This document provides a summary of the following subjects: intracellular pathogens, bacteria, human kinome, Salmonella thyphimurium, structure-activity relationship (SAR), molecular mechanisms of Salmonella, small GTPases, intracellular transport in Salmonella, Phosphoinositides, endosomes, AKT, R...
Experimental Cell Biology II: Host factors
Pathogens are becoming more resistant against Intracellular pathogens
antibiotics, in this summary we are going to look Facultative intracellular pathogens that live
at the functional role of host proteins in the inside the host cell are Mycobacterium
intracellular trafficking of bacteria. The reason tuberculosis and Salmonella typhimurium.
for this is to find out how bacteria can be killed Salmonella is becoming more resistant to
inside the host, without killing the host. antibiotics
The human kinome
This is the complete set of kinases
present in a certain cell or organism.
Kinases are enzymes that can phos-
phorylate (add phosphate groups to
other molecules). Kinases are
involved in removing phosphate
from ATP -> ADP and transfer it to
another molecule to initiate signal
transduction. These kinases can be used as drug
targets for kinase inhibitors.
Certain ways a bacterium can protect itself from
antibiotics are: Salmonella thyphimurium
• They pump the antibiotics out of the cell Salmonella thyphimurium causes an infection of
• They break the antibiotics down the gut epithelium and can survive inside the
• They can mutate the drug target so the
host cell. It is protected from the immune system,
antibiotics can’t bind to them.
but also from antibiotics because they can modify
Therefore the host can serve as a target to
eliminate the infection. For example, HIV can the behaviour of the host cell (transport/
enter the host cell by using the CCR5 or CXR4 transcription). But the processes that are
receptor. After entering, the virus can express regulated by Salmonella could be used as a drug
reverse transcriptase so that the virus can target.
replicate inside the cell. When the CCR5 or Salmonella uses its fimbriae to attach to the
CXCR4 receptors are blocked, the virus can not surface of the epithelial cells and engages in a
enter and therefore can’t replicate. type-3 secretion system (TTSS). First, a needle-
Thus, CCR5 blockers, maraviroc can be used as a shaped structure is formed. The tip of the
drug to eliminate the infection. Also, CXCR4 structure creates a pore on the cell surface after
blockers could be used, but they are highly toxic placing a protein. Afterwards, the needle-shaped
for the host itself. Some people don’t even have structure protrudes into the host cell using the
the CCR5 receptor, so the drug will not work. pose and injects its proteins inside. When the
(Peleg, A. Y. et al. N. Engl. J. Med. 362, 1804– proteins enter the cytosol they bind to their
1813 (2010).) binding partner, this can be a Rho-GTPase (Rad51
or CDC42) and activate them. After the
activation, actin polymerization takes place so
that the Salmonella bacteria can be engulfed and
taken up by the host cell. After this, the GTPase
will be inactivated by another Salmonella protein.
, Salmonella uses a second TTSS that pokes To find a compound that can inhibit a kinase,
through the membrane of an endosome and data mining in literature is used. This revealed a
injects 30 effector molecules inside the list of compounds that show inhibition of various
cytosol. Salmonella recruits Rab7 which kinases that lead to the inhibition of Salmonella
makes sure that the endosome acidifies and replication. H-89 showed a 50-fold reduction in
allows Salmonella to replicate. This way the number of bacteria in the host cell, but also
Salmonella stays safe from the immune Rp-cAMP showed this but was not present in the
system and antibiotics (because of the low list with siRNA.
pH). A marker that can be used to follow the Sadly, H-89 inhibits multiple kinases and is not
maturing of the early endosome into the late specific, which could lead to an off-target effect.
endosome is the protein LAMP that is But this also means that the inhibiting effect on
attached to the late endosome. Salmonella might be caused by a different kinase
which makes it not suitable as a drug.
siRNA is used to inhibit the production of
certain proteins. In the negative control, you
can see the replication of Salmonella in green
and the late endosome in red with LAMP as
the fluorescent marker. In the positive
control, the Rab7 protein production is Structure-activity relationship (SAR)
inhibited by siRNA, which prevents the This is an approach to finding relationships
acidification of the endosome and the between the chemical structure and biological
Salmonella dies. The only problem with this is activity of a compound. Hereby, the chemical
that Rab7 can’t be used as a drug target yet, derivatives of H-89 were created by
since there are no GTPase inhibitors available modifications such as adding methyl groups. The
currently. next step was to see if these changes affected the
Instead of trying to target Rab7, they tried to cells, where red would be a good inhibition and
target kinases. There were siRNAs developed white would be no inhibition. The list of kinases
of all human kinases and added to knock- was used to see if there was a match.
down models that were infected with The new compounds
Salmonella. A list was made of all kinases that were added to the
affect Salmonella replication. qPCR and model cells and the
western blotting were used to confirm if the replication of bac-
protein production was reduced by the siRNA. teria was again
The next step was to find a molecule that can observed. Some
block these kinases. Kuijl, C. et al. Intracellular modified compounds
bacterial growth is controlled by a kinase network showed significant
around PKB/AKT1. Nature 450, 725–30 (2007)) effects on the Salmonella replication and others
showed less. When testing these kinases they
want to inhibit, they expect to see a similar
pattern, which would confirm the right kinase
with an inhibiting effect.
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