The document summarizes a presentation on inducing a cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus through co-cultivation with the actinomycete Streptomyces rapamycinicus. Co-cultivation led to the activation of a previously silent polyketide synthase gene cluster and the production of novel prenylated polyketides, including Fumicyclines A. Deletion of the polyketide synthase gene confirmed its involvement in biosynthesis. While co-cultivation induced pathway expression, inhibition of histone acetyltransferase did not, suggesting the bacterium alters fungal epigenetic regulation. Understanding secondary metabolism in A. f
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The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
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This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
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Streptococcus pyogenes (group A β-hemolytic Streptococcus (GABHS)) is the common cause of acute bacterial pharyngitis also known as strep throat or sore throat. Recorded cases of GAS bacterial pharyngitis are 15-36% in children and 5-15% in adults. School aged children in seasons of spring and winter harbor the bacterium up to 20% whereas this rate is much lower in adults. On a global scale, over 616 million new cases of GAS pharyngitis occur every year. Not only is this bacterium responsible for hundred millions of pharyngitis cases but also approximately 10,000 to 15,000 cases of invasive GAS cases are reported annually in the United States, accounting for 10% to 13% mortality rate (http://www.cdc.gov/ncidod/dbmd/abcs/forinvasiveandnoninvasivediseaseincidence). Acute infections can lead to rheumatic fever and post-streptococcal glomerulonephritis (kidney inflammation), which distress children worldwide with disability and death, if antibiotic treatment fails or if the disease is left unattended. Rheumatic fever and rheumatic heart disease are known to be the leading causes of cardiovascular death during the first five decades of life in underdeveloped countries mainly concerning children. Tissues contributing to the GAS nosocomial infections are upper respiratory tract, skin, vaginal and anal area, although latter cases are rare.
GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
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This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
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GAS has several surface proteins and produces numerous extracellular products that facilitate permeation and successive evasion of the host’s immune system. Streptococcal pharyngitis results from the proliferation of GAS in the pharynx. Virulence associated factors enable S. pyogenes to attach to host tissues, elude the immune response, and spread by penetrating the host tissue layers followed by colonization.
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Bacteria Induced Cryptic Meroterpenoid Pathway in Pathogenic Aspergillus fumigatus
1. A Presentation On
Bacterium Induced Cryptic
meroterpenoid pathway in the pathogenic
Aspergillus fumigatus
By Debanjan Chatterjee (NIPERA1719NP03)
12/15/2018
2. Bacterium Induced Cryptic meroterpenoid pathway in the
pathogenic Aspergillus fumigatus
Publication- Chemical Society of Europe
Date of issue – 13/07/2013
Impact Factor- 3.3
22/15/2018
3. Part 1
• Introduction
• Method for the activation of cryptic gene
• Process for the analysis of Co-cultivation
Part 2
• Spectral Study
• Verification of Compound
• Proposed Pathway
Part 3
• Conclusion
• Limitations
• Future prospective
32/15/2018
4. Microorganisms produce a multitude of bioactive natural products, many of which
have found application in medicine or have served as leads for the development of
new drugs.
Bioinformatic analysis of the genome sequence of A. fumigatus revealed the
presence of 14 genes coding for polyketide synthases (PKSs) and 14 genes encoding
nonribosomal peptide synthetases (NRPSs).
Despite this huge biosynthetic potential, only a few of these encoded pathways
could be correlated to secondary metabolites e.g gliotoxinand pseurotin.
It appears that most biosynthetic pathway genes are silent or are only ex- pressed at
very low levels in the absence of specific triggers and/or conditions
42/15/2018
5. Method For the activation of cryptic gene
Co-culture of cultured A. fumigatus with the actinomycete
Streptomyces rapamycinicus.
• Co-cultivation of the model organism Aspergillus fumigatus with a soil-
dwelling actinomycete leads to the specific activation of a cryptic poly-ketide
gene cluster .
• Altering the fungal gene expression of bacterium specifically by inducing a
histone modification through the action of the main histone acetyltransferase
inhibitor (anacardic acid) complex Saga(Spt-Ada-Gcn5-acetyltransferase) .
52/15/2018
6. For inducing silent biosynthetic
pathway they cultured
A .fumigatus with the
actinomycete streptomyces
rapamycinicus .
Through electron microscopy
(scale bar 100μ ,20μ, 10μ, 1μ) , it
reveals that the bacterium &
fungus establishes an
intimate contact , in which
bacterium filaments are
attached to mycelia .
The researchers also done the
same observation
S.rapamycinicus & A.nidulans
(another species of A.
Fumigatus )
62/15/2018
7. For elucidating transcript level interaction they
done a full genome microarray
Impact of bacterial co-cultivation on fungal gene
expression
Representative view of A. fumigatus full genome
oligonucleotide microarray.
Wild-type and co-culture cDNA were labeled
with Cy5 and Cy3 dyes, respectively.
The zoomed picture shows increased expression
as indicated.
Architecture of a cryptic secondary metabolite gene
cluster in A. fumigatus and northern blot analysis.
Putative gene functions.
fccB : metallo-b- lactamase domain protein.
fccR : C6 transcription factor, putative gene .
fccC: FAD-dependent monooxygenase, putative gene.
fccA: polyketide synthase.
fccD: dimethylallyl tryptophan synthase.
fccE : NAD-dependent epimerase/dehydratase.
AFUA_7G00110 and AFUA_7G00190 are flanking genes
that are not coexpressed.
Northern blot – for analysing gene expression in mutant
Total RNA from A. fumigatus is (—) and A. fumigatus
coincubated with S. rapamycinicus (+) was analyzed.
72/15/2018
8. Metabolic profiling of the co-culture by
HPLC (High Performance Liquid
Chromatography)
a complex set of related compounds is found
with UV absorption maxima between 396 and
424 nm that are not produced in the axenic
fungal culture .
Although the separation of the major
metabolites proved highly challenging because
of tautomerism and instability, they did succeed
in obtaining a pure sample of a congener with
the molecular mass of 424 Da.
A molecular composition of C24H24O7 They
determined it by HRMS (High resolution Mass
spectrometry ) ESI (Electrospray ionization )
this is a soft ionization technique in Mass
Spectrometry .
LC-HRMS (ESI) analysis pointed to a number of aromatic polyketides possibly present in several
tautomeric forms, as indicated by the simultaneous occurrence of hardly separable peaks of identical
molecular composition . They also found intense yellow colour of the co-culture , which pointed to a
presence of aromatic chromophore .
82/15/2018
9. Spectral Study
1. fumicyclines A 2.fumicyclines B
The 1D NMR spectra showed the presence of
several aromatic carbon atoms , two keto
functions, three methylene carbon atoms,
and three methyl carbon atoms .
The DEPT135(Distortionless Enhancement
by Polarisation Transfer) measurements, 13 of
the 20 carbon signals were assigned as
having no attached protons, thus indicating a
highly substituted aromatic system.
H,H COSY (Correlated Spectroscopy) of H-
16 and H-17, together with diagnostic
HMBC((Heteronuclear Multiple Bond
Correlation) couplings , confirmed the
structure of the dimethyl allyl moiety.
A coupling constant of J = 2.0 Hz between H-
6 and H-8 revealed their meta relationship.
HMBC correlation of the hydroxy proton H-
7-OH with C-6, C-7, and C-8 established the
substitution pattern of the left-hand ring.
Long-range HMBC couplings of the
methylene protons H- 4 with C-2, C-5, C-10a,
and C-11, as well as of the methylene protons
H-11 with C-2 and C-13, confirmed the
tetracyclic skeleton of 1 .
Further HMBC correlation between the
methyl protons H-15 and C-13 and C-14 finally
elucidated the structure of Fumicyclines
92/15/2018
10. Verification of the compound
For verification the proposed relationship of the
induced PKS(poly ketide synthetase) gene cluster
and after the formation of the prenylated derivative,
they deleted the PKS gene (fccA) in the genome of
A. fumigatus.
Comparative metabolic profiling of wild-type and
mutant cultures clearly showed that the formation of
1. This compound unequivocally demonstrated the
involvement of the fcc locus in meroterpenoid
biosynthesis.
Compound 1 is the dehydration product of a
tricyclic congener—fumicycline B , which they
elucidate in 1DNMR process previously
Compound 2 is being characterized by
HRMS (ESI) and MS/MS analyses (previously
stated )
They isolate the compound having molecular
mass 424 Da & molecular formula C24H24O7
The researches stated that with a molecular
mass of 448 Da and a molecular formula of
C26H28O9, could also be detected by the
analysis of the HRMS (ESI)/MS , as data
suggested the presence of an additional
acetyl group (possibly at position 2) .
102/15/2018
11. Proposed Biosynthetic Pathway
This is a plausible biosynthetic
model starts with assembly of
the decaketide backbone by
fccA, that is polyketide
synthetase
The metallo-b-lactamase-type
thioesterase (encoded by
fccB) could participate in offloading of
the polyketide.
The polyphenol would then undergo
hydroxylation (by the FAD-dependent
monooxygenase) and prenylation (by
the dimethylall- yl tryptophan
synthase) yeilding 2 compound .
Compound 1 , that is the crypted
prenylated compound is the
results from a spontaneous cyclo-
condensation reaction,….. Is our
aimed product in this
meroterpenoid formation pathway .
112/15/2018
12. S. rapamycinicus is able to alter gene expression in A. fumigatus .
Because S. rapamycinicus interferes with the epigenetic regulation of
gene expression in the related fungus .
S. rapamycinicus were able to induce a previously silent polyketide
synthase pathway in the important human pathogenic fungus
A. fumigatus, and this led to the discovery of a previously unreported
prenylated polyketide.
Targeted deletion of the PKS encoding gene unequivocally established
the involvement of the cryptic gene locus in the biosynthesis of the
meroterpenoid.
Over-expression of pathway-specific regulatory gene uncovered a
second way to access the new natural product.
122/15/2018
13. In this study the researchers could not detect any transcription gene of
PKS(Poly Ketide synthease)through micro-array or Northern Blotting
after addition of the histone acetyl transferase inhibitor anacardic acid .
The increased knowledge of secondary metabolite of A. fumigatus might
contribute to the understanding of pathobiology of this human pathogen,
that accounts for a high number of serious infections in humans .
132/15/2018