69
Q

what is the purpose of forming a legionella-containing vacuole (LCV)?

A

legionella protects itself from phagocytic killing and also creates a niche productive for replication

70
Q

what are the two main virulence factors of legionella?

A

Dot/lcm type IV secretion system

Dot/lcm substrates

71
Q

what is the Dot/Icm type IV secretion system?

A

comprised of about 25 proteins

has amino acid similarity to tra conjugation systems involved in DNA transfer

required for acquisition of ER-derived membrane by LCVs, prevention of maturation of LCV’s into lysosomes and intracellular growth of legionella

transports many substrate proteins across bacterial inner and outer membranes through host vacuole

72
Q

what are the major components of the Dot/Icm type IV secretion system?

A

substrate-associated chaperones (IcmS, IcmW, LvgA) - these interact with substrates called effectors which are transported through system and kept folded properly so they can interact properly with secretion system

ATPase (made of the proteins DotL, DotM, DotN)

Inner and outer membrane proteins: DotC, D, F, G, H)

proteins that make a pre in phagosome membrane (probably)

73
Q

what is listeria monocytogenes?

A

gram-positive food-borne pathogen

low incidence of disease

mainly affects immunocompromised, elderly or pregnant women

consequence of infection include meningitis and abortion

74
Q

what are the main virulence proteins involved in the intracellular life of Listeria monocytogenes?

A

LLO

PlcA

PlcB

Hpt

ActA

75
Q

what mediates lysis of the phagosome and escape to the cytosol by listeria?

A

mediated by listeriolysin O (LLO)

76
Q

what is listeriolysin O (LLO)?

A

cholesterol dependent, pore forming toxin which binds to membranes and undergoes cholesterol-dependent oligomerisation leading to pore formation in the lipid bilayer

needed for lysis of the phagosome in cultured cells (also for lysis of double membrane vacuole formed after cell-to-cell spread)

activated by low pH as LLO has acidic pH optimum ensuring it exerts its pore-forming activity primarily in phagosome

77
Q

pores made by LLO are too small for bacteria to escape phagosome, so how does listeria escape into the host cell cytoplasm?

A

through bacterial enzymes PlcA and PlcB which act after LLO to allow bacterial escape into cytoplasm

78
Q

what is PlcA?

A

is a phospohatidylinositol-specific phospholipase C, which hydrolyses phosphatatidylinositol (Pl) in the outer leaflet of phagosome membrane

79
Q

what is PlcB?

A

a broad-specificity phospholipase C that hydrolyses most phospholipids including phosphatidylcholine (PC) which is present in both leaflets of the phagosome

80
Q

what are the food sources which allow cytosolic growth (replication) of listeria in host cell cytosol?

A

glucose 1-phosphate (carbon source)

this requires the Hpt membrane protein which transports glucose 1-phosphate (G1P) which enters the glycolytic pathway

so host G1P is the main food source for listeria

81
Q

what is the intracellular food source for listeria glucose 1-phosphate (G1P) derived from?

A

G1P is derived from glycogen through host glycogen phosphorylase

82
Q

how does Listeria move inside human cells?

A

addition of actin monomers at the plasma membrane interface generates protrusive force that drives eukaryotic cell motility

listeria subverts human actin to stimulate movement of bacteria inside human cells

so basically by adding actin monomers between bacterial pole and actin filament

83
Q

what is a toxin?

A

a microbial factor that damages the host

84
Q

what are the three main kinds of bacterial toxins?

A

surface-actin toxins e.g. TSST

pore forming-actin toxins

A/B toxins e.g. cholera toxin, diphtheria toxin

85
Q

how do surface-actin toxins work?

A

doesn’t go into the cytoplasm just interacts with receptor triggering signalling event which alters cell physiology

86
Q

how do A/B toxins work?

A

go into cells by binding to receptor then going in via endocytosis

87
Q

what is S. aureus?

A

gram-positive cocci which colonise skin and anterior nases

causes skin infections, medical implant-associated infections, invasive disease (endocarditis), necrotising fascitis, toxic shock syndrome (TSS)

88
Q

what is toxic shock syndrome (TSS)?

A

caused by toxin produced by S. aureus (TSST)

TSST is a super antigen which stimulates T cells causing uncontrolled IL-2 release leading to septic shock (decreased tissue perfusion and oxygen delivery)

89
Q

how does super antigens like TSST cause uncontrolled cytokine release?

A

superantigen binds to MHC/TCR complex even when it has an antigen which isn’t specific to pathogen

superantigen binding the complex will activate shit loads of T cells and thus shit loads of cytokines produced

this much cytokine causes blood vessels to leak so key organs don’t get enough oxygen thus organ failure

90
Q

does TSST provide any benefit to S. aureus in anterior nares?

A

TSST prevents development of protective antibodies against the toxin and against S. aureus

so superantigens tend to be selected for cause they interfere with normal immune function increasing organism chance of survival and transmission for both organisms

91
Q

what are A/B toxins?

A

have an A domain and B domain and these can be both on one single protein or different proteins (which would mean encoded by different genes)

example are diphtheria toxin, cholera toxin and anthrax toxin

92
Q

what is the function of the B domain on A/B toxins?

A

binds to receptor on surface of the host cell and helps A domain translocate into host cytoplasm (by inducing endocytosis of toxin when it binds receptor)

93
Q

what is the function of the A domain on A/B toxins?

A

has the toxin activity, this domain is translocated into host cytoplasm either from an endosome or from the ER

so basically B domain gets the toxin into the cell and then A domain fucks shit up

B domain driver A domain shooter

94
Q

what is diphtheria toxin?

A

prod by Cornybacterium diptheriae

causes diphtheria, an upper respiratory tract infection

damages mucosal cells in throat resulting in pseudomembrane comprising of bacteria, inflammatory cells and fibrin

serious complications include suffocation (pseudomembrane blocks air passage) or damage to internal organs following release of toxin into blood

95
Q

how does diphtheria toxin kill cells?

A

by ADP ribosylating human elongation factor 2 (EF2) thereby blocking protein synthesis

96
Q

outline the process of diphtheria toxin entering the host cell and killing it?

A

A and B domain bound by disulphide bond and bind to receptor on human cell which triggers endocytosis so that toxin is now in endosome in host cell

endosome starts to acidify causing disulphide bond to break separating domains

separated B domain makes pore in endosome membrane allowing A domain to get into cytoplasm

A domain covalently modifies key protein involved in protein synthesis (EF2)

97
Q

how does the A domain covalently modifying EF2 block protein synthesis?

A

it ADP-ribosylates it which inhibits the binding of EF2 to tRNA meaning that the ribosome translocation step of protein synthesis cannot take place

98
Q

why is diphtheria toxin very specific?

A

it ADP-ribosylates the dipthamide residue (modified histidine) in EF2

dipthamide residue is an amino acid which is the target of diphtheria toxin and EF2 is the only thing in mammalian cell with dipthamide on it

so EF2 the only thing in mammalian cell that the toxin can modify (by adding ADP ribose to it) i.e. v specific

99
Q

what is the diphtheria vaccine?

A

highly effective

the immunogenicity in the diphtheria vaccine is a toxoid (inactivated toxin) generated by formula treatment

often combined with tetanus toxin and products from Bordetella pertussis to make a trivalent vaccine (DTaP) that prevents three diseases (diphtheria, tetanus and whooping cough)

100
Q

what is cholera toxin?

A

produced by vibrio cholerae, causes cholera

kills cells by ADP ribosylating a host GTPase called Gs-alpha. This modification of Gs-alpha results in cAMP accumulation in the host cell resulting in secretion of ions and large amounts of water into lumen of intestine

101
Q

how does cholera toxin cause diarrhoea?

A

in intestine body maintains isotonicity between lumen and cells in blood streams electrolyte flow v regulated

A subunit of toxin (mayb B too) gets into cell and stimulates prod of signalling molecule cyclic-AMP (cAMP) by activating the enzyme adenyl cyclase

cAMP accumulation causes efflux of Cl- and bicarbonate from blood vessels to lumen and also blocks import of sodium

accumulation of all these enzymes in lumen of intestine makes it hypotonic thus water flows into intestine to maintain tonicity thus diarrhoea

102
Q

how does cholera toxin activate adenyl cyclase?

A

by covalently modifying regulatory protein called G protein

a subunit of the G protein complex is Gs-alpha. Cholera toxin puts an ADP-ribose molecule on a residue in Gs-alpha causing it to be always active (it normally regulated to be active or not active) and increasing its activity

this why you get uncontrolled production of cAMP

103
Q

what is site on Gs-alpha that is modified by cholera toxin?

A

an arginine

lots of proteins have arginine so specificity of toxin cannot be purely due to arginine

104
Q

how does adenyl cyclase control chloride movement?

A

adenyl cyclase regulates a chloride transporter called CFTR and interacts with it to activate it such that CFTR transports chloride ions to outside the cell

105
Q

what is CFTR?

A

cystic fibrosis trans-membrane conductance regulator

is the chloride transporter regulated by adenyl cyclase

106
Q

what is the structure of cholera toxin?

A

AB5 hexameric structure i.e. there are five different molecules of B protein which interact with the A protein

this forms the complex that gets into cells

107
Q

how many genes encode cholera toxin?

A

two different genes - one encoding A and one encoding B

108
Q

how many genes encode diphtheria toxin?

A

a single gene

109
Q

how does cholera toxin get internalised and delivered into the host cytoplasm?

A

doesn’t make a pore in endosome, instead undergoes retrograde transport to move to Golgi apparatus and then ER

retrogade transport involves toxin taking advantage trafficking pathways in human cell

once in ER it uses normal pores (for protein transport usually) to get into cytoplasm

ER has quality control system for misfiled proteins, transports them to cytoplasm for degradation, toxin exploits this

110
Q

what is a good way that toxin genes are spread between bacteria?

A

many bacterial toxins (incl diph and chol toxins) are encoded by lysogenic bacteriophages

these phage-borne genes can be transferred between bacteria thus spreading virulence properties

phage injects DNA, this is integrated into chromosome, if bacteria gets stressed it can excise this DNA and phage goes elsewhere

111
Q

what is the receptor for the phage carrying cholera toxin gene?

A

a type four pilus and adhesin called TCP

this usually allows vibrio cholerae to attach to human intestinal epithelial cells

only bacteria expressing TCP will be infected by phage so you either have both TCP and toxin or neither

112
Q

why would it be energetically wasteful for bacteria without TCP to produce cholera toxin?

A

because for the toxin to be effective the bacteria has to be able to adhere to intestinal epithelial cells

113
Q

how do toxins such as cholera toxin, diphtheria toxin and toxic shock syndrome toxin benefit pathogens?

A

cholera toxin may aid in spread of bacteria through diarrhoea

all the other ones noone is really sure how

maybe they have targets other than people

114
Q

what are some characteristics of biofilms?

A

resistant to cell loss (cells in biofilm tight together so hard to detach)

tolerance to antibiotics (cells interior of biofilm inaccessible and/or metabolically dormant)

resistance to phagocytosis and killing by macrophages or neutrophils

115
Q

why does antibiotic tolerance develop in some bacteria in biofilms?

A

stress responses (e.g. low nutrients) causes physiological changes in some bacteria allowing them to not be killed by antibiotics

this may involve bacteria undergoing change in gene expression e.g. turn of efflux pump

tolerance is physiological not genetic change like antibiotic resistance

116
Q

what are persister bacteria?

A

tend to occur in stationary phase of growth and are genetically identical to normal cells

117
Q

how do persister bacteria often play a role in relapsing S. aureus infection?

A

host immune system kills regular cells and persisters in planktonic state but cannot kill cells in biofilm

antibiotics penetrate biofilm matrix killing majority of cells but leaving behind persisters in biofilm

once antibiotic treatment done persisters resuscitate and grow thus infection relapse

118
Q

what are some bacteria that make medically relevant biofilms?

A

pseudomonas aerginosa (lungs of cystic fibrosis patients)

streptococcus mutans (tooth surface)

staphylococcus epidermidis (medical implants)

staphylococcus aureus (medical implants)

119
Q

what are the stages in formation of an S. aureus biofilm?

A

attachment: involves a bacterial surface protein called fibronectin binding protein (FnBP) and other adhesins that bind serum proteins

maturation: involves bacteria growing and getting glued together by PIA (polysaccharide intercellular adhesin)

detachment: involves Agr system, phenol-soluble modulins (PSMs)

120
Q

what is the role of fibronectin binding protein (FnBP) in the attachment stage of S. aureus biofilm formation?

A

fibronectin in blood coats the surface of medical implants

FnBP on the surface of S. aureus mediates binding of bacteria to fibronectin coating the medical implant

121
Q

what is the role of PIA (polysaccharide intercellular adhesin) in the maturation of S. aureus biofilm formation?

A

bacteria secretes PIA which is a positively charged polymer comprised of modified (partially de-acetylated) N-acetylglucosamine

PIA causes extracellular matrix that causes bacteria in biofilm to stick together as generally bacterial surface neg charge

122
Q

what is the role of phenol soluble moulins (PSMs) in structuring of S. aureus biofilms?

A

structuring is the loss of regions in biofilm to form fluid filled channels which allow delivery of nutrients to deeper layers of biofilm

PSMs not expressed homogeneously so only expressed in certain regions of biofilm and these are the bit which fall off to form channels

123
Q

why can PSMs fall off biofilms?

A

PSMs use surfactant activity to structure biofilms as they are amphipathic (hydrophobic and hydrophilic side) peptides so hydrophobic region interacts with biofilm to form micelle which allows solubilisation of it

124
Q

what does the final detachment stage of biofilms require?

A

PSMs but mechanism not really known

125
Q

when are PSMs expressed and what is their expression controlled by?

A

PSMs become expressed after maturation of biofilms

their expression is under control of the Agr “quorum sensing” system

126
Q

what are the two stages of biofilms promoted by PSMs?

A

structuring and detachment stages

127
Q

explain the Agr quorum sensing system which controls expression of PSMs?

A

AgrC (sensor kinase) autophosphorylates his residue then AgrA (response regulator) takes that phosphate off and transports it to aspartate residue which activates and stimulates gene expression allowing S. aureus to sense density hence “quorum sensing”

S. aureus secretes density signal to enviro and at certain threshold senses this and activates AgrC/AgrA system

when activated AgrA binds P2 promoter activating expression of itself, AgrC, AgrD (encodes precursor of autoinducer peptide (AIP)), and AgrB (encodes membrane proteins which processes AgrD to active form and transports to outside cell)

AgrA also turns on P3 promoter which turns on gene encoding regulatory RNA which regulates expression of PSM and toxin genes

128
Q

when is lots of auto inducer peptide (AIP) produced?

A

not much being produced during attachment phase but once critical density threshold reached (during maturation phase) and AgrC/AgrA system activated everything is ramped up and lots of AIP produced

129
Q

AgrC/AgrA is an example of a __________ system?

A

two-component regulatory system

130
Q

what is the mechanism of gene expression regulation by RNAIII (the regulatory RNA turned on by AgrA)?

A

the shine delgarno (SD) sequence normally interacts with 16s allowing ribosome to initiate translation

genes encoding the toxin HIa and PSMs induced in expression (translation) by RNAIII

however SD already base paired with another region not 16s so at low cell density toxins/PSMs can’t be translated well

but when RNAIII synthesised (high cell density) it has nucleotides which complement region of SD so that SD now accessible and ribosome can bind SD initiating translation of toxin and PSM genes

131
Q

different S. aureus groups make distinct AIPs, what does this mean?

A

an AIP from one group can interfere with an AIP from another group

group 1 AIP will only bind and activate group 1 AgrC (so there is specficity between the two)

group 1 AIP can however bind group 2 AgrC but NOT activate it

it does however prevent group 2 AIP from binding it meaning diff S. aureus groups can compete through interfering with each others quorum sensing

MICR223 (7 decks)

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