Streptococcus pyogenes is a pathogenic bacterium that causes significant infections in humans. It interacts with the host plasminogen system by expressing plasminogen receptors on its surface. When plasminogen binds to these receptors, it is activated into plasmin, a protease that assists in the spread of the bacteria. The objective is to characterize the interaction between the streptococcal surface dehydrogenase (SDH) enzyme produced by S. pyogenes and the host urokinase-type plasminogen activator receptor (uPAR). Recombinant SDH was purified and analyzed using size-exclusion chromatography and SDS-PAGE. Assays found no direct interaction between SDH and uPAR, suggesting SDH does not mediate

1. SharaCarolinaMontoyaBarreneche
BIOLOGÍA MOLECULAR
Tercer semestre1-2019

2. INTRODUCTION
Streptococcus pyogenes (GAS)
The streptococcus family includes many species
of Gram-positive cocos, being some of them
important for human illnesses. Streptococcus
pyogenes which means pus producer; is a
facultative anaerobic microorganisms that have a
fermentative metabolism; they lack catalase
which differentiates them from Staphylococcus
and they can be typically found in pairs or short
chains.
Streptococcus with clinical value are classified
according to the polysaccharide antigens in their
wall, in this case, the pyogenes is identified as
an A group streptococcus(GAS).
The human being is the only natural reservoir,
and the microorganism causes significant
infections in the throat, the genital tract, rectum,
skin, and underlying soft tissue.
Plasminogen
Plasmin is a broad spectrum serine protease that
degrades fibrin, extracellular matrices, and
connective tissue.
Is a secreted blood zymogen that is activated by
proteolysis and converted to plasmin and
angiostatin. Plasmin dissolves fibrin in blood
clots and is an important protease in many other
cellular processes while angiostatin inhibits
angiogenesis

3. RELATION BETWEEN PLASMINOGEN AND
STREPTOCOCCUS
A large number of pathogens express plasminogen receptors which immobilize plasminogen on the
bacterial surface. Surface-bound plasminogen is then activated by plasminogen activators to
plasmin through limited proteolysis thus triggering the development of a proteolytic surface on the
bacteria and eventually assisting the spread of it.
Plasminogen receptors and activators are expressed by group A, C, and G streptococci. GAS
interact with plasminogen either by direct binding with specific surface proteins or indirectly by
sequential binding of fibrinogen and plasminogen.
The host hemostatic system plays an important role in systemic infection. The interplay between
hemostatic processes such as coagulation and fibrinolysis and the inflammatory response constitutes
essential components of host defense and bacterial invasion.
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4. OBJECTIVE
.
“We prepared recombinant SDH tetramer. After
purification and crystallization, we determined its crystal structure,
and characterized its interaction with the extracellular domain of
uPAR (referred as soluble uPAR, suPAR) through three canonical
assays.”

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MATERIALS AND METHODS:
The separation of macromolecules in an electric field is called electrophoresis, uses a
discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate
(SDS) to denature the proteins. The method is called sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and is one of the most widely used
laboratory methods to separate biological macromolecules such as proteins and nucleic
acids, according to their electrophoresis mobility which is a function of the length,
conformation and charge of the molecule.
SDS is an anionic detergent, meaning that when dissolved its molecules have a net
negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in
proportion to its relative molecuar mass. The negative charges on SDS destroy most of
the complex structure of proteins, and are strongly attracted toward an anode (positively-
charged electrode) in an electric field.

6. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because
the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation
of proteins is dependent almost entirely on the differences in relative molecular mass of
polypeptides. In a gel of uniform density the relative migration distance of a protein (Rf) is
negatively proportional to the log of its mass. If proteins of known mass are run simultaneously
with the unknowns, the relationship between Rf and mass can be plotted, and the masses of
unknown proteins estimated.
Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the
relative abundance of major proteins in a sample, and to determine the distribution of proteins
among fractions.
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7. Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice,
the process often involves combining the DNA of different organisms. The process depends on the
ability to cut and re-join DNA molecules at points which are identified by specific sequences of
nucleotide bases called restriction sites. DNA fragments are cut out of their normal position in the
chromosome using restriction enzymes (also called restriction endonucleases) and then inserted into
other chromosomes or DNA molecules using enzymes called ligases to produce new genetic
combinations that are of value to science, medicine, agriculture, and industry.
This describes the process of copying fragments of DNA which can then be used for many different
purposes, such as creating GM crops, or finding a cure for disease. There are two types of gene
cloning: in vivo, which involves the use of restriction enzymes and ligases using vectors and
cloning the fragments into host cells (as can be seen in the image above). The other type is in vitro
which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA
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8. Recombinant DNA technology has made it possible to isolate one gene or any other segment of
DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in
highly specific ways, and reinsert the modified sequence into a living organism.
In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule
and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The
small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are
plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells; They are
small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra
DNA that is spliced into them.
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9. RESULTS
Figure 1.
According to previous studies the recombinant
Streptococcal surface dehydrogenase (SDH) was
produced in Escherichia coli BL21 (DE3) cells.
After purification through affinity and size-
exclusion chromatography, the protein achieved
to a high purity (>99%).
SDH was fractioned at12.5 ml on the calibrated
size-exclusion column, corresponding to
approximately 150 kDa, while its bands located
near 37 kDa on SDS-PAGE . Based on these
results, purified SDH was obtained as tetramers,
which was consistent with other reports
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Size-exclusion chromatography and SDS-PAGE analyses of recombinant SDH.
Lanes 1 and 2: The target protein under reducing condition with b-mercaptoethanol; Lane M:
Protein marker.

10. Figure 4.
High affinities yield stable complexes in vitro
and can be obtained by gel filtration
chromatography. The results showed that there
were no complexes formed in the mixture as
each protein was separately eluted.
suPAR was not observed on native-PAGE
probably due to its glycosylation. In the mixture,
there was no band of the complex compared to
each single protein.
Moreover, although suPAR was associated with
ATF-coupled column, SDH wasn't eluted with
suPAR in the meantime, suggesting that there
was no strong interaction between them
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Characterization of the interaction between SDH and suPAR.
(A): Gel filtration chromatography analysis.There were no complex peaks in the mixture. The
result of SDS-PAGE was labeled and not shown.
(B): Native-PAGE analysis.. There was no complex band compared to each single protein.
(C): In vitro pull-down analysis. There was no SDH eluted with suPAR as shown on SDS-
PAGE.

11. DISCUSION
AUTHOR CONCEPT YES OR NO
F. Blasi, P. Carmeliet
“uPAR, also known as urokinase receptor or
CD87, is a multidomain (DI-DIII)
glycoprotein and anchored on cell membranes
via glycosylphosphotidylinositol (GPI).
It is an essential member in the plasminogen
activation system”
V. Pancholi, G.S. Chhatwal
“The multifunctional SDH actually
contributes to GAS infection and survival in
host cells.”
H. Jin, Y.P. Song, G. Boel, J.
Kochar, V. Pancholi
“SDH interacted with host urokinase-type
plasminogen activator receptor (uPAR),
which mediated GAS infection into human
pharyngeal cells.”
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12. CONCLUSIONS:
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• Bearing in mind that the Streptococcus pyogenes affects many tissues in the human
body, finding their weakness involves finding the solution or treatment for the illnesses
that the bacteria causes.
• SDH is a very important enzyme in the metabolism of this kind of bacteria, and that´s
why is considered an important target in this investigation, unfortunately, is necessary to
deepen the studies even more to find the real importance in this cell, how it´s involve in
the infections and how to fight it.

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