Identification of Francisella tularensis virulence factors by differential protein expression KWUN W

1
Identification of Francisella tularensis
virulence factors by differential protein
expressionKWUN WAH WEN, TIMOTHY CUMMINS, AND
EDWARD COLLINSDepartment of Immunology,
University of North Carolina, Chapel Hill, NC
27599
Abstract
III) Generation of FT transposition knockouts
II) Two dimensional fluorescence differential gel
electrophoresis (2D-DIGE)
F. tularensis mutants were generated by random
transposon insertion. 0.2 ug of bacterial gDNA
was prepared for the EZTNltKAN-2gt insertion
reaction (EPICENTRE). The reaction mixture
included the FT gDNA, transposon, and
transposase. After incubating the mixture for 2 h
at 37oC, electroporation was performed to
transform recA- E.coli competent cells.
Transformants were selected on kanamycin
containing plates.
Purpose This research project aims to identify
virulence factors of Francisella tularensis that
may eventually lead to the development of
pharmaceutical agents targeting the pathogenesis
of the bacterium. Background F. tularensis, a
more virulent member of the Francisellaceae
family, causes tularemia in mammalian host. This
gram-negative, facultative intracellular
bacterium is a potential bioterrorist agent owing
to its extreme virulence, rapid infection,
adapted tolerance to cellular and chemical
stresses, as well as its ability to infect a wide
range of hosts. The microbial pathogen can be
transmitted via zoonoses, animal vectors,
aerosols, fomites, and fecal/oral paths. Despite
the known epidemiology and clinical
manifestations of F. tularensis, details of its
pathogenesis are not well understood. Hypothesis
Our assumption is that using proteomic techniques
and tools, important factors conferring virulence
will be identified. Preliminary data Using two
dimensional fluorescence differential gel
electrophoresis (DIGE), proteins whose expression
in the live-attenuated vaccine strain (LVS) of F.
tularensis was significantly up-regulated after
passaging through macrophage-like murine cell
line J774A.1 were identified and analyzed by mass
spectrometry and their cellular functions
determined experimentally. In collaboration with
Dr. Tom Kawulas lab, we generated a library
composed of random transposon-inserted gene
knockouts of F. tularensis. Methods By making
genomic DNA prep of F. tularensis, designing
oligonucleotide primers, and performing
polymerase chain reactions (PCR), I screened the
transposon insertion library for mutants carrying
disrupted genes of candidate virulence factors.
Results A suitable protocol was established to
prepare F. tularensis genomic DNA from bacterial
lysates. After optimizing the PCR conditions for
the candidate gene C23, I screened a portion of
the transposon library for this gene by PCR using
pooled DNA templates that were mixed in rows and
columns of culture plates. Subsequent gel
electrophoresis revealed a clone that contained
the C23 knockout gene.
IV) PCR screening of transposition KO mutants
that contain C23
(Proof of Principle)
Genomic DNA prep of FT WT and C23 MT. Lysates of
30 clones in the transposon-inserted FT library
were combined in individual rows and columns on a
3x10 plate, their genomic DNA prepared and
subjected to screening by PCR. PCR positive for
C23 mutant DNA are labeled above. The C23 MT
clone is labeled O and the WT ones O.
I) Induction of virulence factor expression
A
PCR analysis of C23 WT and MT gDNA. The amplicon
using the C23 primer pair is 1.1 kb long, whereas
in the transposon insertion mutant, an amplicon
size of 2.3 kb is expected.
Composite image of Cy5-labeled lysates from five
independent gels. H2O2 treated F. tularensis LVS
lysates were labeled with Cy5 and co-separated
along with Cy3 labeled control lysates. Labeled
spots indicate a 2 fold change in expression from
control lysates.
Discussion / conclusion
B
These data support changes in expression patterns
of after passage through macrophages and
treatment with peroxide. We have determined the
identity of some of the proteins including C23
and IglC (data not shown) that have altered
expression patterns. This work provides a proof
of concept that we can prepare F.tularensis WT
and transposon-inserted genomic DNA that will be
used as PCR templates to screen the library for
other candidates of virulence determinants. Each
of these transposition mutants, together with the
wild-type control, will be assayed for altered
susceptibility to F. tularensis infection of cell
line J774A.1 and ultimately of mouse strains
BALB/c and C57BL/6.
PCR using C23 primer pair. Fragments of 1.1 kb
and 2.3 kb were visualized on agarose for pooled
DNA of row A and column 4, suggesting the
presence of C23 MT DNA in some cells belonging to
these row and column.
PCR using the forward primer of C23 and the
reverse primer of the transposon. Only C23 MT DNA
in combined DNA of row A and of column 4 were
amplified. The 500 bp fragment size provided
information regarding the location (1/3 from the
beginning of C23 gene) and orientation of the
transposon insertion. No F. tularensis WT DNA
were amplified because of the absence of
transposon.
The production of virulence factors in
Francisella tularensis was up-regulated by either
A) hydrogen peroxide induction or B) passaging
through macrophage-like murine cell line J774A.1.
References See attached sheet