UNM TVDC UTSA - UNM Tech Call Minutes: 11/28/06
Prepared 11/28/06: Barbara Griffith
Sent to UTSA for review: 11/282006
Reviewed: Karl Klose and Bernard Arulanadam 12/5/06
Distributed to NIAID on: 12/6/2006
Present: Karl Klose, Bernard Arulanandam, Barbara Griffith, Rick Lyons, Vicki Pierson, Marlene
Hammer
Absent: Kristin DeBord, Freyja Lynn, , Joe Breen
Action Items: Karl- email Barbara the milestone completion reports on December 1, 2006
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, 48, 49A 50A
B. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
a. Previously, we have found that cloning large flanking fragments (1.5 kbp)
upstream and downstream of uvrA and uvrB from LVS led to deletions within
cloned region, these were in high copy vectors.
b. We are now creating new low copy vector for mutant construction in LVS/SCHU
S4, utilizing pDS132, this is conjugative, conditionally replicative. This plasmid
has been used in cholera. It is not replicative in Francisella so you mate it in and
it will integrate into Francisella genome because it is not able to replicate outside
the bacterial chromosome. UTSA knows that conjugation works in SCHU S4.
UTSA have changed this vector to introduce six new unique restriction sites.
c. If you orient the gro ELp correctly during the insertion, it will drive sacB
(counterselective marker) and CmR genes in Ft. (will replace with KanR for
SCHU S4). A Francisella promoter is required for the expression of the antibiotic
resistance markers in Francisella. gro ELp has been successfully inserted into
the plasmid, as of end of November. Now have a plasmid to introduce these
mutations into LVS.
d. uvrA and uvrB will be inserted into this plasmid to create LVS mutants
(documented in UTSA TVD notebook #2). UTSA is currently growing the LVS
with the new plasmid inserted, and LVS is slow growing.
e. Vicki: Question- what drives the groEL promoter, what conditions does it respond
to? Karl- it was originally identified as an intra-macrophage activated promoter,
as identified by the Swedish group. GroEl promoter is driven by Ft and is active
in macrophages. UTSA has used groEl Promoter to drive GFP (green
fluorescent protein) in Ft, but it hasn’t been uniform. Karl says that the groEL
promoter is constitutively expressed in Ft bacteria , but isn’t always consistently
expressed in macrophages. Karl has difficulty expressing GroEl Promoter in
whole animals too. UTSA doesn’t have better promoters to choose from. Larry
Gallagher has identified another promoter in their Tn mutagensis study, which
UTSA has successfully used to drive antibiotic resistance genes (kan and erm) in
all the F novicida strains and mutant strains. UTSA calls this other promoter “Fn
promoter” and is more consistently on in F novicida. It is a short sequence.
C. Milestone #48: Characterization of uvrA and uvrB mutant F. tularensis subsp.
novicida strains for intra-macrophage growth
a. Three Ftn strains (uvrA, uvrB, uvrAB) tested for intra-macrophage growth (24 h.
in J774 macrophages).
b. Results indicate mutations in uvrA and/or uvrB do not attenuate intramacrophage growth (documented in UTSA TVD notebook #2). Barbara
indicated that the same result was reported by Cerus had as well.
c. Milestone #48 is completed per Karl.
D. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
a. These are UTSA’s first attempts to make mutants in SCHU S4.
b. We are altering a new mating plasmid (pDS132) for use in SCHU S4 (as detailed
in milestone 43). Before using this plasmid in SCHU S4, UTSA had to change out
the antibiotic resistance marker. pDS132 is chloramphenicol resistant so UTSA
is changing this out since in USA can’t use chloramphenicol resistant selection in
SCHU S4. UTSA will probably change out chlormaphenicol for kanamycin
resistance.
c. Rick: Can you use chloramphenicol resistance in LVS?- Karl- You can use any
antibiotic resistance marker in LVS as there are no restrictions in the USA. LVS
is restricted only due to natural erm resistance. UTSA uses erm resistance in
novicida and SCHU S4. LVS is naturally erm resistance so if don’t use Kan in
LVS then you are restricted to Tet or chloramphenicol that you would normally
not use in SCHU S4. Then UTSA is forced to making many constructs that are
specific to specific Francisella strains, which is less convenient.
d. We are introducing sacB into our SCHU S4 mutagenesis vectors (high copy
number vector) downstream of Ftp to provide a counterselectable marker. Crystal
has gotten the sacB into the vector as of the end of November, which will be the
counter selectable marker.
e. (Value added) We have been adapting the Targetron (intron targeting system of
Sigma) to work in Ft, our initial target was beta-lactamase, and it appears to have
been knocked out in Ft novicida; we are currently attempting in SCHU S4.
Targetron doesn’t rely on any host factors; it is independent of host. UTSA is
trying to get targetron to work in Ft. CDC has allowed this amp sensitivity
substitution to replace amp resistance in Ft at UTSA. F novicida, LVS and
SCHU S4 are all amp resistant. UTSA knows the beta-lactamase that is leading
to their amp resistance. So UTSA targeted beta lactamase with the Targetron
system. Targetron appears to have worked in F. novicida and are optimistic that
it will work in SCHU S4. UTSA is validating that the created mutants made amp
sensitive are truly intron insertions. UTSA designed the adapted Targetron
system to work in all Francisella strains. So if it did work in novicida, UTSA will
quickly make a SCHU S4 amp sensitive version.
f. Amp sensitive SCHU S4 would open up possibilities for additional mutagenesis
to knock out other genes without cloning/ selection/ counter selection etc, and
Targetron may facilitate mutant construction in SCHU S4/LVS (documented in
UTSATVD Notebook 3).
E. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains-Results Update
a. Determine the LD50 of Ft subsp. novicida uvrA mutant
1. LD50 of uvrA in the intranasal infection model (BALB/c mice) is
calculated to be less than 10 CFU (Fig. 1).
2. The virulence of uvrA is not significantly reduced compared to
wild-type strain. UvrA is not attenuated at all.
3. Figure 1: Survival of mice infected with Ft subsp. novicida uvrA
uvrB mutant . Groups of BALB/c mice (female, 6-week old) were
challenged intra-nasally with 4 doses (10, 50, 250, and 1250
CFU) of uvrA uvrB to determine LD50 of this strain. All the
animals succumb to infection by day 5, with the double mutant of
F. novicida. There is no attenuation in the single mutants or the
double mutants.
b. Measured intra-macrophage (J774) survival of uvrA uvrB Double Mutant
1. The double mutant replicated to similar levels as the wild-type
strain.
F. Goals for the next month.
a. Milestone #16: completed.
b. Milestone #39: completed.
c. Milestone #48: completed.
d. Milestone #43:
1. Construct pDS139 plasmid with gruel for use in LVS/SCHU S4
2. Clone uvrA::Kan into pDS139 derivative, has mated with LVS
(Karl says done at end of November already)
e. Milestone #49A:
1. Clone iglC in pDS139 derivative
2. Clone sacB into SCHU S4 mutagenesis plasmids (Karl thinks
Crystal has completed this at the end of November)
3. Continuing to work on Targetron system.
f. Milestone #51:
1. New milestone starting for double mutants with attenuating
mutations that will knock out the virulence so they can be used
as vaccine strains.
2. Construct uvrA::Kan; iglC::ermC double mutant first
g. Milestone #50:
1. Determine bacterial load and dissemination of the double urvA
uvrB double mutant in mice.
2. Characterize the efficacy of Ft novicida Δiglc vaccination + LVS
LPS in protection against Schu S4 intranasal challenge. Bernard
showed preliminary results in the contract submission, where
used F novicida iglC mutation and characterized in for protection
against wild type F novicida itself.
3. Will vaccinate animals with F novicida IglC mutation and try to
boost the response by administering the LVS LPS from John
Gunn, looking for a cocktail vaccination approach against SCHU
S4. Bernard hopes to get preliminary results regarding how the
iglC mutation in SCHU S4 may behave. The F novicida iglC
mutant is known to protect well against Fn challenge but doesn’t
protect against SCHU S4 challenge. Karl-maybe this is an LPS
specific effect. If the novicida mutant was expressing SCHU S4
LPS, you might get protection. So perhaps, we could coimmunize with LVS LPS and the novicida mutant, we may get
protection against SCHU S4. Karl thinks this experiment has not
been done. Rick- Wayne has used purified LPS and this exact
experiment hasn’t been performed. Karl thinks it would be good
to determine whether the efficacy of novicida strains could be
broadened by providing the LPS along side the attenuated strain.
G. Next UTSA Tech Calls:
a. December 19, noon-1pm MT, 1-2pm CT, 2-3pm ET
b. January 23 (4th Tuesday; exception in Jan due to 1/2/06 being holiday)
H. Additional Discussion
a. Milestone completion reports- Jirong and Jeff are working on these and will send
to Barbara on Dec 1.
b. Karl : traveling to Cerus in December (12/20 to 12/22/06)
c. Karl: Jirong Liu, post doc, is leaving Karl’s lab and will be replaced on TVDC.
Jirong is returning to Beijing, China for biotech development opportunities