Discovery of Antibiotics Flashcards
(81 cards)
1
Q
Give the 7 types of natural products?
A
Antibacterial Anti fungal Anticancer Antimalaria Anti parasitic Immunosuppressant Herbicide
2
Q
Give an example of a of an antibacterial, an anti fungal,
an anticancer, an antimalaira and an anti parasitic?
A
Penicillin G Aphotericin Doxorubicin Artemisisin Avermectin
3
Q
Give an example of a herbicide?
A
Bialaphos
4
Q
Give an example of an immunosuppressant?
A
Rapamycin
5
Q
Define natural products?
A
A small molecule produced by a living organism, a secondary or specialised metabolite produced by a microorganism (fungi, bacteria, arches) or plant.
6
Q
Who developed the magic bullet?
When and what was it?
A
Paul Ehrlic (1854-1914) Salvarsan, a selective toxicity treatment for syphilis.
7
Q
Who discovered penicillin?
A
Fleming, 1928. Originally termed ‘mould juice’.
8
Q
What did Waksman discover?
A
Streptomycin, 1943.
First effective treatment for TB.
Saved Bob Dole, a 1926 republican president nominee.
9
Q
Describe filamentous acitnomycetes?
A
Soil bacteria, produce geosmin (soil smell), produce a number of natural products: Streptomyces make approx. 60% of all clinically important antibiotics in use today
(Actinorhodin, Coelimycin).
Sporalting bacteria.
10
Q
Give the life cycle of sporulating bacteria and state at which point natural products are produced?
A
- Spore dispersal
- Spore germination
- Outgrowth of substrate feeding mycelium
- Formation of reproductive aerial hyphae
- Chromosome segregation and separation
- Spore formation.
Formation of reproductive aerial hyphae: antibiotics are produced to protect nutrients released from dead substrate mycelium (normally at this point where the natural products are produced to outcompete there neighbours).
11
Q
Describe the Waksman Platform?
A
A method of natural product discovery.
- Isolate actinomycete bacteria
- Antibacterial bioassay screen (assay ability to inhibit growth)
- Small scale fermentation
- Bioactivity-guided fractionation (and purification)
- Chemical characterisation.
12
Q
When was the golden era of antibiotic discovery?
A
1908-1962
13
Q
Why has there been decline in antibiotic discovery?
A
- Low profitability (acute vs. chronic conditions)
- Short window of use (e.g. development of resistance)
- R&D is time consuming & expensive and there are issues with rediscovery
- Regulatory issues
14
Q
What happened from 1962-2011?
A
Golden age of medicinal chemistry and innovation gap.
15
Q
What are we now entering?
A
The natural product renaissance: the new era of natural product discovery ad engineering.
16
Q
What was the longitude prize 2014?
A
£10 million for creating a cost-effective, accurate, rapid and easy-to- use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time.
17
Q
What does the natural product renaissance include?
A
Bioprospecting, genome mining, exploiting biosynthetic dark matter, natural products from the uncultured (Ichip and eDNA), combinational biosynthesis.
18
Q
What are the most common classes of natural products?
A
Polyketides, non-ribosomal peptides and lantipeptides.
19
Q
What is bioprospecting?
A
Looking for new natural products in under explored environmental niches.
20
Q
Complete: novel bugs=
A
novel drugs
21
Q
Give a few examples of novel niches?
A
Deserts, ants, deep sea
22
Q
What happens as the phylogenetic relatedness decreases?
A
Their chemistry diversity increases.
23
Q
Describe the findings when looking at arid soils?
A
100 soil samples were looked at using a 454 amplicon sequencing A- and KS-domians in order to estimate biosynthetic diversity.
- soils from the same geographic are were more similar
- soils from the same soil type were more similar
- arid (desert) soils were the most biochemically diverse.
24
Q
What are endophytes?
A
Bacteri (or fungi) that live inside pants and do not cause disease. They prime the plant immune system and often produce bioactive compounds that help prevent the plant from becoming infected (antibiosis).
25
Describe the marine environment as a novel niche?
Hugely under-explored. Several new drugs in development from newly discovered actinomytes and mycobacteria, the majority of new compounds were isolated from bacteria living in symbiosis with marine animals.
26
Give some examples of new compounds from the marine environment?
Abyssomicins: polycyclic antibiotics from a new marine acitonomyete genus named Verrucosispora
- inhibits folic acid biosynthesis, old target but new drug
Salinispora sp. (salinosphoramide- anti cancer drug).
27
Give the names of the types of bugs which provide novel niches?
Beewolf digger wasps,
Allomerus trap ants,
Tetraponera Penzigi ants,
Acromyrmex leafcutting ants
28
Discuss Beewolf digger wasps?
Beewovles prey on honeybees to provide food for their larvae housed in an underground chamber. They protect their larvae by covering the brood cell with endosymbiotic actinomycetes harboured in their antennae (the white stuff!).
29
Discuss allomerus trap ants?
Live on plants, never leave. Winged insects get trapped and get eaten. They strip of the leaves from the plants and cultivate a fungus (in relatively pure form) which they then use to build honeycomb structures.
30
Discuss tetraponera penzigi ants?
Plant ants that live on acacia trees in Africa. They live inside hollow thorned structure called domatia. They cultivate a fungus (in relatively pure form) for food and associate with antibiotic-producing. Found streptomyces formica, which led to the discovery that activity of chloramycin increases with the number of chlorine molecules it has.
31
Discuss acromyrmex leaf cutting ants?
They cut the leaves take them to the nest and de wax them and feed them to a fungus that produce what they feed off. The fungi and the ants rely on each other.
Antibiotic-producing actinomycetes protect the host and their food supply.
Newly emerged ants are inoculated and bloom with pseudonocardia within hours. Older ants lose the most of the white covering (to the breast bone) and acquire a more diverse micro biome. Only the older workers leave the next, because they are less prone to infection. Microbes prevent the food supply and ants from disease.
32
How can actinomycetes staring be identified?
```
Identifying actinomycete strains:
1. Culture dependent isolation
2. 16S PCR and sequencing
3. Antifungal bioassays
Genomics and chemistry together.
Bioactivity-guided fractionation
(2 molecules found, both known at the time but the genome sequence was used to find the pathway).
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33
What plays a huge role in modern day natural product discovery bioengineering?
Genome miming
34
What are the genome sequencing platforms?
454 sequencing: doesn’t happen anymore (can’t do it)
Illumina HiSeq (short read platforms)
PacBio: long-read (up to 20kb) makes assembling the genome much easier
Oxford nanopore (currently in data testing), can be done at your desk
35
When was the first bacterial genome sequenced?
1995
36
As of February 2016 how many genomes have now been sequenced?
59,886
37
What annotates complete or near complete bacteria genomes?
Rapid annotation using subsystem technology (RAST).
- high quality
- few errors
- fully automated
38
What does BLAST do?
Basic local alignment search tool, allows query sequences to be aligned against those already present in order to refine annotation.
39
What gives functional information regarding family relationships, biosynthetic pathways etc.?
Pfam
40
What makes it easier to identify genes encoding natural products?
Give an example.
They are clustered (co-located)
eg. Actinorhodin: all the genes are located in the same spot, they jump out the genome.
41
What proves a challenge with secondary metabolites?
Give an example.
The majority are not expressed in the lab
eg. Streptomyces coelicolor: geome projects surprised everyone, only four natural products had been observed in the lab but the genome showed ~ 30 biosyntteic pathways
Lab environment is very different to their natural environment.
42
Describe the status of Streptomyces coelicolor genome project now?
13 years later the community has assigned products for 17/27 pathways (still 10 unknown pathways). Genome sequence helps to predict the molecules in the biosynthetic pathway.
43
Describe the polyketide biosynthetic pathway?
First type 1 polyketide pathway to be discovered.
1. Acyl-coA from the primary metabolite pool
2. Acyl transferase domain which is specific to a specific acyl-coA as a building block 3. Leading to sequential condensation of acyl-coA
4. As the growing molecule gets passed down the assembly line
5. Followed by post PKS processing.
You can look at a type 1 polyketide pathway and predict what is going to happen as the functionality and substarte specficy is well understood. Difficult to know post PKS processing.
44
Describe the non-ribosomal peptide synthesise pathway?
There is an assembly logic to it. Large modular proteins. Similar to polyketide but the difference is amino acids over acyl-coA. Adenyltaion domain is what decides on the amino acis to be utilised
45
Describe lantipeptide synthesis pathway?
Ribosomally synthesised. A very small gene encoding the lantipeptide is translated and all the other little genes have specific little jobs. The detail is not needed, but using bioinformatics you can predict the chemical structure of the final molecule.
46
What is the benefit of looking at genes?
Enhancing in silica identification,
Bioengineering,
Cloning/heterologous over expression.
47
What is the benefits of looking at molecules?
De-replication by genomics,
Enhanced identification of compound in crude extract,
Significantly aids structure determination (help chemists).
48
Describe a HPLC trace?
A bunch of peaks representing all the molecules in the extract; complex, not as easy to identify.
49
What is the modified Waksman Platform?
1. Isolate actinomcete bacteria
2. Antibacterial bioassay screen
3. De-replication by genomics
3. Small scale fermentation
4. Bioactivity-guided fractionation (and purification)
5. Chemical characterisation 6. Genome guided chemical characterisation
50
What is the automated bioinformatic pipeline and how does it work?
antiSMASH
1. Upload sequence genome
2. Scan genome for biosynthetic genes
3. Extend search to neighbouring genes
4. Sequencing comparison to characterised gene clusters
5. Predict chemical structure when possible
51
Discuss amycolatopsis sp. example?
Bioactivity against Candida albicans.
antiSMASH showed 11 pathways and then the pathway that produced the antifungal compound was detected by rational determination and functional genomics, by making a single adjustement across the five pathways to home in on which cluster is involved to find the correct 1.
52
Discuss pseudonocardia sp. example?
A philamentous atenomyces antifungal.
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Identifying the nystatin P1 biosynthetic gene cluster:
454 genome (back in 2010), which gives 875 contigs (pieces of the genome) makes it very difficult to figure it out.
```
Mass spectrometry data showed that it is similar to a known molecule used in the clinic (Nystatin) and then this pathway was compared to the contigs to try to align into a pathway.
Tandem mas spectrometry was also used to convince themseleves of the differences (an extra sugar (mannose) on their molecule, a glycosidase transferase).
53
Discuss the streptomycin albus S4 example?
Identification of the antimycin biosynthetic pathway:
(Example of taking something where you have the structure but nobody knows the biosynthetic pathway.)
454 genome looked at and a threonine was found. This suggests a non-ribosomal peptide synthetase assembly logic.
Found to be a hybrid non-ribosomal peptide synthetase/ polyketide synthase pathway.
54
What are the culture-dependent approaches of finding new natural products?
Plate count,
Phosphate quagmire,
Iron throne,
Isolation chip
55
Describe the great plate count anomaly?
Looking at an environmental sample under a microscope you see a lot more things in comparison to culturing them on a plate. This is because only ~ 1/100 microbes grow in culture due to the standard practice of autoclaving agar and phosphate buffer together to make solid growth media. It is thought to generates byproducts that kill many cells and prevent us from cultivating many of the "unculturable" microbes.
56
How do you solve the plate count anomaly?
The phosphate quagmire: the simple act of autoclaving the agar and the phosphate separately (Ps medium) allows the cultivation of many more bacteria from soil, sediment, and river water samples.
57
What could also contribute to the reduced cultivation?
Many bacteria produce their own siderophores and are dependent on other molecules.
Siderophores in the culture medium allow for a greater cultivation of microbes than without.
58
What is a benefit of the Ichip?
It mimics the natural environment
59
Describe the Ichip?
For high-throughput in situ cultivation of uncultivable microbial species. Works by having a soil sample diluted in a low agar concentration and you would dip the chip into the sample solution and one microbe would ideally fit in each well of the chip. You would sandwich the chip in a clamp-like devise with a semi-permeable membrane either side and place it back in the natural environment.
60
Give an example of a natural product discovered using Ichip and explain the process?
Teixobatin from a newly cultured beta-proteobacterium.
Use Ichip to gain a soil sample from a grassy field in Maine which had extracts from 10000 isolates.
Found to bind to lipid II (cell wall precursor) and lipid III (teichoic and precursor) inhibiting them.
61
What are culture-independent approaches of finding new natural products?
Metagenomics: coined by Jo Handelsman in 1998 (Handelsman et al. 1998. Chem Biol 5:R245-249)
DNA is extracted directly from the environment (eDNA) and is sequenced and/or cloned into model hosts for detailed analysis.
62
What are the challenges with sequence-based metagenomics?
The assembly of shotgun sequenced eDNA is challenging. Particular true for PK and NRPS (erythromycin) pathway due to their modular architecture- difficult to assemble across modular biosynthetic pathways.
63
Describe the successes of sequence-based metagenomics?
Looking at lichens: metagenomics revealed nosperin may be produced in lichen symbiosis.
Looking at tunicate (coral reef): petellazole is involved in chemical defence of the tunicate.
Looking at sponge: already new that any natural products could be isolated from this sponge, fluorescence-assisted cell sorting (FACs) of sponge extrancts and DNA sequencing resulted in the identification of new candidate genus ‘Entotheonella’ that is talented produces of natural product.
64
Describe the successes of sequence-tag metagenomics?
From a soil sample, there was a nice correlation between phylogenetic relatedness of KS domains (for PKS pathways) and A domains (for NRPS pathways) and the molecules their respective pathways encode.
65
What is an environmental DNA library?
A DNA library made from an environmental sample.
66
How do you make an eDNA library?
1. DNA isolation
2. Clone
3. Transform expression host and screen
Limitations: to screen 100, 000 you need over 1000 96-well plates, lot sof time and lots of space
67
How do you overcome the limitations of eDNA libraries?
Use sequence driven enrichment which allows you to place more than one sample in each well
or
Functional enrichment of eDNA libraries: PPtase screening.
68
What are the underlying principles of PPtase screening?
PPtases are rather promiscuous.
Some (not all) biosynthetic pathways have their own discrete PPtase.
Siderophores chelate iron and are required for growth under low iron conditions.
Siderophores biosynthetic proteins require phosphopantetheine groups in order to be functional.
If you knowck out the PPtase, only those harbouring a PPtase will permit growth on low iron growth media.
69
How is secondary metabolism regulated?
Complex.
Global activators: activate proteins that act on multiple different genes (very large replicon).
Global repressors: repress the expression of multiple genes in the genome.
Cascade from global control to a cluster-situated regulatory protein (1 or more). Ultimately everything passes through the cluster regulated proteins, therefore a big emphasis on trying to turn them on.
Still being figured out
70
What two typed of methods are there of waking up sleeping genes?
Global and targeted.
71
What are the global methods of waking up sleeping genes?
Chemical genetics,
co-cultivation,
ribosome engineering.
72
What are the targeted methods of waking up sleeping genes?
Delete repressors,
activate activators,
promoter exchange.
73
Discuss chemical genetics?
Not new but a lot of buzz about it. The general idea is to enlist a certain reposes through chemical in a culture media. This response could be; signal availability/transport/modification, global/specific regulation, antibiotic biosynthesis.
74
Give an example of how chemical genetics have been used to wake up sleeping genes?
N-acetyl glucosamine addition which switches on the production of certain genes- a response.
ARC2 found to be a triclosan like product which enhances the yield of actinorhodin (triclosan inhibits fatty acid biosynthesis).
Use of epigenetic modifiers:
Using the histone complex superstructure which is controlled by histone acetyltransferases (acteylases and deacteylases). Histone deacetylase inhibitors (HDAC)- this permentalty allows the DNA to be unwound from the protein, opening up and presenting the promoters
Eg. Sodium butyrate.
75
Discuss co-cultivation?
Microbes don’t live in isolation, every pathway has evolved as a purpose, useful for the bacteria. They are expressed in line with their purpose. Switching on pathways can be done by growing with the natural bacterial which would live side-by-side.Not used very often but very powerful and easy to do.
76
Discuss ribosome engineering?
You can take a strain and grow it on sub-lethal doses of antibiotics.
These mutations can alter the transcription/translation profile of some strains (working via the ‘alarmone’ ppGpp) to switch-on gene clusters.
eg. rpsL (ribosomal protein S12) is resistant to streptomycin
77
Discuss deletion of repressors?
Delete the repressor genes in an effort to activate. There are often multiple so multiple deletions and activation of activators may be required- laborious.
78
Discuss activation of activators?
Clone the regulatory proteins and put it in the organism to prompt activation.
79
Discuss promoters exchange?
Engineer a biotic cassette with a strong promoter and recombineer it into the genome upstream of the target to switch on.
80
What methods are there to understand and manipulate natural product biosynthesis?
Mutagenesis and modification of actinomycetes,
Targeted cloning of biosynthetic pathways,
Assembly of biosynthetic pathways,
Bioengineering
81
Why is genetic manipulation of acitomycetes difficult?
No heat-shock transformation,
No electroporation,
The way exogenous DNA is mobilised to actinomycetes is via conjugal transfer from E. coli,
Mutagenesis relies on having a genomic DNA library (e.g. a cosmid library),
Modifications are made to the cosmid in E. coli using recombineering
