UNM TVDC UTSA - UNM Tech Call Minutes: 12/19/2006
Prepared 12/19/2006: Barbara Griffith
Sent to UTSA for review: 12/19/2006
Reviewed: Karl Klose and Bernard Arulanadam 1/23/2006
Distributed to NIAID on: 1/23/2006
Present: Karl Klose, Barbara Griffith, Vicki Pierson, Marlene Hammer, Joe Breen
Absent: Bernard Arulanandam, Rick Lyons
Action Items:
 Karl is at Cerus 12/19 and 12/20/06; will convey the scope of the discussions when Karl
returns
 Barbara has milestone completion reports for MS 16 &39 from UTSA and will be reviewing
 Karl will ask Jeff Barker to provide UNM the MS 48 completion report
The meeting was recorded for the purposes of the minutes.
A. Milestone Progress: University of Texas San Antonio
a. Update on F. tularensis attenuated vaccine strain construction and evaluation
(TVD Team).
b. Active milestones during last reporting period: 43, 49A, 51, 50A.
B. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
a. We have created new low copy vector for mutant construction in LVS/Schuh4,
pKEK1090, this is conjugative, conditionally replicative, with six new unique
restriction sites, and FT groELp promoter to drive sacB (counterselective
marker) and CmR (will replace with KanR for Schuh4).
b. uvrA::Kan and uvrB::Kan will be inserted into this plasmid to create LVS
mutants (documented in UTSA TVD notebook #2). Jirong is working on this
currently and UTSA will have more to report next month.
C. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
a. We have attempted to put iglC into mating plasmid (pKEK1090) but so far
unsuccessful following ligation, we are repeating and screening colonies. Crystal
is working on this currently.
b. We introduced sacB into our Schuh4 mutagenesis vector downstream of Ftp to
provide a counterselectable marker, however, it had additional DNA inserted due
to incorrect oligonucleotide, oligo has been reordered and this construction is
underway again. Have pUC based plasmids to introduce foreign DNA into
SCHUS4; got Sac B in and could not the plasmid back out after homologous
recombination in the chromosome.
c. (Value added) We have adapted Targetron (intron targeting system of Sigma) to
work in Ft, our initial target was beta-lactamase, and it has been knocked out in
Schuh4 as well as in novicida. Targetron originally was designed for E. coli and
other salmonella so UTSA got it to work in Ft for the contract. The Targetron
specifically targets genes and works in F. novicida and F. tularensis. Targetron
targets the specific gene that you re-target the intron towards. Targetron is not
host specific; it doesn’t require host recombination machinery to interact with the
vector. The Ft recombination is so much less efficient in SCHU S4 and LVS it is
in F. novicida. Targetron doesn’t rely on the host recombination machinery; user
orders special oligos to re-target the plasmid.
d. AmpS Schuh4 will open up possibilities for additional mutagenesis vectors, and
we will also use Targetron to knockout iglC in Schuh4 (documented in UTSATVD
Notebook 3). Have to order special oligos directed to iglC. Should work better
than prior strategies but will continue both strategies in parallel. Could be
publishable. By the creation of an amp sensitive SCHU S4 strain, UTSA now
has an amp sensitive basic strain to then knock out additional genes. This allows
the usage of an additional marker and markers are limited in SCHU S4. In
general, one can only use Kan and erm resistence in SCHU S4, but Karl also
requested to use amp resistance as a marker as well, from CDC. Karl got this
permission, and UTSA now has an amp sensitive SCHU S4 strain to use. This
opens up genetic opportunities in Francisella.
e. Question: Is Targetron new? It is fairly new. It was designed for use in E coli and
salmonella, but researchers already had many options for knocking out genes in
these two bacteria that Targetron wasn’t used that much. So,
Targetron hasn’t achieved wide spread usage to date. Getting Targetron
expression in a bacterial strain involves an RNA molecule and a protein that
helps that RNA molecule integrate into the chromosome. UTSA had to redesign
Targetron to express in Francisella. Karl is pleased that Targetron worked with
beta lactamase to make SCHU S4 amp sensitive. Sigma wants Targetron to be
more widely used and they have helped Karl.
f. Question: Joe- are there limitations to the modified oligos required by the
Targetron system? Sigma knows that plasmids can easily be shared. Sigma is
making their profit by using the Sigma computer site to design the specialized
oligos needed for the Targetron intron system. It is a Type 2 intron with
preferred target sites for insertion. The software picks out sites within the gene
that are good for intron insertion. The user needs the Sigma algorithm for the
oligos to direct the insertion. Sigma charges for use of their algorithm to design
the oligos. Then UTSA purchases the oligos separately from any vendor, not just
Sigma. Sigma licenses Targetron from some other source. Sigma thinks that
Targetron will be as powerful as the Gateway system has been for Invitrogen.
The best aspect of the Targetron is that the construct works with same site of
insertion in novicida as SCHU S4. It works with the same efficiency too. It works
across the bacterial strains.
g. Question: Are other Francisella labs using the Targetron system? Joe Petrosino
of Baylor is thinking about using for Ft as well. Currently, Joe is waiting for Karl
to get it to work.
D. Milestone #51: Construction of F. tularensis subsp. Novicida uvrA + pdpD, iglA,
glB, iglC, iglD and uvrB + pdpD, iglA, iglB, iglC, iglD strains.
a. UTSA will construct all ten of these strains, by moving uvrA::Kan and uvrB::Kan
mutations into strains with single ermC insertions in pdpD, iglA, iglB, iglC, and
iglD. Each of these mutations in the single genes in the pathogenecity island are
associated with erm C resistance. UTSA can then move the uvrA and uvrB
genes into these markers and knock the original marker out. Will make all the
constructs because won’t be that hard, rather than consulting with Rick to
prioritize for novicida.
b. We have all the single mutant strains except iglB, so we are constructing an
iglB::ermC mutant. Jirong is working on iglB currently.
c. Oligos were designed to create iglB::ermC by overlapping PCR, as we have
made all other mutants in novicida. Can move uvrA , uvrB as long as associated
with a different marker. Jirong is working on the uvrB:Kan this month. Jirong is
making the iglC erm mutant too.
d. Individual fragments isolated, mutation will be created in next period and
recombined back into Ftn chromosome.
e. UTSA will take all five of the single mutants (pdpD through iglD) and will combine
in uvrA, uvrB mutations into the strains. UTSA will have 10 novicida strains with
uvrA and uvrB mutations as well as the attenuating mutation. This will be
equivalent to a vaccine strain that also has uvr genes so that Cerus can work on
the new strains.
E. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains-Results Update
a. Monitored replication and dissemination of Ft subsp. novicida uvrAuvrB double
mutant.
1. Bacterial burden were determined in the lungs, liver and spleen
of infected BALB/c mice (Fig 1) at 24, 48 and 72 hrs post
intranasal vaccination.
2. The uvrAuvrB double mutant replicated rapidly in the infection
site (lungs) and quickly disseminated to the liver and spleen by
48 hr and 72 hr (Fig.1).
3. Replication and dissemination of the double mutant in BALB/c
mice is similar to the wild-type strain. So there’s no defect in
virulence with the uvrA uvrB double mutant.
b. Evaluation of the efficacy of Ft novicida Δiglc (made before he contract started) +
LVS LPS vaccination in protection against Schu S4 challenge.
1. The iglC mutant in novicida is a very effective vaccine against
later novicida wild type challenge. Bernard has characterized the
iglC mutant in novicida very well. The novicida protected fails to
protect against SCHU S4 challenge. Maybe this is due to the
differences in the LPS of the novicida and SCHU S4.
2. Groups of BALB/c mice (6 mice per group) were vaccinated with
Δiglc (106 CFU per mouse via intranasal (i.n.) route) and purified
LVS LPS (50μg per mouse via Intraperitoneal injection). These
mice received an additional boost with Δiglc (106 CFU, i.n.) +
LVS LPS (25µg, i.p.) two weeks after the first immunization and
rested for three weeks before being challenged i.n. with either
300 CFU or 3000 CFU of the Schu S4 strain.
c. This experiment did not show any measurable degree of protection or extended
time to death (Fig. 2).
d. The idea of using LPS was to examine if boosting the antibody response at the
time of vaccination would induce a noticeable difference in protection.
e. These results provide a baseline to establish the efficacy of using attenuated
novicida strains to protect against Schu S4. Results: there is no difference in
protection with LVS LPS. Novicida doesn’t protect from SCHU S4 challenge.
F. Goals for the next month.
a. Milestone #16: completed.- UTSA gave UNM a completion report
b. Milestone #39: completed.- UTSA gave UNM a completion report
c. Milestone #48: completed.- Karl thinks that Jeff has drafted it and will ask Terry
to send it to UNM.
d. Milestone #43:
1. Clone uvrA::Kan and uvrB::Kan into pKEK1090, mate into
LVS. (Jirong has already cloned the genes and is mating into
LVS)
e. Milestone #49A:
1. Clone iglC in pKEK1090.
2. Clone sacB (correctly) into Schuh4 mutagenesis plasmids.- had
issues with oligo last time so have new oligo
3. Design oligos to clone iglC target into Targetron vector.
f. Milestone #51:
1. Construct iglB::ermC single mutant in novicida progress by
Jirong.
g. Milestone #50:
1. Isotyping of anti-Δiglc antibodies in sera of mice immunized with
Δiglc + LVS LPS. – From an immunological stand point, it
doesn’t make sense that the induction of antibodies against the
LPS of SCHU S4 had no protective value in the challenge
experiment. Bernard wants to know what type of antibody was
made and will determine from fluids saved from the animals.
2. Determine the kinetic growth and clearance of the Ft novicida
Δiglc mutant in target organs after i.n. vaccination. How long
does the bacteria persist in the host tissues? If the vaccine
strains die out too quickly in the host tissues, then you don’t get
good induction of the protective responses. We know the iglC
mutant does engender a protective response against the wild
type novicida challenge; therefore, how long did the bacteria
persist in the tissues relative to other strains that don’t protect
against a wild type challenge. The vaccine bacteria may die too
quickly to engender an immune response.
G. Next UTSA Tech Calls:
a. January 23 (4th Tuesday; exception in Jan due to 1/2/06 being holiday).
H. Additional Discussion
a. Karl is at Cerus 12/19 and 12/20; will convey the scope of the discussions when
Karl returns.