1. Title: Detailed analysis of protein-protein interactions within the type-III secretion system of the
deadly human pathogen, Chromobacterium violaceum. Kaitlin Zoccola & Shanna James.
Chromobacterium violaceum is a Gram negative motile bacterium exhibiting hybrid morphology
between a rod and a sphere [1]. The organism primarily inhabits soil and water indigenous to tropical and
sub-tropical regions [2]. A characteristic feature of C. violaceum is its ability to produce the purple
pigment, violacin, in response to auto-inducers. Manipulation of the violacin pigment has shown
antibiotic, anticancer, and anti-viral activity. Despite the positivity, C. violaceum is a rare opportunistic
pathogen with a high mortality rate in humans and other mammals due to antibiotic resistance. The
pathogen induces serious sporadic infection within the host organs and literature suggests the bacterium
could pose a threat in the future. The current research investigates whether pathogenicity is
linked to a type-III secretion system (T3SS) [3,4,5].
T3SSs have evolutionary relationships with bacterial flagella; however, flagella
proteins have evolved for locomotion and T3SS proteins have evolved for virulence [4]. As
shown in figure 1, T3SSs have only been witnessed in Gram negative bacteria
(characterized by an additional outer membrane) and are analogous to a molecular syringe
protruding from bacterial cells. The apparatus resides in the bacterial cell wall and
assembles to transfer type-III anti-host effector proteins directly into a host eukaryotic cell
[3]. T3SSs are composed of the following five components: a secretion apparatus and
needle complex, translocation apparatus, regulators, chaperones, and effector proteins [3,4].
The secretion apparatus is a multi-protein structure that extends across the inner/ outer
membranes and periplasm of a bacterial cell and functions by the hydrolysis of ATP. When
assembled, the needle complex, translocators, and the secretion apparatus shuttle effectors
from the bacterial cytosol directly to the host cytoplasm [2,5,6]. The translocation apparatus
consists of two translocator proteins secreted though the needle-like surface projection
which form a pore in the plasma membrane of a eukaryotic cell [7,8]. Once the effector proteins enter the
eukaryotic cell, they work by paralyzing host function and causing disease. Some common T3SS
containing organisms include pathogenic strains of E. coli, Salmonella, and Chlamydia [7].
In 2003, the Brazilian Nation Genome Project Consortium sequenced the genome of C.
violaceum. Unusually, they discovered two adjacent T3SSs, now termed Chromobacterium pathogenicity
islands 1 and 2 (Cpi-1 and Cpi-2). Genes on the loci of both Cpi-1 and Cpi-2 are evolutionary related to
those in the T3SSs of Salmonella enterica. Based on this evidence, it has been proposed that Cpi-1 injects
effectors into host cells and Cpi-2 is required for survival within macrophages [6]. The focus of this
research will be on Cpi-2.
Even though much is known about T3SSs of other bacteria nearly nothing is known about these
systems in C. violaceum. This grant proposes research that aims to analyze the putative components of
Cpi-2 to determine the possible protein-protein interactions that take place within the T3SS apparatus.
This information will allow us to identify possible future targets for antibacterial agents and antigenic
components that may be used in vaccines. The following proteins have been chosen for experimentation:
CV2574 (chaperone protein), CV2575 (translocator/effector), CV2576 (translocator/effector), CV2578
(class II secretion system chaperone), CV2588 (needle complex), csa Q-U (secretion system apparatus),
and csaN (secretion system apparatus). Molecular techniques such as Polymerase Chain Reaction (PCR),
a unique molecular cloning method, and the Yeast Two-Hybrid (Y2H) assay will be used to record
information about protein–protein interactions [9,10]. To date, all genes encoding for the proteins of
interest have been amplified by PCR, ligated into vector pDONOR221, and transformed into competent
E. coli cells. We require funding in order to proceed with transferring our genes into Y2H vectors for
screening [10]. Once our protein-protein interaction map is constructed based on data collected from the
Y2H screen, it will potentially explain how Cpi-2 assembles itself across the polar and nonpolar regions
of the C. violaceum cell wall. It will also provide evidence that the proteins of interest can indeed form a
functional type III secretion apparatus. Further research built on the outcome of this study may explain
how C. violaceum’s effector proteins interact with host cells to ultimately cause disease.
Figure 1- Schematic
representation of the Yersinia
T3SS in a bacterial cell wall [3]

2. Materials and Methods
DNA Manipulations
Eleven genes were chosen: csaQ, csaR, csaS, csaT, csaU, csaN, CV2588, CV2576, CV2574, CV2575,
and CV2578, due to their relevance to the proposed inner membrane components and needle apparatus
respectively. In order to amplify the genes, Polymerase Chain Reaction (PCR) was performed. PCR
works by using specifically designated primers to amplify a particular gene of interest. The primers were
designed with large overhanging sequences known as attB sites, which originate from the bacteriophage
lambda, a bacterial virus that inserts its DNA into the bacterial chromosome. The attB sites are required
to utilize the unique features of the Gateway cloning system, which is a novel cloning method that allows
shunting of genes between different plasmid vectors. The PCR products were analyzed via gel
electrophoresis to ensure that they were of the correct size and purity.
The Gateway Cloning System
The Gateway cloning system is a unique cloning method that enables us to quickly and accurately transfer
DNA sequences in and out of different vectors whilst maintaining the correct reading frame for protein
expression [9]. Unlike traditional methods, the Gateway cloning system eliminates a number of
intermediate steps that are time consuming and have the potential to incorporate inaccuracies. Following
the PCR reaction, the eleven genes were purified and used in the first cloning step - the BP reaction to
shuttle them into the pDONR221 vector. Currently we are analyzing whether this step was successful.
Once confirmed by DNA sequencing we will perform the LR reaction as follows. The LR reaction is
catalyzed by the LR Clonase II enzyme, which functions to shunt the initial gene from the pDONR221
and insert it into a destination vector. A typical LR reaction mixture consists of 1 μl of pDONR221+gene
of interest, 0. 5 μl destination vector, 2 μl LR Clonase II enzyme, and 6.5 μl of buffer. The reactions will
be incubated for 1 hour at 37o
C before being transformed into competent bacteria for replication. We will
perform LR reactions in order to move our genes of interest into the Yeast Two-Hybrid vectors,
pDEST22 and pDEST32, to screen for protein:protein interactions between expressed genes.
Yeast Two-Hybrid System
The yeast two-hybrid system works by exploiting the Gal4 transcription factor in yeast that allows for
utilization of galactose; it can only be activated when two protein domains, the gene activating domain
(AD) and the DNA-binding domain (DBD) are in association with each other. Thus in order for the Gal4
to be activated, both the AD and DBD must bind together to recruit the RNA polymerase, for gene
expression. We can utilize this mechanism to test if two proteins interact by fusing them to the AD and
DBD and then expressing them in yeast. The pDEST22 and pDEST32 vectors enable us to make such
fusion proteins with our genes of interest. We will co-transform the recombinant DNA into the yeast
strain MAV203, which is auxotrophic for leucine, tryptophan, uracil, and histidine. The yeast is first
plated on vector selective media that does not contain leucine or tryptophan, this ensures the vector and
the colonies have both the pDEST22 and pDEST32 vectors. Positive protein: protein interactions will be
tested with screening media that lacks uracil and histidine. If the proteins interact, the Gal4 activator will
be turned on and the yeast will be able to grow on the selective media [9, 10]. We will be able to test
many interaction combinations of our genes of interest using this system. The data generated will allow us
to build an accurate model of the Cpi-2 T3SS apparatus and pave the way for further work.
Timeline:
October: Harvest pDONOR plasmids containing our genes of interest and send for sequencing
November - December: Perform LR reactions, prepare yeast media and perform co-transformations
January - April: Perform yeast two-hybrid assay, analyze data and plan further work

3. References:
1. Betts, Helen J., Chauduri Roy, R., Pallen, Mark J. An analysis of type –III secretion gene clusters
in Chromobacterium violaceum” Trends in Microbiology, 2004
2. Betts H. J, Annotation and bioinformatics analysis of two type-III secretion system gene clusters
in Chrombacterium violaceum in “Type III secretion: From sequence to consequence” Ph.D.
thesis, Birmingham University, UK, 2005
3. Betts, H.J., et al., Bacterial secretion systems, in Bacterial-Epithelial Cell Cross-Talk Molecular
Mechanisms in Pathogenesis. Cambridge University Press: p. 60-98, 2006
4. Erhardt, M., Namba, K., and Hughes, K.T. Bacterial Nanomachines: The flagellum and Type III
injectisome. Cold Spring Harbor Perspectives in Biology, 2010.
5. Brazilian National Genome Project Consortium. The complete genome sequence of
Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability.
Proceedings of the National Academy of Sciences, 2003
6. Miki, T., Iguchi, M., Akiba, K., Hosono, M., Sobue, T., Danbara, H. and Okada, N.
Chromobacterium pathogenicity island 1 type-III secretion system is a major virulence
determinant for Chromobacterium violaceum-induced cell death in hepatocytes. Molecular
Microbiology, 2010.
7. Coburn, B.I. Type III secretion Systems and Disease. Clinical Microbiology, 2007
8. Cornelis,G. R. The type III secretion Injectosome. Nature Reviews 2006
9. Invitrogen. Gateway Technology manual. Version E May 13, 2010
10. Fields, S., & Song, O. K. A novel genetic system to detect protein-protein interactions. Nature,
1989

4. Undergraduate Research/Project Budget Form
Supplies Source of
Item
Quantity Unit
Cost
Total
Cost
Source of Funds Justification
LR Clonase
II
Invitrogen 20 reactions $214.00 $214.00 Grant Catalyzes the ligation
of the entry clone to
the plasmid vector
Sequence
Reaction
GeneWiz 20 reactions $8.00 $160.00 Grant Used to check
pDONR221
constructs are free
from errors
Plastic Petri
Dishes
Fisher
Scientific
1 case of 500 $90.00 $90.00 Grant Used to grow, culture,
and store bacteria on
agar
Plasmid
MiniPrep Kit
Fisher
Scientific
50 reactions $73.44 $73.44 Grant Isolates plasmid DNA
(pDONR221,
pDEST22/32) from E.
coli cultures
3AT
Chemical
Sigma 50 g $40.00 $40.00 Grant Used as a selective
marker in the Yeast
Two- Hybrid Assay
Carbenicillin RPIcorp 1 g $40.00 $40.00 Grant An antibiotic used to
select for specific
recombinant DNA
molecules.
Kanamycin RPIcorp 5 g $20.00 $20.00 Grant An antibiotic used to
select against certain
bacterial cells.
Equipment Source of
Item
Quantity Unit
Cost
Total Cost Source of Funds Justification
10µL
Micropipette
Fisher
Scientific
1 $300.00 $300.00 Grant Device used to measure
aliquots of liquid in a
precise manner and
required for all steps of
setting up reactions.
Grand Total: $937.44
We request the maximum amount of $800.00 to cover these expenses. Additional costs will be met using
Dr. Hampikian’s grant money from other sources.

5. Addendum:
My name is Kaitlin Zoccola and I am currently a junior at Clarion University of Pennsylvania. I
am a double major in Medical Technology and Molecular Biology/Biotechnology with a minor in
Honors. Out of my first four semesters, I maintained a 4.0 and was a representative of the Dean’s List.
During my freshman year, I was inducted into the Phi Eta Sigma National Honors Society Program and
was a member of the Clarion University Dance Team. During my sophomore year, I was admitted into
the Honors Program and was the recipient of the 2014 ASCUP scholarship. That same year, I was
inducted into the Tri Beta Biological Honors Society and was offered a work study position as an
assistant to the pre-professional committee. I was also trained in Cardiopulmonary Resuscitation and how
to correctly handle an Automated External Defibrillator. Currently, I serve as President of an
organization known as Peer Biology Mentors and I hold a teacher’s assistant position for an introductory
freshman biology course (Freshman Biology Seminar). Some other clubs I am involved with are Students
Honors Association and Health Careers Club.
My Career objectives include graduating from Clarion University with a B.S. in Molecular
Biology and completing my clinical year at Saint Vincent Hospital as well as receiving my certification as
a Medical Technologist. After receiving my certification, I plan to receive acceptance into a Medical
School where I will hopefully gain the qualifications to orient myself along the path of becoming an
Oncologist.
Undergraduate research has fined tuned my skills and expanded my horizons in the medical field.
The protocols for my current research have allowed me to master aseptic techniques, the advance process
of molecular cloning, and electrophoresis. It has given me the opportunity to access and gain hands on
experience with technology that I may not have gained experience with until my clinical or medical years.
Research is not only giving me hands on experience, but it is also given me the ability to think
scientifically, approach problems with an open mind, and ask questions about particular processes.
Research has allowed me understand certain labs and biology courses to a greater extent. In all,
undergraduate research has added several skills and prepared me to enter my future with a higher level of
understanding.
It would be an honor to receive the financial assistant of this Undergraduate Student Grant. The
money received would allow the project to be continued and allow all members to gain experience with
current technology and protocols. Not only would it benefit the members, but it would have the potential
to raise awareness for the medical treatment of a dangerous pathogen and allow an increased
understanding of type III secretion systems. The consideration of this application is greatly appreciated.