Download Mechanisms of the Calcium Oscillation Inducing Toxins a

Mechanisms of the Calcium Oscillation Inducing Toxins -Haemolysin and
Cytolysin A
Keira Melican
Humans and bacteria share a fascinating relationship, bacteria acting as both our friends and
foes. Beneficial bacteria that live within our body help keep our system clean and healthy.
Unfortunately some bacteria are capable of excreting potent toxins (poisonous proteins).
Invading environmental bacteria as well as some of the bacteria that normally reside in our
bodies produce toxins that may have many harmful effects on the body’s cells.
This project concentrated on two toxins excreted by a bacterium called Escherichia coli,
haemolysin (HlyA) and cytolysin A (ClyA). HlyA is excreted by many bacteria that cause
urinary tract infections. It forms pores in the membranes of our cells causing them to rupture
and die. Recently, another action of this toxin has been described. In lower concentrations it
affects the internal calcium concentration in cells with which it comes into contact. The
periodic increases and decreases in calcium concentration caused by the toxin, termed
oscillations, in turn trigger the cell to produce more proteins that stimulate inflammation in
the area, recruiting the body’s defence cells.
My study focussed on the way bacteria excrete this toxin as they enter and invade the
kidney. It was possible to identify areas in a rat kidney where bacteria gathered together,
causing infection. Using a technique called laser capture microdissection (LCM), areas of
obvious bacterial infection were cut out and removed. Analysis of RNA extracted from these
areas provided evidence of bacterial infection, but as yet no evidence for the excretion HlyA.
These early experiments involved late stages of infections, so maybe the HlyA toxin is only
excreted at the beginning of a bacterial invasion and is not needed once the bacteria are inside
the tissues.
The ClyA toxin is excreted from bacteria in little ‘bubbles’ made up of the bacteria’s outer
surface, called outer membrane vesicles (OMVs). When human cells come into contact with
OMVs carrying ClyA they respond in one of two ways. If the concentration of toxin is high,
the cells burst and die, if the concentration is low calcium oscillations are observed similar to
those seen in response to HlyA. In this study I investigated whether OMVs act differently
depending on modifications made to the outermost layer of E. coli, the lipopolysaccharide (a
specific combination of sugar and fat molecules). If the body senses LPS interacting with its
cells it triggers its defence mechanisms, as it knows bacteria are present. LPS is a rather large
structure with many sections. Here different sections of the LPS were removed to see whether
a shorter or longer LPS affected the action of the OMVs carrying the ClyA toxin. A machine
that measures weight using sound frequency allows for very small measurements to be carried
out, seeing how vesicles behave when they interact. So far these experiments have shown that
the presence of the ClyA toxin within the OMVs indeed alters the behaviour of the vesicles. A
shorter LPS structure appeared to increase production of both vesicles and toxin, without
affecting the activity of the toxin. The ClyA protein appeared to bind strongly to cholesterol
in our experiments. Such binding would prevent toxin activity. There are areas of the cells
membrane, termed lipid rafts, that have very high concentrations of cholesterol. Future
experiments will address the possibility that vesicles bind to these specific cell areas.
Degree project in biology, University of Uppsala, autumn/spring 2004/2005
Examensarbete i biologi, 20 p
Biology Education Centre, Uppsala University and Microbiology Tumour Biology Centre, Karolinska Institutet,
Nobels väg 16, Solna
Supervisor: Agneta Richter-Dahlfors