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Bacterial Pathogenicity > Streptococcus pneumoniae and cell wall > Flashcards

Streptococcus pneumoniae and cell wall Flashcards

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1
Q

Pneumo pathogenesis

A

gram-positive bacteria (monoderm)

prolayelipsoid - oval shape; pointed at one end

point allows it to always land on their side
- land on point and fall over
- virulence factor are all in mid section

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2
Q

Diseases caused by pneumo

A

Pneumonia
Meningitis
Sepsis
Otitis Media (middle ear infection)

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3
Q

Pneumonia symptoms

A

Shaking chill
Fever
Cough
Discomfort

Can be very subtle but onset of severe is abrupt

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4
Q

Pneumo virulence

A

Causes 1.2 million deaths worlwide a year
- under reported due to people with underlying illnesses e.g. cancer

Mainly immunocompromised groups i.e. >65 or <5

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5
Q

Major pneumo virulence factors

A

Polysaccharide capsule

Pneumolysin
- really large
- damages the heart

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6
Q

Pneumo reservoir/transmission

A

Direct contact with respiratory secretions

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7
Q

Pneumo treatment/prevention

A

Antibiotics
- penicillin or other beta-lactams

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8
Q

Pneumo niche

A

Nasopharynx
- top of throat, back of nose

60% squamous epithelium
40% ciliated columnar cells

Lymphocytes buried in submucosa along with seromucous glands - make mucus

Lymphocytes constantly patrolling

NOT A STABLE NICHE

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9
Q

Pneumo food source

A

Mucus and glycoproteins on epithelial cell surface

40-50% genome is dedicated to metabolism of sugar

Has N-poor diet so retained genes required to make all basic amino acids from scratch

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10
Q

Brain: Meningitis

A

pneumo can leave nasopharynx through hole at top of spine that allows spinal cord into brain

Only soft tissue between then - gets into brain meninges

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11
Q

Middle ear: Otitis Media

A

Pneumo travels down Eustachian tubes in middle ear
These tubes control pressure in ear by periodically openining and shutting
Open into nasopharynx

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12
Q

Lungs: Pneumonia

A

Contantly inhaling air that passes over nasopharynx

If droplets are released off of surface they can be inhaled into lungs

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13
Q

Blood: Spesis

A
  1. Taken into lungs and cause local infection
    Lungs covered in blood vessels so gives direct access to blood
  2. Nasopharynx is covered in very fine set of capillaries
    Can get direct access if they start to invade
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14
Q

Why does pneumo become invasive?

A

Due to stress of being in part of the body it’s not meant to be in
- not designed for it

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15
Q

Pneumo carriage

A

Carriage lasts only weeks/months

Does not have a stable niche so needs to be passed on frequently

Falls somewhere between commensal and parasite

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16
Q

Challenges of building cell wall

A

Have to build from inside out
While mainting shape
Without compromising integrity
At maximum possible rate

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17
Q

Role of cell wall

A

Bacteria have high internal osmotic pressure due to concetrated contents

Pressure is balanced by tensile forces provided by PG cell wall

If cell wall compromised, cell explodes

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18
Q

PG cell wall composition

A

Repeating dissacharide units crosslinked together by short peptides

Made from precursor: Lipid II

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19
Q

Lipid II

A

Lipid-linked precursor that contains dissacharide and pentapeptide stem - ending in D-ala-D-ala

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20
Q

PG formation

A

Transglycosylase activity (TG)
- polymerase lipid II into glycan strands

Transpeptidase activity (TP)
- cross-links strands
- removes D-ala at end of penta-peptide strand

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21
Q

Penicillin mode of action of PG

A

Irreversible competitive inhibition of transpeptidase activity required to build cell wall
- Is a chemical mimic of D-ala leaving group
- capable of binding TP domains

Forms intermediate cross-link via covalent bond
- lock protein in inactive state

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22
Q

PBPs

A

Identified biochemically as they are all covalently modified by penicillin

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23
Q

Bacterial cell growth

A

Grow through repeated cycles of cell elongation and division

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24
Q

Problem with genetic approaches

Study These Flashcards
A

Cells have to survive in order for them to be studied further
If cells die there is no way of identifying gene

AND
Genetic redundancy

25
Conditional phenotype
Permissive and Non-permissive conditions to get around problem of cells having to survive to be studied Acheived by: Changing composition of media (add salt or Mg2+) Temperature + or - antibiotics
26
Forward genetic approach for identifying divisome components
Use conditional phenotypes E.coli random mutagenesis using nitrosoguanidine Select for cells that don't divide in non-permissive condition Use phage complementation
27
Forward genetic approach: Phage complementation
Use lambda phage containing random chunks of WT E.coli genome Create library of phage, each with different genome section Introduced phage to E.coli mutants; grow in non-permissive condition Those that survive have been complemented Compare complementing phage to identify gene
28
Identification of components of divisome
ALL identified via forward genetic approach EXCEPT ZapA and B - very small protein so hard to get mutants through random mutagenesis
29
Ordered assembly of divisome components
Combination of GFP-tags to Fts proteins show ordered assembly 1. Formation of FtsZ ring 2. FtsZ recruits all other cell division proteins to division site FtsQLB > FtsK > PBP enzymes + FtsW > FtsEX + FtsN
30
FtsZ
Tubulin homologue Form Z-ring around circumference of cell - provides protein platform for other components
31
FtsK
involved in DNA segregation
32
PBP enzymes + FtsW
start PG synthesis
33
FtsEX + FtsN
involved in hydrolysis need to cut PG to insert new material
34
FtsZ as drug target
Challenging as it is a tubulin homologue Tubulin is very important for function within eukaryotic cells Compounds targeting FtsZ can be used to augment existing anti-microbial compounds - used as enhancer for antibiotic action - along side penicillin
35
Reverse Genetic Approach for identifying components of cell elongation
MreB identification using inducible promoters for permissive and non-permissive conditions Directed mutagenesis to KO mreB in B. subtilis Put mreB on inducible promoter controlled by Xylose + Xylose = MreB ON - Xylose = MreB OFF
36
Mre
Murein formation gene cluster E MreB, MreC and MreD All essential genes in B. subtilis
37
MreB function
Identified using tagged MreB proteins Get localised small patches Patches navigate around circumference of bacterial cell wall Move to sites of high cell curvature and synthesis new cell wall
38
MreB as drug target
A22 used to target MreB Lose localisation of MreB Cells start to swell and eventually lyse Challenge is that MreB is actin homologue - actin is essential for eukaryotic cell structure and stability
39
Developments in genetic sequencing
Improved sequencing gives access to massive amounts of genome and phylogenetic data Can be a route to identifying novel drugs
40
Identifying antimicrobial compounds from Actinomycetes family of bacteria
Used genetic sequencing and ignored ones that have gene for vancomycin Identified two novel antibiotics: Complestatin Corbomycin Both target cell wall hydrolases
41
Genetic redundancy
Bacteria have two or more pathways towards same goal More important the process is, the more likely it is to have multiple different pathways Have to deliberately prevent one of the pathways to make all genes in other pathways 'essential'
42
Synthetic lethality
Combination of genetic mutations that are lethal to cells In E.coli: PBP1a and PBP1b are a synthetic lethal pair - remove one and the other becomes essential KO PBP1b, now PBP1a is essential but so is everything upstream of it that is required for its function
43
Synthetic lethal screen 1
Used parental strain of E.coli with chromosomal deletions of genes lacZYA and ponB (PBP1b) and an unstable plasmid Plasmid has defective ori (without selection this plasmid will get progressively lost from population), genes for lacZYA, AmpR and ponB - expression controlled by Plac promoter
44
Synthetic lethal screen 2
E.coli strain undergoes transposon mutagenesis Library mutants grown on media containing LB, X-gal and IPTG (Plac inducer) Three different types of colonies formed: white (LacZ -) blue-sectored blue (LacZ +)
45
Significance of LacZ results in synthetic lethal screen
Solid blue colonies indicate unstable plasmid has been taken up Suggests transposon mutagenesis interrupted a gene required for PBP1a activity Took up plasmid because plasmid PBP1b now essential
46
Tested blue colonies from synthetic lethal screen
Blue colonies tested in prescence and absence of IPTG 5 IPTG dependent colonies 3/5 had insertions in ponA (PBP1a) 2/5 had insertions in yram (LpoA)
47
Converse PBP synthetic lethal screen
Identified 7 IPTG-dependent mutants with insertions within ycfM (LpoB)
48
LpoA and LpoB
Found in outermembrane (gram -ve bacteria) Activate PBP1a or 1b by reaching through PG layer
49
LpoA and LpoB as drug target
Easier to reach as drugs only have to travel through one membrane BUT no outermembrane in gram +ve bacteria
50
What factors regulate PBPs in monoderms?
CozE - PBP1a MacP - PBP2a
51
What PBPs are a synthetic lethal pair in pneumo?
PBP1a and PBP2a
52
Next-gen sequencing
Transposon sequencing - sequence a load of transposons to work out what insertion profiles look like
53
Benefits of next-generation sequencing
Has removed the challenge of needing cells to survive - can detect 'absence' of a cell signature Can capture millions of transposon insertion events in one dataset
54
Next-gen sequencing for identifying PBP regulators in pneumo
~200,000 Transposons applied to pneumo mutant lacking pbp1a - grow cells - next-generation sequencing - map reads to genome Any genes that kill cell when KO are synthetically lethal to PBP1a
55
CozE
Regulator of PBP1a Keeps PBP1a in regulated state at cell division site
56
PBP1a toxicity in pneumo
KO both pbp1a and cozE Add plasmid containing pbp1a gene in - pbp1a expression controlled by inducible zinc promoter (Pzn) + Zn = WT restored Over time cells swell and eventually lyse Without CozE, PBP1a carried out inappropriate cell wall synthesis
57
CozE as drug target
Works in lab well to induce cell death BUT if combined with penicillin the actions would cancel each other out
58
MacP
Synthetically lethal with PBP1a Responsible for activation of PBP2a If MacP 'OFF' = PBP2a OFF - cells shrink and die - cells keep dividing but get smaller and smaller until they lyse
59
MacP as drug target
Easier to drug than CozE BUT not deadly enough Slow death may allow them time to respond and induce mutations

Bacterial Pathogenicity (4 decks)

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