2013 Annual Report - Jesus College - University of Cambridge

BACTERIOLOGY I Jesus College Annual Report 2013 31
caused within the intestine, resulting in
loss of water and diarrhoea, the death of
Salmonella results in the release of
endotoxins, which in turn causes the
associated enteritis.
I primarily study the methods Salmonella
Typhimurium utilises to invades our cells, in
particular looking at how it is able to
manipulate normal cellular processes to its
own benefit. To induce invasion
Salmonella interferes with the protein
skeleton (actin cytoskeleton), which is
responsible for maintaining the integrity
of the cell, as well as being involved in
movement and induced uptake. Actin is
regulated by a series of proteins, and
Salmonella is able to use its effectors to
either activate or directly mimic those
found in the host. The means by which
macropinocytosis is induced involves a
cascade of protein activation eventually
leading to generation of actin rich ruffles
(lamellipodia) that are required to drive the
whole process.
Aside from washing my hands around
forty times a day my time is spent
employing a number of methodologies to
elucidate the precise mechanisms by which
invasion occurs. Through the use of
biochemical techniques I attempt to
recreate the platforms in which the actin
polymerisation takes place. By purifying
combinations of bacterial effectors, and
host proteins potentially involved, I try to
artificially generate actin and discover
precisely which components are required.
This involves the use of beads that mimic
the plasma membrane of cells, to which I
anchor the proteins of interest, these are
then incubated with a rich extract (made
from pig brains). These beads are washed,
the proteins recruited are identified, and
the extent to which actin is generated is
assessed. By using a combination of
chemical inhibitors and mutated proteins I
am able to pinpoint which proteins interact
with each other, and how Salmonella may
be able to interfere in these processes.
Once Proteins of interest have been
identified I move to more cell biological
approaches, using microscopy I visualise
the localisation of fluorescently tagged
host proteins in cells. Using tagged
Salmonella I am also able to assess
whether or not there is an interaction
between the bacteria and the protein of
interest. Often, proteins directly involved
in the process of uptake are recruited to the
invasion site, and are also sometimes
found on the surface of the
macropinosomes that the Salmonella
creates. By knocking out, mutating, or
enhancing the activity of proteins that
appear to be important, it possible to
assess their contribution to invasion, by
quantifying the efficiency of Salmonella
uptake.
Although not directly attempting to
discover a means to prevent Salmonella
infection, the work I carry out endeavours
to identify key components involved in the
invasion process. As many bacteria are
becoming resistant to modern antibiotics,
pinpointing new potential drug targets is
essential to prevent serious outbreaks of
disease in the future.