University of Texas San Antonio
F. tularensis attenuated vaccine strain construction and evaluation
10/5-6/09 Annual TVDC Meeting
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Progress on milestones to date:
Milestone #16: Create luciferase expressing F. tularensis LVS
COMPLETE 4/30/06
Milestone #39: Creation of uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE 8/31/06
Milestone #43: Creation of uvrA and uvrB mutant F.
tularensis subsp. holarctica (LVS) strains
COMPLETE 8/31/07
Milestone #48: Characterize uvrA and uvrB mutant F.
tularensis subsp. novicida strains
COMPLETE
Milestone #51: Construction of F. tularensis subsp. novicida
uvrB + pdpD, iglA, iglB, iglC, iglD strains.
COMPLETE
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Milestone #49: Construction of mutant F. tularensis subsp.
tularensis strains
COMPLETE AS OF LAST MONTH!
Milestone #50: Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis,
and LVS strains
COMPLETE
Milestone #52: Construction of mutant F. tularensis subsp.
tularensis strains containing recA mutations
ONGOING
Milestone #53: Immunologic characterization of F.
tularensis subsp. tularensis strains
ONGOING
Milestone #54: Construction of mutant F. tularensis subsp.
tularensis strains
ONGOING (Just started)
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Our laboratory is focusing on the generation and characterization
of live attenuated F. tularensis subsp. novicida, tularensis, and
LVS strains for their vaccine potential
We have performed targeted disruption of various genes, including:
vgrG
iglCD
These are virulence genes located in pathogenicity
island
nadM
involved in NAD synthesis, mutant isolation
unsuccessful
transcriptional regulator important for virulence
in Fn
restriction enzymes that inhibit genetic recomb.
in Ftt; inactivation will facilitate genetic manipulation
FTT0748
FTT1579
FTT0523
recA
This gene facilitates genetic recombination; its 4
inactivation will stabilize potential vaccine strain
We have spent a considerable amount of time developing
and optimizing techniques for genetic manipulation of
Francisella tularensis with SUCCESS!
Major accomplishments have included:
1. New optimized targeted mutagenesis of Ft novicida
2. New plasmid-based mutagenesis of Ft holarctica (LVS)
3. New technique to mutagenize Ft tularensis and Ft holarctica
“Tulatron”
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Some of the things we have learned about genetic
manipulation of F. tularensis:
Use of Ft promoter to drive antibiotic resistance
is essential (both ermC and KanR work well)
Amount of flanking homology is critical for Ftt and Fth
(>=1 kbp)
Second recombination (loss of plasmid) does not
occur at high enough frequency without counterselection
Tulatron works well in Ftt and Fth
Transformation efficiency in Ftt still low, inhibits genetic
recombination, we are working at reducing this.
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•RNA loops EBS1 and EBS2
basepair with specific sequences
in target
•LtrA recognizes flanking sequences
•EBS1 and EBS2 can be “retargeted”
to recognize sites in target gene
•LtrA site preferences within target gene
are identified by Targetron computer
algorithm, which designs appropriate oligos
to retarget intron to your gene
Tulatron contains:
1. Ft promoters to drive
Antibiotic resistance
Intron RNP
2. Ft ori
3. ts mutation
4. Ec ori
5. KanR
6. lacZa “stuffer”
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Tulatron has worked to simultaneously inactivate both copies
of iglC, iglD, and vgrG found in duplicated FPI in Ftt.
We have successfully inactivated iglC1 + iglC2, iglD1 + iglD2,
and vgrG1 + vgrG2
IglC is most upregulated protein during intramacrophage growth.
IglC, IglD, VgrG are required for phagosome escape, intramac.
growth
VgrG is involved in secretion of proteins into mac. cytosol
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Ftt FPI mutants are highly attenuated in vitro and in vivo:
Intramac survival of iglD1 iglD2
3hrs
24hrs
10 MOI 100 MOI
iglC1 iglC2
CFU / well (log10)
CFU / well (log10)
6
5
4
3
2
1
0
10 MOI 100 MOI
Schu S4
6
3hrs
24hrs
5
4
3
2
1
Intramac survival of vgrG1 vgrG2
CFU / well (log10)
Intramac survival of iglC1 iglC2
10 MOI 100 MOI
iglD1 iglD2
F. tularensis tularensis strain
10 MOI 100 MOI
Schu S4
7
6
5
4
3
2
1
3hrs
24hrs
10 MOI 100 MOI
vgrG1 vgrG2
Schu S4
LD50
(intranasal in mice)
iglC1 iglC2
>9 X 105
iglD1 iglD2
>4.8 X 106
vgrG1 vgrG2
>1.9 X 106
<10
Schuh S4 WT
10 MOI 100 MOI
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However, mice infected with FTT FPI mutants via intranasal route
(105-106 CFU) were not protected against subsequent Schuh S4
challenge via intranasal route (0% survival following challenge
with 80-800 CFU)
We substituted 2 other mutations in place of 2 FPI mutants
since this was not a productive avenue for vaccine strains
using SchuhS4 as a platform.
1. FTT0748: Identified by Weiss et al. (PNAS 104:6037) in
STM screen in Ftn. Required for suppression of IL-1b release in
infected macrophages, ~103 decrease in virulence (s.c.), encodes
transcriptional activator.
We constructed a FTT0748 mutant in Schuh S4 via Targetron.
We tested this mutant for virulence in mice via intranasal route,
NOT ATTENUATED.
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2. nadM: We identified a C-terminal insertion in nadM of
Ftn as being attenuated for virulence.
Huang et al., Structure 16:196
•
Ft NadM is bifunctional
enzyme, N-terminus has NMNadenylyl-transferase activity (aa
1-174) and C-terminus has ADPribose pyrophosphatase activity
(aa 200-347)
TN
•
Tn insertion inactivated
ADPRP domain, not essential
NMNAT domain
•
The ADPRP domain is predicted to be
involved in recycling NAD, but no specific function yet
determined
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The Fn nadM::Tn insertion (ADPRP) strain is
attenuated for virulence:
Intranasal inoculation∆nadM,
of mice
CFU 8.9 x 10
6
3
5
Survivors
•
nadM
104 CFU
4
3
WT WT, CFU 1.1 x 10
103 CFU
2
∆nadM, CFU 8.9 x 105
3
1
nadM
106 CFU
nadM/pnadM
104 CFU
KKF357, CFU 9.7 x 103
0
1
2
3
4
5
6
7
Days post-inoculation
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9
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We attempted to construct nadM (Cterm) mutation
in Schuh S4.
(One year later!): We tried different Targetrons
targeted to Cterminus, could identify insertions
in pool, but could never isolate pure mutant
We once saw what appeared to be “pure” mutant
by PCR, but this strain failed to grow upon
subsequent sub-culturing.
Because targetron insertion sites were not identical to
Tn insertion site in Ftn, we tried to move Tn insertion
from Ftn into Ftt via mating plasmid and
counterselection, but also failed by this technique (!)
Our conclusion: C-terminus of NadM may also be
essential in Ftt, mutation is impossible to achieve.
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•
We have opened new milestone (54) to create new
attenuated Schuh S4 mutants based on literature,
contract.
Our first two mutations: lpxF and atpC
LpxF is a Lipid A 4’-phosphatase (Wang et al PNAS
104:4136).
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•
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Mice infected s.c. with 106 CFU Ftn, all lpxF infected
animals survived (highly attenuated), attracted more
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neutrophils, higher pro-inflammatory cytokines.
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•
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AtpC is ATP synthase subunit
(Kraemer et al IAI 77:232).
atpC Ftn mutant is attenuated for
virulence via aerosol route
(FTN1645):
We have begun constructing
lpxF and atpC mutations in
Schuh S4 strain.
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We cannot transform SchuhS4 with linear DNA to
achieve recombination in chromosome.
We still have issues with low-level transformation
in Shuh S4 inhibiting genetic manipulation, this may
contribute to lack of recomb. with linear DNA.
Gallagher et al. (JBact 190:7830) inactivated four
different restriction enzymes in Ft novicida which
resulted in 5 log increase in transformation efficiency
Three of these four enzymes appear to be
pseudogenes in Schuh S4, only FTT1579 is intact.
One additional Ftt-specific restriction enzyme,
FTT523.
We are creating mutations in FTT1579 (already done)
and FTT523 to increase SchuhS4 transformation
frequency
We will also clone lRed genes into Ft plasmid to
facilitate recombination of linear DNA into
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chromosome.
Plans for next six months:
Milestone 52:
• Inactivate two restriction enzymes (FTT1579/FTT523)
in Schuh S4
• Test resulting strain for increased transformation
• Clone lRed recombinase genes in Ft plasmid
• Test transformed Schuh S4 for increased
recombination
Milestone 54:
• Create lpxF Schuh S4 strain
• Test lpxF Schuh S4 strain for virulence in mice
• Create atpC Schuh S4 strain
• Test atpC Schuh S4 strain for virulence in mice
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Problems encountered (last year):
Inability to knockout nadM gene in Schuh S4
Corrective action: we tried many different ways, times,
even (briefly) identified mutant, only to ultimately fail to
Isolate this mutant
Conclusion: this mutation is lethal in Schuh S4 (difference
between planned and actual progress).
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Milestone 50-A
Immunologic characterization of F.
tularensis subsp. novicida, subsp. tularensis
and LVS
F. novicida uvrA, uvrB
Double mutant
F. novicida uvrA+pdpD
F.novicida uvrB+pdpD
iglA, iglC, iglD
In vitro Growth
In vivo Bacterial Burden
LD50 determination
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Red: completed
Blue: Steps in the milestone
LVS: uvrA, uvrB
Schu4: iglC, iglD,
vgrG,
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
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Key Achievements
We evaluated the protective efficacy of additional mutants against
Francisella infection and host immune responses induced by the
vaccination.
 Mutants: 3 mutants were studied: ΔiglB (U112),
ΔiglD (SCHU S4), ΔvgrG (SCHU S4)
 Virulence: All mutants are attenuated in mice via intranasal (i.n.) challenge.
 Immune responses: All mutants stimulate significant amount of antibody.
 Protective efficacy:
SCHU S4 mutants (ΔiglD, ΔvgrG) provided minimal protection against
SCHU S4 i.n. challenge.
U112 ΔiglB provided partial protection against SCHU S4 challenge
(Cong et al.).
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Oral Immunization with KKF235 (ΔiglB of U112)
Provided Partial Protection Against SCHU S4 Challenge
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Milestone 53-A
Immunologic characterization of F.
tularensis SCHU S4 mutant strains
recA
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Green: in progress
Red: completed
Blue: Steps in the milestone
recA double mutants
In vitro Growth
In vivo Bacterial Burden
LD50 determination
nadM,
ipxF, atpC
In vitro Growth
In vivo Bacterial Burden
LD50 determination
Further immunological characterization
based on initial screen
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Key Achievements
We evaluated the virulence and protective efficacy of additional SCHU S4
mutants against wild type pulmonary challenge and host immune responses
induced following vaccination.
 Mutants: 3 mutants were studied: ΔrecA (KKT11), DFTT1579
(restriction enzyme; KKT19), and ΔrecAiglC (KKT23)
 Virulence:
 (1) ΔrecA and DFTT1579 replicated similarly in J774
macrophages to wild type SCHU S4, and showed no attenuation in
mice via intranasal challenge.
 (2) ΔrecAiglC double mutant showed minimal growth in J774
and is attenuated in mice by pulmonary challenge.
 Immune responses: Intranasal immunization with ΔrecAiglC stimulated
significant amount of antibody but failed to protect mice against
pulmonary SCHU S4 challenge.
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