Reed Lectures Flashcards
1
Q
2 properties of a bacterial pathogen
A
- can colonize host
- causes disease
Many bacteria in the environment don’t cause disease
Not all strains of a pathogen will cause disease
2
Q
Define virulence. Give a major determining factor for virulence
A
Ability to cause disease; host susceptibility
3
Q
Define virulence factor, and give an example
A
Any strategy that promotes disease-causing properties/causes virulence
ex. toxins, secretion systems
4
Q
T or F: infection always leads to disease
A
False. Asymptomatic ppl are called carriers (colonized by bacteria, but show no symptoms of disease)
5
Q
Define bacterial pathogenesis
A
Mechanisms through which bacteria cause disease
6
Q
Why is it important to study bac path?
A
To development prevention strategies (Vaccines) and treatments (Antibiotics)
7
Q
3 bacteria that Robert Koch studied
A
B. anthracis
M. tuberculosis
V. cholerae
8
Q
List Koch’s postulates
A
- Microbe must be associated with symptoms of disease and must be present at site of infection
- Microbe must be isolated from disease lesions and be grown in pure culture
- Inoculation of isolated bacteria into healthy host needs to generate the same disease
- The same microbe must be re-isolated from the inoculated host
9
Q
Give limitations to Koch’s postulations
A
- Not all infections lead to clinical symptoms; host susceptibility determines virulence and whether disease develops or not
- Cannot grow many bacteria in pure culture due to complex nutritional/environmental needs for growth
- To test the 3rd postulate, you need a perfect animal model that perfectly replicates the disease seen in humans. Often, this is not the case. The strain of bacteria causing disease X in humans may not cause the same symptoms in mice. Therefore, may need to change the mouse (ex. mutant mice), or use another strain of bacteria to emulate similar symptoms
10
Q
Give 2 molecular biology techniques that can be used to identify presence of microbes in carriers
A
- PCR - identifies bacterial DNA
2. IHC - identifies bacterial proteins
11
Q
Who developed the molecular Koch postulates?
A
Stanley Falkow
12
Q
List the molecular Koch postulates
A
- A suspected virulence factor (gene) should not be present in the avirulent strain.
- Disrupting the virulent gene should attenuate its virulence. Reintroducing WT gene should reconstitute virulence
- Putting putative virulent gene in non-virulent bacteria should cause it to become virulent
13
Q
Give limitations to the molecular Koch postulates
A
- virulence may be multifactorial - requires multiple genes/a gene cassette for full virulence to occur
- disrupting bacterial metabolism/biochemical pathways can also attenuate virulence - are such housekeeping pathways virulence factors?
14
Q
uropathogenic E. coli (UPEC) causes what infection?
A
UTI (urinary tract infection)
15
Q
What cell type does UPEC infect?
A
bladder epithelium
16
Q
Give 4 ways that UPEC can interact with host cells
A
- Attachment via pili
- rearrange host cell cytoskeleton by interacting with actin
- interact with host signalling pathways
- form microcolonies
17
Q
T or F: coordinated expression of virulence facts is essential for full virulence and disease
A
T
18
Q
UPEC expresses Type __ pili. It is required for what?
A
Type 1. Needed for colonization, invasion, persistence.
19
Q
Where do IBCs form? nucleus, vesicles, or cytoplasm?
A
cytoplasm
20
Q
What are IBCs
A
Intracellular bacterial colonies
21
Q
Are filamentous bacterial cells (UPEC) proliferating or non-proliferating?
A
Proliferating - allows them to evade killing by neutrophils
22
Q
What are quiescent intracellular reservoirs? (QIR) What type of host cell are they established in?
A
Non-replicating bacteria form QIR for long term survival. Established in transitional cells below uroepithelium.
23
Q
Name 1 toxin that UPEC secretes for nutrient acquisition
A
alpha-haemosylin (HlyA) - lyses host cell to release nutrients
Also a way for UPEC to exit 1 cell and spread to another
24
Q
T or F: UPEC doesn’t need coordinated expression of virulence factors to cause disease
A
F - expression of proteins/virulence factors needed to cause disease are organized into 3 main steps: entry, survival + proliferation in cell, exit.
25
T or F: Knocking out 1 virulence factor may lead to avirulence, but it may still lead to full disease
F - full disease requires combined effects of each virulence factor. Ability for bacteria to cause disease may be attenuated if one virulence factor is missing/mutated/disrupted
26
Is a single virulence factor known to be essential for virulence sufficient to cause full disease?
NO. Full disease requires combined effects of each virulence factor
27
T or F: Sec and Tat secretion pathways are present in Gram negatives only
F. they are common to both Gram + and -
28
What does Tat stand for?
Twin arginine translocation
29
What are the Sec and Tat pathways?
General bacterial secretion pathways used to transport proteins across cytoplasmic membrane.
30
T or F: many proteins are secreted through the Sec and Tat pathways
T
31
T or F: many proteins secreted by Sec and Tat systems are secreted outside the cell
F. Most proteins secreted through these systems are destined for periplasm or cytoplasmic membrane
32
The _____ is required for proteins to be localized to a specific location
signal sequence
33
T or F: 2 step pathways are commonly required in Gram + bacteria to secrete proteins out of the cell
F. commonly required in Gram NEGATIVE bacteria. The first step often involves Sec or Tat, while the second step (secretion out of cell) involves another secretion system
34
Are Sec and Tat pathways active or passive transport systems?
Active
35
The general Sec pathway transports folded or unfolded proteins?
unfolded
36
The protein transported by Sec pathway often have a signal sequence at C or N terminal?
N
37
T or F: The general Sec pathway is not essential to gram + and - bacteria.
F - ESSENTIAL for survival bc most proteins are transported through this pathway
38
What provides the energy for active transport in Sec pathway?
SecA - an ATPase, and proton motive force
39
T or F: Tat pathway is a passive transport system.
F - ACTIVE
40
Tat pathway transports unfolded or fully folded proteins?
Fully folded; bound to cofactors
41
T or F: Tat pathway is present in all bacteria
F
42
Proteins that go through Tat pathway must have what signal?
twin-arginine motif on N terminus
43
T or F: Many proteins that enter general Sec pathway are bound to cofactors
F - many proteins entering TAT pathway are bound to cofactors
44
Energy to transport proteins in Tat pathway comes from where?
Proton motive force
45
List 4 Gram + secretion systems
General Sec Pathway (essential)
Injectosome
Sortase system (SrtA)
T7SS/ESX
46
The sortase system anchors proteins to the ___ of Gram+ bacteria
cell wall/precursor peptidoglycan
47
The sortase systems recognizes proteins coming through the ___ pathway with a ____ motif
Sec; LPxTG
48
T or F: The LPxTG motif is present at the N terminus of the protein.
F - present at C terminus. The N terminus contains the signal sequence
49
Which Gram + secretion system transports small proteins ~100 AA long?
T7SS/ESX
50
Which Gram + secretion systems transports proteins with WxG motif?
T7SS/ESX
51
T or F: T7SS-exported proteins often dimerize
T
52
Give a major pathogen that contain the T7SS
M. tuberculosis
53
Is the Sortase system in Gram + or -?
+
54
Is the ESX system in Gram + or -?
+
55
T or F: injectosome transports things across cell wall directly into host cell cytoplasm
T
56
T or F: T7SS forms a channel.
T
57
Give the functions of ESX-1, ESX-3, ESX-5
ESX1; pokes holes in phagosomes to transport bacterial proteins into cytoplasm; essential for bacterial survival in phagosomes
ESX3: Fe acquisition
ESX5: involved in host immune invasion; secretes mycobacteria specific PE and PPE protein families
58
Define exoproteins/exotoxins
Proteins secreted outside the bacterium and acts on host cell
59
Define effector proteins
Bacterial proteins secreted directly into host cell cytoplasm ; mediated by T3, 4, and 6 SS
60
T or F: Effector protein secretion is dependent on Sec pathway
F: independent
61
Are all Gram - secretion systems essential?
No - this makes them good targets for antibiotics bc they're not under pressure to mutate to survive the drug
62
Flagella is used in which types of bacterial motility?
Swarming and swimming
63
What is a peritrichous bacterium?
Bacterium with several flagella all over its body
64
T or F: The flagella is a simple structure
F
65
T or F: All genes involved in structure, regulation, and function of flagella are considered virulence factors
T
66
T or F: T5SS are thought to have evolved from flagellar systems
F: T3SS
67
Motile bacterial cells run or tumble more frequently when they are moving towards chemoattractants?
Run
68
T or F: flagella expression can be regulated
T. Also, flagella can switch from polar states to peritrichous states.
69
Primary lesions are associated with latent or active TB?
Latent
70
The greatest number of Mtb bacteria are found in which structure? Why?
Cavitary lesions; lots of oxygen
71
List the main factor that induces Mtb dormancy in latent TB:
Availability of oxygen
72
Granulomas contains lots/little oxygen? What is the word for lacking oxygen?
Very little; granulomas are hypoxic
73
Does Mtb require oxygen for growth?
Yes
74
What evidence showed that Mtb needs a period of adaption to adapt their metabolism to anaerobic conditions?
Putting Mtb into anaerobic chamber with rapid depletion of O2 caused Mtb death
75
What does the plateau on a bacterial growth chart mean for Mtb?
Oxygen and nutrients have been depleted
76
T or F: ATP synthesis levels go down to 0 in dormant, non-growing Mtb
F
77
T or F: MTb can regulate their expression of ATP synthase (the protein complex)
T
78
Are dormant Mtb still metabolically active?
Yes - they still produce ATP, but at low levels
79
What was being searched for in a whole cell screening assay?
Compounds with antimycobacterial activity
80
What does Bedaquilline do?
Inhibits ATP synthesis in Mycobacteria
81
How was Bedaquilline discovered?
Whole cell screening assay
82
Why is INH (drug) ineffective at killing latent Mtb?
It targets glycolic acid synthesis. Glycolic acid synthesis is the main component in cell wall, and synthesis is only active when bacteria are replicating (ie. not in dormant state)
83
Humans also have ATP synthase, so why is BDQ safe to use in humans? Wouldn't BDQ also target human ATP synthase?
There are AA differences in binding site between human and Mycobacterial ATP synthase. The binding site on human ATP synthase is not correct, therefore BDQ has low affinity for it.
84
Genes related to ________ are highly induced in Mtb inside macrophages
lipid catabolism
85
The upregulated genes for lipid catabolism are commonly found in which pathways?
Beta oxidation pathway
| Glyoxylate cycle
86
Which enzyme that is part of the glyoxylate cycle is highly upregulated in Mtb that have infected macrophages?
isocitrate lyase (ICL)
87
What is the name of the process that breaks down fatty acids?
Beta oxidation
88
What is the product of beta oxidation?
Acetyl-CoA
89
What is the ultimate result of beta oxidation?
ATP synthesis
90
List the basic steps in generating ATP from beta oxidation
1. FA and lipids are catabollized via beta oxidation to produce acetyl-coA
2. Acetyl-coA enters TCA cycle and is oxidized to CO2
3. NADH and ATP are generated during TCA cycle; NADH enters ETC to produce more ATP
91
What is important about the glyoxylate cycle that allows Mtb to use FA as their only source of E?
No carbons are wasted/oxidized to CO2, therefore C can be redirected to making biosynthetic precursors needed for making AA, etc.
92
Why is the glyoxylate cycle attractive as a drug candidate to target Mtc?
Glyoxylate cycle doesn't occur in humans
93
Which opern encodes a major cholesterol import system in Mtb?
Mce4
94
Will knocking out mce4 operon prevent Mtb from infecting people?
no
95
How does Mtb induce a foamy macrophage phenotype?
By altering the signalling and transcriptional regulation in macrophages
96
What is a foamy macrophage phenotype?
High in cholesterol, lipid vesicles
97
Name 2 key virulence traits
1. motility via flagella
| 2. metabolic shifts/adapting metabolism
98
What is the major source of energy and Carbon molecules for Mtb inside the host?
Fatty acids
99
What are the 3 essential components of a T1SS
1. ATP binding cassette (ABC) transporter protein
2. Membrane fusion protein
3. Outer membrane factor
100
Where is the ABC transporter protein located?
Inner membrane of Gram negatives
101
What does the membrane fusion protein do?
Bridges IM to OM
102
Give an example of a outer membrane factor
TolC
103
What does TolC do?
Multipurpose pore-forming protein
104
What does the T1SS do?
transports substrates in one step across inner and outer membrane of the Gram negative bacteria
105
What does the T2SS do?
Translocates proteins from periplasm to extracellular space (transport across outer membrane)
106
Which Gram - secretion system transports proteins across the outer membrane?
T2SS
107
Which Gram - secretion system transports proteins across both IM and OM?
T1SS
108
Which systems bring proteins into the periplasm?
General Sec or Tat pathways
109
T or F: T2SS transports unfolded proteins across the OM
F - transports folded proteins
110
What type of proteins are generally secreted by T2SS?
Enzymes
111
Give a classic example of a T2SS substrate
Cholera toxin (in V. cholerae)
112
The T2SS secretion apparatatus consists of 12-15 ___ proteins, the genes of which are organized into __ operon
core; 1
113
What are the 4 main components of a T2SS?
1. OM channel aka secretin
2. IM platform
3. Secretion ATPase
4. Pseudopilus
114
The T2SS transports exoproteins or effector proteins?
Exoproteins
115
Where are the genes for structural components of T3SS usually encoded on?
Plasmids and pathogenicity islands
116
T or F: T3SS can be horizontally acquired. Explain
T - genes coding for T3SS structural components are on plasmids, which can be horizontally transferred
117
T or F: evolutionarily distinct bacteria can have similar T3SS
T, due to horizontal transfer of T3SS genes
118
What are the 3 components of a T3SS?
1. base complex/basal body
2. needle component
3. translocon
119
T or F: the translocon is essential for secretion of exoproteins
F - but it is essential for passage of effector proteins through host membrane
120
Are T3SS always intact on the bacterial membrane?
No. Assembly is regulated - is only formed when bacteria comes in contact with host cell
121
T or F: folded proteins are secreted through needle of T3SS
F - the proteins are unfolded, and are also unfolded in bacterial cytoplasm
122
How do unfolded proteins (transported by T3SS) stay as indvl proteins and not aggregate together?
They are bound to chaperones
123
What do T3SS effector proteins do in the host cell?
Modulate host signalling pathways to promote bacterial survival and establish an infection
124
In T3SS, what causes bacteria to sense whether or not is has contacted a host cell
Tip complex
125
List 2 functions of the tip complex
1. senses whether bacteria is near host cell
| 2. regulates transport of effector proteins
126
What is the significance of eukaryotic-like domains in T3SS effector proteins?
They modulate host signalling pathways
127
Which Gram neg secretion system is structurally similar to conjugative DNA transfer apparatus?
T4SS
128
T5SS substrates have what type of domain?
beta-barrel domain
129
T6SS evolved from the structure of which microbe?
Contractile tail of bacteriophage
130
What are the 3 components of T6SS?
1. Outer tube - VipA/VipB
2. Inner tube - HcP
3. puncturing device - VgrG
131
What induces the T6SS to puncture target cell?
Contact with target cell
132
Name a Gram neg bacteria that has the T6SS
P. aeruginosa
133
What happened if Tse2 is injected into a cell lacking Tsi2?
The cell lacking Tsi2 dies bc Tsi2 protein confers immunity against Tse2 toxin
134
List the 4 domains of T5SS substrates. Which domain is not present in all substrates?
1. Translocator
2. Linker
3. Passenger
4. Protease - not present in all substrates
135
What is twitching powered by?
Type IV pili
136
What is swarming powered by?
rotating flagella
137
What is swimming powered by?
rotating flagella
138
If flagella is turning in CW motion, is the cell running or tumbling?
tumbling
139
If flagella is turning in CCW motion, is the cell running or tumbling?
running
140
What are the 3 substructures of the flagella?
1. Basal body
2. filament
3. hook
141
What is the basal body and what does it do?
A series of protein rings
| Anchors flagella to cell envelope
142
What is the filament and what does it do?
Polymerized flagellin subunit that acts as propeller
143
What is the hook and what does it do?
Connects basal body to filament and serves as a joint
144
Where does the energy for flagellar motion come from?
Proton gradient across the cytoplasmic membrane
145
Which secretion system is crucial for assembling the components of the flagella that lie outside the cytoplasmic membrane?
T3SS
146
CheY alters its binding affinity for ___
FLiM = flagellar motor switch protein
147
If CheY is not phosphorylated, is the cell running or tumbling?
CCW - running
148
If CheY is phosphorylated and bound to flagellar motor switch protein, is the cell running or tumbling?
CW - tumbling
149
What are the chemoreceptors in bacteria that regulate flagella motion called?
methyl-accepting chemotaxis proteins
150
What does the MCP do?
Binds chemoattractants and is methylated or demethylated by CheA or CheB, depending on the concentration of chemoattractants
151
What is the autokinase part of the MCP called?
CheA histidine kinase
152
When is the CheA histidine autokinase turned on?
When there is high concentration of repellent
153
What happens where there is a high concentration of chemorepellent?
CheA histidine autokinase turns to ON conformation
Autokinase phosphorylates MCP
P group transferred to CheY
CheY-P induces CW motion of rotor --> tumbles
154
What happens when there is a high concentration of chemoattractant?
CheA histidine autokinase is OFF
MCP is not phosphorylated
CheY is not phosphorylated
CheY induces CCW motion of rotor --> runs
155
If flagellins are antigenic, how do they escape detection by host's immune system?
Flagella expression is regulated so they're not expressed once inside host
156
Flagella biosynthesis is regulated by ___
phase variation/antigenic variation
157
FljA is a repressor of ___ in which bacteria?
FliC, Salmonella
158
List 2 properties of Chlamydiae
1. Gram negative
| 2. obligate intracellular pathogens
159
How many species in the Chlamydiaceae family are pathogenic to humans and animals?
11
160
What are the 2 major species of Chlamydia that infect humans?
C. penumoniae
| C. trachomatis
161
What are the 2 serovars of C. trachomatis?
1. ocular --> trachoma
| 2. genital --> STD
162
The 2 C. trachomatis serovars are _____-trophic pathogens that infect ocular and genital ___
epithelium, mucosa
163
Is trachoma a preventable infection?
Yes
164
Which regions of the world do ocular serovar C. trachomatis infections occur? Why?
Developing countries; lack of sanitation and clean running water
165
Are people infected with C. trachomatis gential serovar often symtpomatic or asymptomatic?
Asymptomatic
166
Are there antibiotics available for C. trachomatis infections?
Yes - azithromycin, doxycycline, eythromycin
167
T or F: Chlamydia genomes are large with manygenes encoding for metabolic enzymes
F - small and lacking many metabolic enzymes, which is why their obligate intracellular pathogens - reliant on host for metabolic requirements
168
Chlamydia codes for which secretion systems?
T5SS, T3SS, T2SS
169
The Chlamydia life cycle is ____
biphasic
170
What is the infectious form of Chlamydia called?
Elementary body
171
List 3 properties of the elementary body
extracellular
metabolically inert
infectious
172
What is the non-infectious form of Chlamydia called?
reticulate body
173
What are 3 properties of the reticulate body
non-infectious
metabolically active
intracellular
174
How does the reticulate body replicate?
Binary fission
175
What happens after the reticulate body replicates?
Differentiates back into elementary body and causes host cell lysis to exit cell
176
T or F: plasmids are not found in Chlamydia
F - plasmids are highly conserved
177
How do elementary bodies enter the host cell?
They secrete translocated actin recruiting protein (TARP), a T3SS effector.
TARP causes actin to be recruited to entry site by activating Rac-dependent signalling cascade
178
How is the cytoskeletal framework around the RB --> EB dismantled to allow the EB to exit cell?
EB secrete CPAF = chlamydial protease activity factor
179
How many ORF are on Chlamydial plasmids?
8
180
Which ORF on Chlamydial plasmids code for many putative virulence factors?
Pgp4
181
What is Pgp4?
A trancriptional regulator of genes that are candidate virulence factors
