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Characterisation of Salmonella Abortusequi Strains Harbouring Defined Mutations in aroA, htrA and phoP/Q Genes
Dr. Bhoj R singh, Principal Scientist (VM)I/C Epidemiology; Centre for Animal Disease Research and Diagnosis
Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, UP, India. TeleFax +91-581-2302188
Note: The work was conducted in 1999 at IAH Compton, Newbury, UK under Guidance of Dr. Tim Wallis.
• Salmonella enterica ssp. enterica serovar Abortusequi (S. Abortusequi) is a major cause of abortion in horses and of mortality in foals born from infected mares.
• Killed S. Abortusequi vaccines afford only partial immunity for 3-6 months.
• Aim was to generate defined mutants of S. Abortusequi and to characterize their behavior in vitro and in inbred mice.
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Attenuating mutations
• aroA is involved in shikimate biosynthesis.
• aroA mutants are auxotrophic for certain aromatic compounds which are at limited supply in mammalian tissues .
• htrA , a stress response gene encodes a periplasmic protease responsible for degradation of aberrant proteins which got accumulated during growth at >37C.
• htrA mutants are more sensitive to hydrogen peroxide thus less efficient to survive in macrophages .
• phoP and phoQ encode a two component regulator controlling a few virulence genes .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Mutants of S. Abortus-equi
• Mutants were made in a wild type S. Abortusequi strain isolated in 1969 from an aborted fetus as well as in virulence- plasmid cured background .
• Virulence plasmid was detected by PCR using two sets of primers for spvA and also by midiprep .
• Out of 4 clinical isolates only one contained the plasmid .
• Virulence plasmid was cured using principle of incompatibility with pLL6 , a temperature sensitive plasmid which is lost at 42 ºC
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
aroA mutation
• 1.676 kB segment having full length aroA was amplified from S. Typhimurium by PCR and TOPO-TA cloned.
• A 392 bp fragment was deleted from ORF of aroA by Hinc II digestion
• The aroA gene harbouring the deletion was then subcloned into a positive-selection suicide vector, pCVD442 which carries sacB and ampR genes .
• Construct was introduced into S. Abortusequi under antibiotic selection through conjugation with E. coli S 17.1pir
• Integrated vector was removed from Salmonella under sucrose selection (sacB) .
• Mutant strains were confirmed by PCR and southern blotting .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
htrA mutation
• 1.758 kB segment having full length htrA was amplified from S. Typhimurium by PCR and cloned into pUC19 digested with Sph I and Sac I.
• A 608 bp fragment was deleted from ORF of htrA by Hinc II digestion.
• htrA gene harbouring the deletion was then subcloned into pCVD442.
• Construct was introduced in to S. Abortusequi under antibiotic selection through conjugation with E. coli S 17.1 pir.
• pCVD442 was removed under sucrose selection (sacB) .
• Mutant strains were confirmed by PCR and southern blotting .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
phoP and phoQ mutants
• Made only in plasmid free background .
• Tn10 mutations transduced using bacteriophage P22.
• Used for only in vitro studies .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Characterization of mutants
• In vitro studies
• Intracellular survival and LDH release assays in: J 774 cells
• Bovine alveolar macrophages
• Horse blood mononuclear cells
• In vivo studies in inbred mouse model for: Pathogenicity
• Survival
• Antigenicity
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Observations ( mouse model-6w/o Balb/C females )
ORAL INOCULATION
• Doses S787 (wild type ) 2.8 x 108
• aroA787 (aroA mutant) 3.0 x 108
• htrA787 (htrA mutant) 3.2 x 108
• D787 (aroA & htrA mutant) 3.4 x 108
• Mice inoculated with wild type bacteria were lethargic, had rough coat and huddled together within 24 hours of inoculation but all recovered in next 24 hours .
• Mice inoculated with mutant strains showed no signs of illness.
• All mutants (but not wild type Salmonella) were cleared by 24 days post infection (DPI) .
• aroA mutants were never recovered from any of the visceral organ .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Intra-peritoneal inoculation (IP)
• Doses were 2 Log lower than used for oral inoculation .
• All mice inoculated with wild type got sick within 24 hours of inoculation and 4 developed general symptoms compelling for euthanasia .
• aroA and double mutants persisted in mice after 24 DPI but not at 30 DPI in livers and spleens .
• htrA mutants were isolated from liver but not from spleen up to 7th DPI .
• From kidneys , wild type and aroA mutants could be isolated up to 14th DPI .
• All mutants but no wild type Salmonella could be isolated up to 24th DPI from uteri of mice .
• Uteri of mice inoculated with wild type Salmonella were congested while from mice inoculated with mutants were full of clear fluid without any apparent congestion .
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Anti S.Abortus-equi titres in sera and uterine fluids of mice at 30th DPI
• -------------------------------------------------------------------------------------------------------------------------------------• Test Antigen Uterine fluid from Mice Serum from Mice
inoculated with inoculated with
• Heat killed aroA 787 htrA 787 D 787 aroA 787 htrA 787•
• Agglutination O 80 160 1280 20 0 0 • H 160 320 1280 0 0 0
•
•ELISA Sonicate 5120 20480 51200 2560 1280 160
••
••
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Recovery of Salmonella Abortusequi and mutants from different organs of orally inoculated mice at various time intervals
Organ Strains Detection of Salmonella after
7 DPI 14 DPI 24 DPI
Kidneys S787 2 (3 ) 1 ( 3 ) 1 ( 3 )
Kidneys aroA787 0 0 0
Kidneys htrA787 0 1 ( 3 ) 0
Kidneys D787 0 0 0
Uterus S787 0 0 0
Uterus aroA787 1 ( 3 ) 0 0
Uterus htrA787 0 0 0
Uterus D787 0 0 1 ( 3 )
2
3
4
5
6
7
8
S787 aroA787 htrA787 D787
Log1
0Recovery of wild type & mutant S. Abortusequi from
livers of IP inoculated mice
3 DPI
7 DPI
14 DPI
24 DPI
30 DPI
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
2
3
4
5
6
7
8
S787 aroA787 htrA787 D787
Log
10Recovery of wild type & mutants of S. Abortusequi from
spleens of IP inoculated mice
3 DPI
7 DPI
14 DPI
24 DPI
30 DPI
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
2
3
4
5
6
S787 aroA787 htrA787 D787
Log1
0Recovery of wild type & mutants of S. Abortusequi from
kidneys of IP inoculated mice 3 DPI
7 DPI
14 DPI
24 DPI
30 DPI
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
2
2.5
3
3.5
4
S787 aroA787 htrA787 D787
Log1
0Recovery of wild type and mutants of S. Abortusequi
from liver of orally inoculated mice
7 DPI
14 DPI
24 DPI
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
S787 aroA787 htrA787 D787
Log
10Recovery of wild type & mutants of S. Abortusequi from
spleens of orally inoculated mice
7 DPI
14 DPI
24 DPI
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
-10.0
-5.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
3 hour 12 hour
% L
DH re
leas
eLDH releaseby bovine macrophages on infection with plasmid cured mutant strains of S. Abortusequi (10 bacteria per cell)
PC
PC aroA
PC htrA
PC aroA+htrA
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
3 hour 12 hour
% L
DH
rele
ase
LDH release by bovine alveolar macrophages on infection with S. Abortusequi strains (10 bacteria
per cell)WT
PC
aroA
htrA
aroA+htrA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
3 hour 12 hour
Num
ber o
f bac
teria
in Lo
g10
Intracellular survival of plasmid cured mutants of S. Abortusequi in bovine alveolar macrophages
PC
PC aroA
PC htrA
PC aroA+htrA
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.001.002.003.004.005.006.007.00
3 hour 12 hourNum
ber o
f bac
teria
in Lo
g10
Intracellular survival of wild type and mutant S. Abortusequi strains in bovine macrophages (10 bacteria
per cell)
WT
PC
aroA
htrA
aroA+htrA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
-5.0
0.0
5.0
10.0
15.0
20.0
2 hour 12 hour 24 hour 30 hour
% L
DH re
leas
eLDH release by bovine macrophages on infection with wid
type and mutants of S. Abortusequi (1 bacteria per cell)
WT
PC
aroA
htrA
aroA+htrA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
-5.0
0.0
5.0
10.0
15.0
20.0
2 hour 12 hour 24 hour 30 hour
% L
DH
rele
ase
LDH release by bovine alveolar macro phages on infection with plasmid cured mutant strains of S. Abortusequi (1
bacteria per cell)
PC
PC aroA
PC htrA
PC aroA+htrA
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
2 hour 12 hour 24 hour 30 hourNum
ber o
f bac
teria
in L
og10
Intracellular survival of wild type and mutant strains of S. Abortusequi in bovine alveolar macrophages (1
bacteria per cell)
WT
PC
aroA
htrA
aroA+htrA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
2 hour 12 hour 24 hour 30 hour
Num
ber o
f bac
teria
in Lo
g10
Intracellular survival of plasmid cured mutants of S. Abortusequi in bovine alveolar macrophages (1 bacteria
per cell)
PC
PC aroA
PC htrA
PC aroA+htrA
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
05
1015202530354045
3 hour 12 hour 24 hour
% L
DH re
leas
eLDH release by J774 Cells on infection of wild type and mutant strains of S. Abortusequi (10 bacteria per cell)
WT
PC
aroA
htrA
htrA+aroA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
05
1015202530354045
3 hour 12 hour 24 hour
% L
DH re
leas
eLDH release by J 774 cells on infection with plasmid cured
mutant strains of S. Abortusequi (10 bacteria per cell)
PC
PC aroA
PC htrA
PCaroA+htrA
PC phoP
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 2 3
Num
ber o
f bac
teria
in L
og10
Intracellular survival of plasmid cured mutants of S. Abortusequi in J 774 cells (10 bacteria per cell)
PC
PC aroA
PC htrA
PCaroA+htrA
PC phoP
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 2 3
Num
ber o
f bac
teria
in Lo
g10
Intracellular survival of plasmid cured mutants of S. Abortusequi in J 774 cells (10 bacteria per cell)
PC
PC aroA
PC htrA
PCaroA+htrA
PC phoP
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
2 hour 12 hour 20 hour
Num
ber o
f bac
teria
in L
og10
Intracellular survival of wild type and mutant strains of S. Abortusequi in J 774 cells !) bacteria per cell)
WT
PC
aroA
htrA
htrA+aroA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
-70.0
-60.0
-50.0
-40.0
-30.0
-20.0
-10.0
0.0
10.0
20.0
8 Hour 30 Hour 48 Hour
% L
DH re
leas
eLDH release by horse blood mononuclear cells on
infection of plasmid cured mutants of S. Abortusequi (10 bacteria per cell)
PC
PC aroA
PC htrA
PC aroA+htrA
PC phoP
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
-35.0
-30.0
-25.0
-20.0
-15.0
-10.0
-5.0
0.0
8 Hour 30 Hour 48 Hour
% L
DH re
leas
eLDH release by horseblood mononuclear cells on infection
with wild type and mutant strains of S. Abortusequi (10 bacteria per cell)
WT
PC
aroA
htrA
aroA+htrA
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
2 hour 8 hour 30 hour 48 hour
Nu
mb
er
of
bacte
ria i
n L
og10
Intracellular survival of wild type and mutants of S.Abortusequi in horse blood mononuclear cells
WT
PC
aroA
htrA
aroA+htrA
PC aroA
PC htrA
PC aroA+htrA
PC phoP
PC phoQ
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
Conclusions
• S. Abortus-equi aroA and htrA mutants are attenuated in Balb/c mice.
• Mouse model is not ideal for Salmonella Abortusequi. High doses were needed to produce disease.
• No correlation between macrophage lysis in vitro and virulence in mice.
• aroA htrA double mutant is over-attenuated.
• htrA mutant was the most immunogenic.
• Effect of plasmid curing on virulence unclear.
• Mutants present in uteri at 24 Days Post Inoculation.
BR Singh, i/c Epidemiology , CADRAD, IVRI, Izatnagar
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