1. Future Directions
Elucidating the role of the Chromosomal Type III Secretion
System structural protein SsaV
Jackson O. Osaghae-Nosa1*, Miqdad O. Dhariwala1, Gilbert A. Cortes1 and Deborah M. Anderson1
1Department of Veterinary Pathobiology, University of Missouri-Columbia
Abstract
Methods
Introduction
1. Dieye, Yakhya, Keith Ameiss, Melha Mellata, and Roy Curtiss. "The Salmonella Pathogenicity
Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica
serovar Typhimurium." BMC Microbiology 9.1 (2009): 3. Web. 12 Apr. 2015.
<http://www.biomedcentral.com/1471-2180/9/3>.
2. Harding, Robert. How the Plague Is Transmitted. Digital image. Nature. Nature Publishing Group,
2014. Web.
3. Plague Worldwide. Digital image. Centers for Disease Control and Prevention. N.p., 2013. Web.
Results
References
Growth Curves
Cultures of EO-55 and CO92 pCD1- were grown in 5mL of Heart Infusion Broth (HIB) overnight at 26⁰C.
These cultures were then diluted to an OD600 of 0.50 and were monitored over the course of 12 hours
at both 26⁰C and 37⁰C. An OD600 was taken at every hour as well as at a 24 hour time point. An OD600
correlates to approximately 1.0X10^9 colony forming units (CFU) of Yersinia pestis.
Polymixin B sensitivity
Cultures of EO-55 and CO92 pCD1- were grown in 5mL of Heart Infusion Broth (HIB) overnight at 26⁰C.
15 ml conical tubes at 8 different concentrations of Polymixin B were then infected with 1x10^6 CFU.
An OD600 was taken 16 hours after incubation in 26⁰C for the lowest three concentrations supporting
growth.
Intracellular Survival
A phagocytosis assay was performed at 2, 8, and 24 hours post infection. Cultures were grown in 5mL
of Heart Infusion Broth (HIB) overnight at 26⁰C. Then, cultures were diluted at 37⁰C for approximately
two hours before infection. RAW 264.7 macrophages were then infected with a multiplicity (of
infection (MOI) of 20. At each time point, macrophages were lysed by exposing them to a 0.1%
solution of Triton for 10min. Viable intracellular bacteria was then plated.
Lactose dehydrogenase (LDH) assay
A LDH assay was performed at 8 and 24 hours post infection. Cultures were grown in 5ml of HIB
overnight at 26⁰C. Then cultures were diluted at 37⁰C for two hours before infection. RAW 264.7
macrophages were then infected with a multiplicity of infection (MOI) of 20.
To determine significance,
two biological replicates
were pooled and a Two Way
Anova with a repeated
measures analysis was run.
No significance was found
at 26⁰C. However, a
significant difference was
determined at 37⁰C.
Yersinia pestis, a Gram-negative rod shaped bacterium, is the causative agent of plague. The bacterium
encodes several virulence factors such as the Type 3 Secretion System (T3SS), Siderophore encoding
pigmentation locus, an altered Lipopolysaccharide (LPS) and an anti-phagocytic capsule among others.
The T3SS of Y. pestis, located on the CD1 plasmid is the most significant virulence factor associated with
the pathogen. It induces apoptosis in mammalian macrophage cells. For this study we used one mutant,
EO-55. It merits further research because it harbors a mutation in a Secretion system apparatus protein
(SsaV). SsaV is a structural protein that is encoded in the chromosomal T3SS. In order to understand
how the chromosomal T3SS interacts with eukaryotic host cells, phagocytic assays were conducted to
study bacterial intracellular survival and uptake. RAW 264.7 cell line were infected with EO-55 and its
parent strain, CO92pCD1-. Furthermore, Lactate dehydrogenase (LDH) assays were conducted to
determine the cytotoxicity of these strains post infection. Preliminary data from our lab suggests that
intracellular survival of Y. pestis is not impacted by mutation of SsaV and our goal is to further assess its
function. This data enhances the understanding of the role of the chromosomal T3SS within Y. pestis.
Hypothesis
The project described was supported by the IMSD EXPRESS Fellows Program (or simply the IMSD
EXPRESS Program) via grant number R25GM056901 from the National Institute of General Medical
Science (NIGMS), a component of the National Institutes of Health (NIH).
Also I would like to thank the members of the Anderson lab for their valuable input and help in
discussing the data and designing experiments.
Acknowledgments
The mutant strain, EO-55, possessing a nonfunctional Chromosomal T3SS has been recognized as a
hypocytotoxic strain compared to its parental strain, CO92 pCD1-. Therefore, we expect the mutant to
induce lower amounts of cell death. With that in mind, we hypothesize that if more cell death is
induced in the presence of the functional Chromosomal T3SS then it must be important for the
pathogenesis of the disease plague.
Conclusion
• The disease plague manifests in three main
forms: bubonic septicemic, and pneumonic
plague.
• The flea vector as well as infected mammalian
hosts are responsible for the transmission of this
lethal disease.
• Historically, the bacteria Yersinia pestis, is
believed to have been the cause of the Black
Death which eliminated 30-60% of European
population in the 14th Century.
• Cases of plague infection still occur in today’s
world with most reported cases occurring in
Congo and Madagascar.
•Several instances of the disease plague have
been reported in the United States in the last
decade.
A mechanism that many gram-negative bacteria possess is the ability
to utilize the type three secretion systems. These bacteria include
Salmonella enterica, Escherichia coli, Shigella flexneri and
Pseudomonas aeruginosa among others. It is a protein appendage
that allows the bacteria to inject virulence proteins, called effectors,
into the eukaryotic host cell. These effectors ultimately manipulate
the cellular functions of the infected host and facilitate the
progression of the infection. For this study we used Y. pestis strains
that were randomly mutagenized by transposon Tn5-lacZ and a
library of 8000 insertion mutants were screened for defects in killing
macrophages with the goal to identify bacterial genes required for
cytotoxicity during infection. Candidates were further screened for
growth defects and for increased sensitivity to Polymixin B. One
mutant, EO-55, harbors a mutation in a Secretion system apparatus
protein (SsaV). SsaV is a structural protein that is encoded in the
• The role of the Chromosomal T3SS is one that is less understood. However, the data shown here
demonstrates that the lack of this T3SS doesn’t impact the survival of Y. pestis.
• Based on the data collected, a mutation in SsaV of the Chromosomal T3SS negatively impacts the
mutant EO-55 when compared to Kim6+, Which also doesn’t have the chromosomal T3SS, at two
and twelve point four hours post infection.
• This data runs counter current with our original hypothesis because we expected to see lower
amounts of intracellular survival when the Chromosomal T3SS was inactive.
• Furthermore, these data may suggest that the chromosomal T3SS contributes to the pathogenesis
of the disease plague particularly within mammalian hosts.
• Conduct an intracellular survival assay using CO92 pCD1- and EO-55.
• The strain, EO-55, has a transposon gene interruption at both YPO0266. To understand if this gene
will give back virulence, we will make a complementation mutant.
• More Cytotoxicity assays will be performed which will measure cell death.
• Fine tune the concentration where Polymixin b inhibits both CO92 pCD1- and EO-55.
• Infect mice with EO-55 and EO-55 complementing mutant
• Determine method of cell death using Electron microscopy
Nine biological replicates were pooled and a comparison T-test was run at each time point. Significant
difference was found between Kim6+ and the other two strains at two and twelve point four hours post
infection. However no significance was found at seventeen point four hours post infection.
chromosomal T3SS. This protein has been characterized on one of the two T3SS of Salmonella. The
T3SS in Salmonella is present on two different loci within the chromosome and uses pH as an inducer
for T3SS-2 assembly and secretion of the effector proteins. SsaV, which is present on T3SS-2 is
responsible for the secretion of effector proteins through the needle and syringe like machinery. SsaV
prevents fusion of the endosome/lysosome with Salmonella-containing vacuoles.
-0.5
0
0.5
1
1.5
2
2.5
-5 0 5 10 15 20 25 30
OD600
Hour
Growth curve at 26˚C
CO92pCD1- EO-55
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
-5 0 5 10 15 20 25 30
OD600
Hour
Growth curve at 37˚C
CO92pCD1- EO-55
0
0.2
0.4
0.6
0.8
1
1.2
CO92pCD1-
(256ug/ml)
CO92pCD1-
(128ug/ml)
CO92pCD1-
(64ug/ml)
E0-55
(256ug/ml)
E0-55
(128ug/ml)
E0-55 (64ug/ml)
OD600
Strains and concentrations
Lowest 3 concentrations supporting growth with
Polymixin B
2 H o u rs In fe c tio n
K
im
6
+

p
h
o
P
E
O
-5
5
0 .0 0
0 .0 1
0 .0 2
0 .0 3
0 .0 4 ***
**
n s
%IntracelularSurvival
1 2 .4 H o u rs
K
im
6
+

p
h
o
P
E
O
-5
5
0 .0 0 0 0
0 .0 0 0 5
0 .0 0 1 0
0 .0 0 1 5
0 .0 0 2 0 ***
***
n s
%IntracelularSurvival
1 7 .3 H o u rs In fe c tio n
K
im
6
+

p
h
o
P
E
O
-5
5
0 .0 0 0 0
0 .0 0 0 5
0 .0 0 1 0
0 .0 0 1 5
0 .0 0 2 0
n s
n s
n s
%IntracelularSurvival
0
10
20
30
40
50
60
70
80
90
100
Max lysis uninfected CO92pCD1- EO-55
Percentcytotoxicity
Percent Cytotoxicity
8 hpi 24 hpi
One experiment was run and no statistics besides standard error have been conducted. Here, the
uninfected are releasing more lactose dehydrogenase than the infected cells.
To determine
significance,
three biological
replicates were
pooled and
statistical test
were run. No
significance was
found at these
concentration.