Transcript Symposium Poster - uospur

Understanding the Potential Fitness Advantage for Intestinal Bacteria
Aeromonas to carry an Immunoregulatory-Protein Coding Gene
Lila Kaye, Annah Rolig, Karen Guillemin
Institute of Molecular Biology, University of Oregon
1. Research Overview and Background
2. The Zebrafish Gut Assay
• Bacteria play an important role in the health of their host
organism; pathogenic lines induce a host immune response, while
commensal (resident) bacteria signal to reduce or prevent such a
response. The ability of a resident organism to communicate with
its host to alter immune system function is a burgeoning topic in
the molecular biology field, offering a potentially rich source of
biotherapeutics to treat illness.
• Gut immune response and inflammation was
measured by counting the number of
neutrophils in fish intestines. Neutrophils are
immune leukocyte cells that are a hallmark of
inflammation. We used a transgenic line of
zebrafish in which Neutrophils are tagged with
a green fluorescent protein (GFP), allowing us
to visually measure innate immune response
in the fish gut.
The Anti-inflammatory Effect of IRP
N e u t r o p h il p e r g u t
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• Zebrafish are a good vertebrate model organism for the study of
host-microbe interactions because we can easily perform
gnotobiotic experiments with high replicability . Additionally,
translucent larvae allow easy monitoring of host cells in real time.
Researchers in the Guillemin laboratory identified a novel
Immunoregulatory Protein (IRP), produced by resident bacteria,
which reduces intestinal innate immune responses in zebrafish
hosts (see adjacent graph). This protein is transcribed from the
1882 gene.
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• Fish Protocol:
1. Fish embryos are fertilized and raised in a
Germ Free (GF) environment. They are free
of bacteria: both pathogens and their natural
colonizing bacterial commensals
Experiments by post-doc Annah Rolig show the
anti-inflammatory effects of isolated and
purified IRP on sox 10 fish, a transgenic
zebrafish line with naturally elevated levels of
gut immune system activity. Introduction of IRP
lowers gut inflammation to levels observed in
healthy WT fish.
• I am investigating whether this 1882 gene confers a fitness
advantage to the Aeromonas species. Humans suffer from high
levels of intestinal inflammation. A lessening of this immune
system activity is something that would benefit many patients
suffering from diseases like IBS and IBD. These conditions often
stem from a disbiosis between the human host and her or his
resident bacteria. Controlling inflammation could be an important
mechanism for commensal microbes to promote host health as
well as their own survival.
0 dpf
Derive germ-free
4 dpf
Inoculate with
microbes
2. At 4 days post fertilization (dpf), the fish are
inoculated with the chosen Aeromonas
strain, and the bacteria are given time to
colonize.
5, 6, or 7 dpf
Dissect gut and
count
neutrophils,
then plate
3. At 5, 6, or 7 dpf, the fish are euthanized, the
guts are dissected from the surrounding
tissue, and the number of GFP neutrophils in
each is tallied. The gut is homogenized,
plated, and the number of colony forming
units (CFU) is tallied
We Hypothesize that a lowered
immune response allows
Aeromonas to more robustly
colonize the gut.
3. IRP Effects on Aeromonas Colonization of the Gut – High Level Persistence After Day 6
• We preformed multiple experiments in which fish were inoculated with the wild type version of Aeromonas (A01) both separately and together with a
mutant strain of Aeromonas in which the 1882 gene that encodes IRP had been deleted (d1882 Ar).
4. Neutrophil Patterns During Colonization – Hidden Effects?
• The colonization defect suffered by the deletion
mutant does not directly correlate with an increase in
gut inflammation. Total gut neutrophil numbers were
remarkably even between all three colonization
groups, both the two monoassociations and
competition.
• Even though we are not able to visualize it now, we
believe there is still an important difference in biology
happening within the gut when Aeromonas is present
versus not.
 There are more wild type A01 eliciting the same
high neutrophil response as the fewer d1882
deletion mutants.
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 The level of gut inflammation per CFU is higher
in colonization by the deletion mutant.
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C o lo n iz in g g r o u p C F U a n d N e u tr o p h ils
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The Competition Index shows
C o lo n iz in g G r o u p a n d T im e
C o lo n iz in g G r o u p a n d T im e
that A01 is able to out compete
 In the competition scenario, A01 and d1882 were inoculated the deletion mutant.
 Alone A01 is slow to establish but populations jump and
in a 50/50 ratio. Both populations resemble each other over
The advantage of wild type
are relatively high after day 6.
time. They colonize quickly, persist for a day in relatively high
A01 seems to increase slightly
 The deletion mutant establishes more quickly, but by day numbers, and on day 7 both populations fall.
as time progresses, with the
7 the population starts to decline.
greatest dominance on day 7.
Compared to their day 7 monoassociation numbers, A01 is
 There seems to be something happening either with the suffering the most, with populations down almost as far as the This day 7 advantage is
bacteria or the fish that allows A01 to persist in higher
consistent with the mono
deletion mutant alone. However, A01 Is still slightly more
abundance than the deletion mutant
association data.
abundant than the deletion mutant
4 - 7 d p f In n o c u la tio n R e s u lts
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( A 0 1 /d 1 8 8 2 ) C o m p e titio n D a y
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Log CI
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C o lo n iz a t io n in A 0 1 : d 1 8 8 2 A r C o m p e t it io n
C F U /G u t
C o lo n iz a t io n in M o n o a s s o c ia t io n
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• In both cases the wild type A01 strain colonized the gut in higher numbers than the d1882 mutant by day 7.
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IR P F o r P e r s is t a n c e A f t e r D a y 6
IRP appears to lower the number of host
neutrophils per bacterium in response to
Aeromonas, thus allowing more robust
colonization and persistence in the gut.
 There is a critical period at day 6 by which A01
must be established and producing IRP in order to
successfully colonize and persist in the gut, otherwise
populations fall to the same level as the deletion
mutant.
This is not a bacterial establishment phenotype:
A01 does not require a 48 hr incubation period
This is not a host development phenotype:
The gut environment does not become intrinsically
more suited to hosting A01 after day 6
IRP is necessary for Aeromonas to
persist abundantly in the gut after day 6
5. Future Directions
Inhibiting Inflammation to rescue
colonization by deletion mutant
•In order to further show that it is the
increased number of neutrophils per
bacterium that causes the deletion
mutant’s colonization defect, fish will
be treated with prednisolone: a drug
that dampens immune response and
neutrophil production in the host.
Creating the compliment Plasmid
•I have been working on creating a mutant
strain of Aeromonas with the1882 gene
deleted from its natural location and reinserted elsewhere in the genome. These
bacteria will be competed against wild type
A01 to show that the colonization defect
suffered by the deletion mutant is solely due
to the lack of IRP, and can be rescued upon reinsertion of the gene
6. Acknowledgements and Gratitude
Thank you to all the members of the Guillemin lab for their support and advice throughout this
process. I would particularly like to extend my gratitude to Karen Guillemin for including me on her
wonderful team, Annah Rolig for inspiring me so deeply, and Sophie Sichel for her guidance.
I would also like to thank the SPUR program and META Center for the opportunity to work full
time this summer with such amazing mentors.
This work was funded by:
NICHD Summer Research Program at the University of Oregon (NIH-R25HD070817)
META Center for Systems Biology at the University of Oregon (NIH- P50GMO989911)