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ExoU: A Poor Clinical Outcome
Kettle Moraine SMART Team: Samantha Cinnick, Maggie Davies, Alexandra
Greene, Anna Henckel, Grant Hoppel, Sridevi Prasad, Matt Wright
Pseudomonas aeruginosa
Teacher: Stephen Plum
Mentor: Jim Feix, Ph.D., Medical College of Wisconsin
Abstract: Pseudomonas aeruginosa, the bacterium which is a major cause of pneumonia and other infections, is especially fatal to cystic fibrosis patients with an excessive build-up of mucous in the lungs. This in turn creates
favorable conditions for the P. aeruginosa to invade and release the protein ExoU. ExoU, one of the key proteins in P. aeruginosa’s invasion process, is a phospholipase which breaks down lipids. If a cystic fibrosis sufferer
acquires P. aerguinosa, the ExoU produced by the bacterium will digest the membranes in the lung cells, leading to poor clinical outcome. ExoU can be found on the surface of the P. aeruginosa bacterium and is transported into
the host cell through the type III secretion system. Next, ExoU kills the host cell by destroying the integrity of the plasma membrane, cleaving the lipids in the membrane. If ExoU is defective or blocked, P. aeruginosa is
completely unable to attack the host cell. Given that the structure of ExoU is mostly unknown, Patatin, a phospholipase similar to ExoU is being studied by scientists in order to develop a better understanding of the structure and
function of ExoU. From this understanding, scientists may then be able to create drugs to lessen the severity of P. aeruginosa infection, thus increasing the chances of survival for infected cystic fibrosis patients.
ExoU’s Significance: Cystic fibrosis is an autosomal
recessive genetic disease, which causes a variety of
symptoms; the defining symptoms being a build up of
mucus in the lungs and pancreas. This mucus can create
serious digestive problems, and also forms the ideal
environment for bacteria such as Pseudomonas
aeruginosa to infiltrate the lungs. ExoU, the protein of
interest for this study, is one of the proteins found in the
virulence mechanism of P. aeruginosa which leads to
massive cell death and multiple system failure.
Targeting Virulence Factors vs. Current
Antibiotics: The current generation of antibiotics used to
treat bacterial infections such as P. aeruginosa target the
bacteria themselves, meaning that those bacteria resistant
to the antibiotics can proliferate even after treatment. These
resistant strains cause numerous issues such as increased
mortality and economic costs associated with developing
new antibiotics. As shown in the graph below, there are
significantly higher levels of antibiotic resistant strains. In
conjunction, current antibiotic are non-discriminatory,
meaning beneficent fauna are also killed upon treatment.
This is especially harmful to P. aeruginosa sufferers, whose
digestive systems are already compromised.
This diagram shows the
structure of Patatin.
Imaging Issues with ExoU: Although this is the
Patatin and ExoU Similarities: In order to
study ExoU, researchers depend on the
study of a homologous protein – in this
case, Patatin. Both Patatin and ExoU are
phospholipases. A phospholipase is an
enzyme that catalyzes phospholipid
hydrolysis for the purpose of membrane
remodeling, production of important
signaling molecules, and digestion of
dietary fat. However, ExoU, a virulence
factor found in P. aeruginosa, destroys the
host’s cell membrane.
The tables to the right show the conserved
regions between ExoU and Patatin in red
and orange.
Antibiotic-Resistant Bacteria: On the Rise!
140,000
130,000
120,000
Estimated Case
protein of interest, ExoU is extremely difficult to study.
The upper limit for structure determination by Nuclear
Magnetic Resonance (NMR) is currently approximately
50 kilodaltons, and ExoU is 74 kilodaltons. There are no
size limits on X-Ray crystallography, but despite
numerous attempts no one had been able to get ExoU
to crystallize. There is evidence that ExoU in its resting
state is partially unfolded which explains its failure to
crystallize ExoU requires a eukaryotic cofactor for
activation which prevents it from being activated inside
a bacterium. So its function invivo is less understood
because of the possible roles of cofactors such as,
ubiquitin and superoxide dismutase.
The above diagrams show a
phospholipase attacking a
lipid micelle.
102,000
100,000
80,000
65,000
60,000
40,000
26,000
16,000
20,000
11,000
12,000
0
Ceftazimide/K. pneumoniae
Ceftazidime/P. aeruginosa
Imipenem/P. aeruginosa
Vancomycin/enterococci
Ampicillin/E. coli
Methicillin/S. aureus
Methicillin/CNS
In a phospholipase, the backbone of
glycine-rich residues stabilize the
phospholipid while the serine and aspartic
acids cleave it from the membrane.
Strains
Biological Significance: Due to continuing bacterial resistance to antibiotics, it will be
necessary for researchers to find different ways to stopping a bacterial infection from
harming patients. Virulence factors are secreted by bacteria in order to keep the bacteria
alive. They can help the bacteria enter the host cell, suppress the immune system, or
obtain nutrition from the host cell. An alternative to current antibiotics is to target different
virulence factors. In P. aeruginosa with the phospholipase ExoU, it may be possible to
permanently bind a peptide to the binding site of ExoU. This would stop ExoU from binding
to lipids and destroying cell membranes. As this does not kill the bacterium, any bacteria
that are resistant to the attempts to target ExoU would still have to compete against the
normal bacterial flora, preventing the selection of resistant strains.
Patatin
active site serine of cPLA2
active site aspartate of
cPLA2
Highly conserved among both groups of phospholipases
Conserved among a subset of phospholipases
Poorly conserved
References:
Feix, James. “ExoU.” PowerPoint Presentation. Medical College of Wisconsin, Milwaukee,
Wisconsin. 17 Nov 2010.
"File: Pseudomonas.jpg." File: Pseudomonas.jpg. Web. 1 Mar 2011.
<http://commons.wikimedia.org/wiki/File:Pseudomonas.jpg>.
A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA) and an NIH CTSA Award to the Medical College of Wisconsin.
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