Module 10 - Bacteria Flashcards
(178 cards)
1
Q
what is the main difference between gram-negative and gram-positive bacteria?
A
gram-negative have a double membrane.
gram-positive have a single but thicker membrane.
2
Q
what kind of vesicles do gram-negative bacteria shed?
A
outer membrane vesicles
3
Q
the thick wall/walls of bacteria allow for what?
A
make it difficult for harmful compounds to get into the bacteria
4
Q
what are the 4 types of mechanisms for gram-negative vesicles formation?
A
- clathrin dependent
- caveolin mediated
- lipid raft
- membrane fusion
5
Q
what can be in gram-negative vesicles to facilitate infection?
A
virulence factors, DNA, RNA, immunomodulatory factors, adhesins
6
Q
how is bacteria entry facilitated in the gut?
A
the stomach’s lower pH triggers periplasmic proteins to oligomerize and insert in the bacteria outer membrane to form a pore. the bacteria can fuse with target cell and depolarize and permeabilize it!
7
Q
what organelle derived from bacteria sheds vesicles?
A
mitochondria
8
Q
what are bacterial secretion systems?
A
Essential membrane embedded multi-protein machineries, enabling bacteria to obtain nutrients, communicate and facilitate disease through the DELIVERY OF VIRULENCE FACTORS out of the bacteria
9
Q
what is special about gram-negative type III, IV, and VI secretion systems?
A
they can cross 3 membranes: double layer bacterial membrane + phagosomal membrane
10
Q
what are the oldest transport machinery systems? what are they used for?
A
Sec and Tat;
mostly for biogenesis of bacterial proteins
11
Q
what membrane can Sec and Tata transport machinery cross?
A
only the first membrane (inner)
12
Q
what are the triple membrane spamming transport machinery system used for?
A
to release virulence factors
13
Q
what are type II secretion systems function?
A
secretes toxins, small
molecules (like cholera toxin), which disables host protein synthesis, leading to lethal infection
14
Q
what are type III secretion systems function?
A
secretes effector proteins, shares similarity to components with flagellar apparatus, evolutionary related. Effectors vary widely in function
15
Q
what are type IV secretion system functions?
A
transfer DNA and proteins (output AND uptake)
16
Q
what are type V and VI secretion system functions?
A
translocate portions of themselves. Beta-barrel channels, or pores.
17
Q
what secretion machineries/systems are also found in gram-positive bacteria?
A
Tat and Sec
18
Q
what way of secretion is often discussed for gram-positive bacteria since we don’t know much about their secretion?
A
passive diffusion
19
Q
what type of cells work to clear the bacteria and develop immunity?
A
professional phagocytic cells
20
Q
name 2 professional degrading cells that degrade bacteria and their different role (APCs)
A
- macrophages: rapidly degrade bacteria before it can replicate or escape, highly efficient
- dendritic cells: slower degradation processes; select antigens to be presented
21
Q
how are MHC class 1 expressed? (steps)
A
- pathogen in cytosol degraded by proteasone
- pathogenic peptides enter ER via TAP
- loaded on MHC 1 & transported to cell surface
- bind and activate CD8+ T cells
22
Q
what type of immune molecule will present the selected bacterial antigens?
A
class II MHC
23
Q
what type of T cells are activated by bacterial antigen on class II MHC?
A
CD4+ T cells
24
Q
how is immune tolerance acquired?
A
by sampling “self” antigen in the thymus
25
where are MHC class I molecules found?
on ALL somatic cells, and in endosomes
26
what pathogens (and how) can be loaded on MHC class I?
- pathogens in the cytosol that get degraded by proteasome
- -> peptides enter ER through TAP
- -> can be loaded on MHC I
27
peptides on MHC class I can activate what kind of immune cells?
cytotoxic CD8+ T cells
28
in what scenario is MHC I found on endosomes?
during cross-presentation
29
where are MHC class 2 molecules found?
on APCs (dendritic cells, macrophages, B cells)
30
what peptides can be loaded on MHC II proteins?
peptides from pathogen in endosomes/phagosomes that have been previously degraded by proteases
31
peptides on MHC class II can activate what kind of immune cells? for what immune process?
cytotoxic CD4+ T cells for antibody production
32
do bacteria spontaneously infect non-phagocytic cells?
no; there is a signal transduction pathway
33
what are the 2 mechanisms via which bacteria infect non-phagocytic cells?
zipper and trigger mechanism
34
explain the zipper mechanism?
surface proteins on bacteria BIND host cell proteins (receptors or adhesion proteins like integrins or cadherins) and that triggers a signaling cascades that remodel lipids and actin to extend the cytosol and allow engulfment
35
explain the trigger mechanism?
it initiates upon bacterial SECRETION of effectors that activate actin and lipid remodeling event
36
what is necessary for the zipper and trigger mechanisms to happen?
modulation of phosphorylated phosphatidylinositol (PIP) (to become PI(3)P
37
what does the presence of PIP in cell membrane do in regards to bacteria infection?
reinforces the recruitment and activation of actin remodeling machinery
38
what type of secretion would be required by trigger mechanism?
type 3 (3 membrane-spanning system)
39
name a bacteria that does zipper vs trigger mechanism of entry
zipper = Listeria internalin (gram +)
trigger = Shigella (gram -)
40
name 3 examples of proteins that bacteria secrete. are they activated?
phospholipid modifiers, kinases, proteases.
often no, they must get activated by the host machinery
41
explain the 3 examples we saw of how secreted proteins/effectors can get activated
1. Rab5:GTP binding to phospholipase A1 (effector) can alter its conformation, activating it
2. protein kinase binding to E2-Ub complex stabilizes its active site
3. protease binding to redox-related protein isomerases can activate its activity
42
bacterial effectors that are injected early can activate what?
internalization of the bacteria
43
bacterial effectors that are injected later can facilitate what?
closure through depolymerization
44
effectors that evolved to evade internalization into macrophages can now ?
allow entry into non-phagocytic cells
45
what can be a function of effectors that are secreted after the bacteria escaped the endosome?
drive bacterial movement through the cytosol
46
explain the example of what happens to salmonella once it entered the cell
It replicates in its own vacuolar compartment.
The vacuoles interact or fuse with organelles via long extensions.
Hydrolytic content is removed through recycling endosome so the bacteria doesn't get degraded.
47
what is the hypothesis of how bacteria (salmonella) escape the vacuole after infection?
via the secretion of a pore forming toxin effector that ruptures the vacuole wall
48
what is SipA? what's its function?
a salmonella effector that acts as an R-SNARE and recruits early endosomal SNAREs syntaxins 8, 7 and 13.
This keeps the bacteria out of the later endosomes
49
name the role of other salmonella effectors other than SipA?
- alter retromer
- alter mannose-6 phosphate transport
(both aim to stop the endosome from becoming hydrolytic)
50
what's a difference between salmonella and shigella?
shigella must escape phagosome to replicate, salmonella can replicate in the vacuole
51
what happens to shagella bacteria after being phagocytosed?
escapes the phagosome after 10-15 min with Rab11-positive recycling endosomes. it lives in the cytosol
52
shigella's escape of the phagosome involves the recruitment of what protein?
Rab11-positive recycling endosome
53
how can the bacteria move through the cell?
it generates actin comet tails
54
explain actin comet tails
actin polymerization machinery is recruited to the bacterial membrane and drives rapid movement through the cell
55
what are 2 things actin comet tails can do, which shows how powerful they are?
- drive movement of bacteria cutting through mitochondria
- drive bacteria directly into the neighboring cell
56
how does the host respond to a phagosome rupture induced by bacteria?
glycans are exposed to the cytosol and recruit Galectins
57
what are galectins and how do they work?
proteins that sense damaged membrane (glycans) and target them for degradation through autophagic mechanism.
They recruit NDP52.
58
what is NDP52 and its role?
autophagic adaptor that recruits Ub E33 ligases to ubiquitinate a broken phagosome membrane and the bacterial surface
59
what other adaptors are recruited with NDP52 after galectins signal damaged membrane?
P62 and optineurin
60
what are P62's and optineurin's function?
P62 binds a ubiquitin ligase Traf6.
Optineurin binds DUBs.
(they sculp the ubiquitin landscape)
61
what do NDP5a, optineurin, and p62 have in common?
they are all recruited to damaged phagosome membrane and all have a LIR domain to interact with LC3
62
what is the final step of removing bacteria by the host cell?
NDP52, optineurin, and p62 LIR domains interact with LC3 which initiates cargo incorporation into an autophagosome
63
how can bacteria escape this highly modulated route to autophagosomes?
with actin tail
64
describe zipper mechanism of bacterial invasion
- contact via surface proteins
- phagocytic cup formation
- closure and retraction
65
describe trigger mechanism of bacterial invasion
- inject effectors by TTSS (secretion systems)
- cytoskeletal rearrangement
- engulf bacteria by vacuole
66
what are the 2 mode of survivals of bacteria?
membrane-bound lifestyle or cytosolic lifestyle
67
what is the advantage of bacterial membrane-bound lifestyle?
it prevents lysosome fusion
68
what are the 2 steps of bacterial evasion from the host cell?
1. actin polymerization
2. destroy the membrane
69
what happens to bacteria when the host cell dies from apoptosis?
Bacteria can escape within the apoptotic bodies and infect next cell this way
70
what is necrosis?
inflammatory cell death from damage, loss of ATP, membrane rupture,
71
what is pyroptosis?
highly programmed form of death where the dying cell expresses and releases specific cytokines to recruit immune cells to help.
(involves transcriptional programs, caspase cascades, etc) (also inflammatory)
72
what is a way that bacteria can force their survival?
some bacteria it can promote host cell survival (activate pro-survival pathways) during infection via different targets
73
what organelles do apoptosis escape of bacteria revolved around?
mitochondria: holds the key
to all forms of cell death
74
how does bacteria evade host death?
different bacterial effectors block apoptotic machinery and activate pro-survival pathways (ex NFKB, MAPKK)
75
what's a bacteriophage?
a virus that parasitizes a bacterium by infecting it and reproducing inside it
76
explain the lytic pathway of virus entry in host bacterial cell?
1. phage attaches to host bacterial cell
2. phage inject its DNA
3. transcription and replication of virus DNA
4. phage assembly
5. cell lysis allows for phage release
77
explain the lysogenic pathway of virus entry in host bacterial cell?
1. same entry as lytic pathway (phage attaches to cell and injects its DNA)
2. prophage formation (virus DNA links with bacterial DNA)
3. replication of bacterial + viral DNA
4. cell division
78
can all viruses go into the lytic pathway?
no
79
what is the advantage of the lytic pathway?
the phage can stay in the cell and wait for the proper conditions to trigger lysis
80
what are the 3 ways how phages can adapt to bacterial anti-viral strategies?
1. Can re-evolve a new receptor binding protein (RBP)
2. Can evolve enzymes to degrade any coats or modifications that impede access to receptors
3. Can express multiple receptor binding proteins to increase flexibility
81
what enzymes can phages evolve that can degrade any coats?
endosialidase or glycosidase enzymes
82
bacterial anti-viral immune system resulted in the identification of what?
Crispr-Cas9 gene editing system
83
phages could be an alternative therapy to what?
antibiotics
84
what are the advantages of using phages instead of antibiotics?
- phages are highly specific and quickly co-evolve to overcome resistance
- they are naturally found in animals and are better tolerated
- they self replicate (single dose treatment)
85
name the 6 steps of the life cycle of a virus in an animal host
1. attachment to cell surface
2. penetration via endocytosis
3. uncoating of virus: viral content is released in the cell
4. biosynthesis of viral genes and proteins via viral RNA in the nucleus
5. assembly of new phage particles
6. release of new viral particles in the ECM
86
viral genomes can code for how many proteins?
2 to 2000
87
how does viral RNA replicated?
it is replicated in the host cell nucleus by the viral RNA polymerase
88
what is the difference between enveloped and non-enveloped virus?
enveloped has the viral capsid in a membrane.
non-enveloped is capsids only.
89
what happens in the infected host cell once the new viral particles are released?
it continues to make new virus
90
give an example of enveloped vs non-enveloped virus
enveloped = west Nile, influenza, HIV, Ebola, SARS-COV2
non-enveloped = rotavirus
91
what are similarities between enveloped and non-enveloped virus?
- DNA or RNA
- single or double stranded
92
how do animal viruses enter cells?
via endocytosis
(pinocytosis, caveolin, lipid raft, phagocytosis, IL2, flotillin, ...)
93
what do viruses bind to infect cells?
surface receptors
94
what does the acidification of the endosome containing the virus trigger?
Acidity generally triggers viral uncoating and/or fusion of the virus with endocytic membrane
95
what happens after the virus fuses with the endocytic membrane?
release of viral genome (NUCLEOCAPSID) into the cytosol
96
do all viruses need to enter in endosome to then release their viral genome?
no; Some viruses can fuse right at cell surface
97
what is corona virus' primary entry pathway?
IL-2
98
does covid replicate in the nucleus or cytosol?
cytosol
99
what can rotavirus cause?
gastroenteritis
100
describe rotavirus
80–100-nm in diameter, three capsid layers with icosahedral symmetry, RNA(+) virus
101
how does rotavirus enter host cell?
in clathrin and non-clathrin mediated endocytosis
102
what molecules are required for rotavirus to release its content in the cytosol?
Rab5, Rab7, Rab9, ESCRT, cathepsins
103
how can pH drop in endosomes trigger viral exit?
can open helical proteins in capsid proteins, or activate viral proteases that cleave viral capsid proteins
104
what can helical/capsid proteins do?
they disrupt the amphipathic peptides in the membrane of acidified endosomes, allowing the capsid to escape the endosome
105
does the capsid always need to escape the endosome to infect the host cell?
no; sometimes only the viral genome need to escape
106
what are the 2 models for viral exit of the endosome?
carpet model and pore formation
107
what is carpet model?
suggests that the viral proteins coats the endosome membrane, and somehow solubilizes it like a detergent.
108
what is the pore formation model?
suggests that viral proteins directly embed into the membrane and generate a pore for viral escape
109
what type of virus is picornavirus?
RNA+ virus
110
explain how picornavirus RNA exits the endosome
pH changes allows:
- the uncoating of the capsid
- RNA release: VP4 is lost, this allows VP1 contact with the endosome membrane where it forms a pore
111
2016 study of genome-wide siRNA screen identified what genes important for rotavirus infection?
- AMPK
- vacuolar ATP synthase
- mTOR
- MAPK
112
explain the different proteins found in SARS-CoV-2 enveloped virus
- Spike proteins: receptor binding & mAB target
- E protein: pore forming
- M protein: membrane protein forming lattice
- lipid bilayer
- N protein: packing the RNA
- ssRNA: 30 kbp
113
explain the 2 types of enveloped virus entry?
1. viral envelope proteins bind specific cell surface proteins for entry & SNARE-like fusion machinery in the viral envelope helps fusion
2. virus is endocytosed while still enveloped and the pH change activates viral fusion proteins once in the cell
114
the fact that viral proteins binds specific cell surface protein says what?
that the virus binding and entry is cell-type specific
115
In addition to initial binding, _______________________ sits in the viral envelope.
SNARE-like fusion machinery
116
describe class I viral fusion proteins
- mostly alpha-helical
- require a cleavage event to reveal the fusogenic region
- Single polypeptide chain
- Activation of viral proteases occurs upon acidic pH.
117
name 3 viruses with class I fusion proteins
Influenza, retrovirus, coronavirus
118
describe class II viral fusion proteins
- mostly beta-sheets
- have an internal fusogenic region
- Are bound to another membrane protein that is often cleaved, allowing activation of the fusion protein
- Includes viral chaperones in a complex of proteins
119
name 3 viruses with class II viral fusion proteins
Flavivirus, Alphavirus, Denguevirus
120
describe class 3 viral fusion proteins
- does not require any cleavage events. - Mix of alpha-helix/beta-sheet.
- Conformational changes are induced by pH change
121
all classes of viral fusion proteins require conformational change, but which classes require cleavage event?
class I and II
122
what must be activated for enveloped virus to enter the host cell?
viral fusion proteins
123
in the influenza virus example, what class of fusion protein is found and how does it enter the host cell?
Class I fusion protein with alpha-helices where HA causes a conformational change that allows cleavage of a fragment of the protein, exposing the fusion peptide that embeds in the host endosomal membrane
124
in the Dengue virus example, what class of fusion protein is found and how does it enter the host cell?
Class II fusion protein with beta sheets: glycoproteins rearrange upon low pH to release the fusion peptides that embed in the lumenal side of endosome membrane and triggers membrane fusion with the viral envelope
125
what is special about SARS-COV2 entry pathways?
it can enter via 2 pathways: endosomal entry or cell surface entry
126
what event is essential for viral fusion of SARS-COV2?
- S1 cleavage of furin site during biogenesis in the Golgi of previously infected cell
- S2 cleavage event (essential for both pathways)
127
via what receptor does covid enter host cell?
ACE2
128
what is TMPRSS2?
protease on cell surface that decide if coronavirus with fuse with the host cell membrane instead of endocytosed
129
what are the steps of the endosomal entry of coronavirus
1. virus binding
2. internalization
3. endosomal acidification
4. cleavage of S2 by cathepsin L
5. membrane fusion
6. uncoating of viral RNA
130
what are the steps of the cell surface entry of coronavirus
1. virus binding
2. cleavage of S2 by TMPRSS2
3. membrane fusion
4. uncoating of viral RNA
131
what do the covid vaccines target?
spike proteins
132
why didn't therapies against covid blocking acidification of endosomes work?
the virus can still infect via cell surface path
133
where can viral genome replication occur?
cytosol or nucleus
134
where do MOST RNA viruses replicate? what about DNA viruses?
RNA: cytosol
DNA: nucleus
135
do viral capsids enter the nucleus?
some do for nuclear viral genome replication!
but capsids can also disassemble at the pore with only the genomes entering the nucleus, OR genome can be transported into the nucleus alone
136
how does viral DNA enter the nucleus?
always through nuclear pores; sometimes still in capsid, sometimes alone
137
what happens to the viral genome once it enters the nucleus?
it replicates! and then it must exit the nucleus
138
how does papilloma virus lead to canceR?
viral genome integrate into the chromosomes
139
how can the virus escape the nucleus?
many ways:
- trigger apoptosis of host cell
- exit via nuclear pore
- fuse with membrane and bud out
140
where does the virus assemble after it replicated its genome?
simpler viruses can assemble in the nucleus, others transport the genome into the cytosol for assembly
141
what does the papilloma (and other viruses) do after replication in the nucleus?
it integrates its viral genome in the chromosomes and leads to cancer
142
what happens if the virus triggers apoptosis of host cell to exit the nucleus?
it can't use the host cell as a "factory" anymore
143
capsids/viral DNA always enter the nucleus via what?
nuclear pore
144
how does the herpesvirus and bacculoviral escapes the nucleus?
herpesvirus vesicles enter the perinuclear space, fuse with outer nuclear envelope and release capsid in cytosol
145
how does herpesvirus become enveloped again after exiting the nucleus?
capsid binds Golgi that is now expressing viral proteins in the membrane, and capsid is internalized or “wrapped”
146
what happens to herpes virus once it is re-enveloped by the golgi?
it now an active virus and gets secreted by the cell
147
how does the "naked" capsid that emerged from the nucleus know to go to the golgi for envelopment?
it binds viral encoded proteins that had been translated in the cytosol and imported into the ER
148
what happens to the viral encoded proteins found in golgi membrane?
- they recycle through TGN/PM
- they are incorporated in the new capsid envelope
149
what is a weird way how viruses can get re-enveloped after replication in the nucleus?
they can stay within the perinuclear space and enter the ER, from which they can egress in different ways
150
why do viruses not fuse back with late endocytic compartments?
the pH is wrong! so viruses can only go in one direction
151
explain apoptotic mimicry
virus acquires host cell markers (phosphatidylserine) that are interpreted as apoptotic fragments and bind directly to receptors on the new host cell
152
what is the principle behind apoptotic mimicry?
apoptotic fragments need to be internalized to ensure cell death is immunologically silent
153
what kind of viruses can do apoptotic mimicry?
enveloped and non-enveloped
154
how does SV40 non-enveloped virus do apoptotic mimicry?
it has capsid proteins that mimic the GAS6 protein that binds phosphatidylserine receptors
155
how can exosomes participate in apoptotic mimicry?
the release of exosomes carrying viral capsids can mimic an enveloped state enriched in PS
156
Internalization of apoptotic fragments drives signaling of what?
anti-inflammatory signaling
157
how do apoptotic fragments drive anti-inflammatory signaling?
activation of apoptotic receptors activate transcriptional responses that:
- inhibit host cell receptors that receive pro-inflammatory signals
- secrete anti-inflammatory factors for neighbouring cells
158
why does apoptotic mimicry trigger anti-inflammatory signals?
helps the virus to propagate without alerting the immune system.
159
what part of the immune system is mostly affected by apoptotic mimicry and anti-inflammatory signaling?
innate immune system
160
how do viruses enter cells?
endocytosis
161
ph transitions are critical for what?
to uncoat or disassemble viruses
162
what is the goal of apoptotic mimicry?
allows efficient uptake and suppress inflammatory signaling pathways in the host
163
what did the paper study?
how can bacteria escape the endosome? looked at sphingomyelin, a lipid enriched in the outer leaflet of the PM
164
what did they do to lysenin?
engineered only its C-terminus domain that binds sphingomyelin
165
how did they test lysenin specificity?
- made PC, PC+SM, and SM liposomes. lysenin only binded in presence of SM
- sphingomyelinase inhibits lysenin binding
166
galectin 8 is a marker of what?
membrane rupture
167
did sphingomyelinase affect galectin binding?
no
168
what is K185A mutation?
lysenin loses the ability to bind
sphingomyelin and did not bind cell surface (confirms that lysenin probe binds SM)
169
what happened to lysenin after salmonella infection?
binds 10-20% of SCV, peaks at 1-2 hours, lost by hour 6
170
what does it mean that lysenin binds SCVs after salmonella infection?
SM is exposed in some SCVs
171
why did they do osmotic shock, lysosome damaging drug (GPN), and listerolysin to endosomes?
to see if these other kind of endosome damage recruit lysenin like bacterial infection, and they do
172
how did they conclude that galectin happens after SM exposure?
listerolysin O damaged to endosome recruits lysenin but no galectin
173
what happened when they overexpressed Sphingomyelinase?
blocks lysenin recruitment, but does not block the nb of bacteria infecting the cell or the replication rate
174
sphingomyelinase not blocking the infection shows what?
flipping is important but not necessary for infection; might just be a signal
175
what did they find studying a strain without T3SS needle?
no lysenin of galectin-8 recruitment = T3SS is involved in damaging the SCV membrane
176
Lysenin recruitment was slow and gradual, Gal8 is abrupt, consistent with ?
the membrane finally breaking and attracting galectin; lysenin is recruited before the membrane breaks?
177
appearance of NDP52 suggests that ?
SCVs are targeted for autophagy
178
The ultimate loss of homogeneity in the Lysenin ring coincided with recruitment of galectin-8, consistent with ?
a break in the SCV membrane resulting in glycan exposure
