Characterisation of salmonella abortusequi strains harbouring defined mutations in aro a, htra and phopq genes

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 .

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

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 17.1λpir
• Integrated vector was removed from Salmonella under sucrose selection (sacB) .
• Mutant strains were confirmed by PCR and southern blotting .

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 λpir.
• pCVD442 was removed under sucrose selection (sacB) .
• Mutant strains were confirmed by PCR and southern blotting .

phoP and phoQ mutants
• Made only in plasmid free background .
• Tn10 mutations transduced using bacteriophage P22.
• Used for only in vitro studies .

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

Observations
( mouse model-6w/o Balb/C females )
ORAL INOCULATION
• Doses S787 (wild type ) 2.8 x 10
8
• aroA787 (aroA mutant) 3.0 x 10
8
• htrA787 (htrA mutant) 3.2 x 10
8
• D787 (aroA & htrA mutant) 3.4 x 10
8
• 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 .

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 .

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

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
Log10
Recoveryof wild type &mutant S. Abortusequi from
livers of IPinoculatedmice
3DPI
7DPI
14DPI
24DPI
30DPI

2
3
4
5
6
7
8
Log10
Recoveryof wild type & mutantsof S. Abortusequi from
spleens of IPinoculatedmice
3DPI
7DPI
14DPI
24DPI
30DPI

2
3
4
5
6
Log10 Recoveryof wild type & mutantsof S. Abortusequi from
kidneys of IPinoculatedmice 3DPI
7 DPI
14DPI
24DPI
30DPI

2
2.5
3
3.5
4
Log10
Recoveryof wild type and mutants of S. Abortusequi
fromliver of orally inoculated mice
7DPI
14DPI
24DPI

2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Log10
Recoveryof wild type & mutantsof S. Abortusequi from
spleens of orallyinoculatedmice
7DPI
14DPI
24DPI

-10.0
-5.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
3hour 12hour
%LDHrelease
LDHreleaseby bovinemacrophages on infection with plasmid
cured mutant strains of S. Abortusequi (10 bacteria per cell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoQ

0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
3 hour 12 hour
%LDHrelease LDH release by bovine alveolar macrophages on
infection with S. Abortusequi strains (10 bacteria
per cell)
WT
PC
aroA
htrA
aroA+htrA

0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
3hour 12hour
NumberofbacteriainLog10
Intracellularsurvival of plasmid cured mutants of S.
Abortusequi in bovine alveolarmacrophages
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoQ

0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
3hour 12hour
NumberofbacteriainLog10 Intracellularsurvival of wild type and mutant S.
Abortusequi strains in bovine macrophages(10 bacteria
percell)
WT
PC
aroA
htrA
aroA+htrA

-5.0
0.0
5.0
10.0
15.0
20.0
2hour 12hour 24hour 30hour
%LDHrelease
LDH release by bovine macrophages on infectionwith wid
type and mutants of S. Abortusequi (1 bacteria percell)
WT
PC
aroA
htrA
aroA+htrA

-5.0
0.0
5.0
10.0
15.0
20.0
2hour 12hour 24 hour 30hour
%LDHrelease
LDH release by bovine alveolarmacro phages on infection
with plasmid cured mutant strains of S. Abortusequi (1
bacteria percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoQ

0.00
1.00
2.00
3.00
4.00
5.00
6.00
2 hour 12 hour 24 hour 30 hour
Intracellular survival of wild type and mutant strains of
S. Abortusequi in bovine alveolar macrophages (1
bacteriaper cell)
WT
PC
aroA
htrA
aroA+htrA

0.00
1.00
2.00
3.00
4.00
5.00
6.00
2hour 12hour 24hour 30hour
Abortusequi in bovine alveolarmacrophages (1 bacteria
percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoQ

0
5
10
15
20
25
30
35
40
45
3hour 12hour 24hour
%LDHrelease
LDH release by J774 Cells on infection of wild type and
mutant strains of S. Abortusequi (10 bacteria percell)
WT
PC
aroA
htrA
htrA+aroA

0
5
10
15
20
25
30
35
40
45
3hour 12hour 24hour
%LDHrelease
LDHrelease by J 774 cells on infection with plasmid cured
mutant strains of S.Abortusequi (10 bacteria percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoP
PCphoQ

0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 2 3
Abortusequi in J 774 cells (10 bacteria percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoP
PCphoQ

0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
1 2 3
NumberofbacteriainLog10 Intracellularsurvival of plasmid cured mutants of S.
Abortusequi in J 774 cells (10 bacteria percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoP
PCphoQ

0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
2hour 12 hour 20 hour
Intracellularsurvival of wild type and mutant strains of S.
Abortusequi in J 774 cells !) bacteria percell)
WT
PC
aroA
htrA
htrA+aroA

-70.0
-60.0
-50.0
-40.0
-30.0
-20.0
-10.0
0.0
10.0
20.0
8Hour 30Hour 48Hour
%LDHrelease
LDH release by horse blood mononuclearcells on
infection of plasmid cured mutants of S. Abortusequi (10
bacteria percell)
PC
PCaroA
PChtrA
PCaroA+htrA
PCphoP
PCphoQ

-35.0
-30.0
-25.0
-20.0
-15.0
-10.0
-5.0
0.0
8Hour 30 Hour 48 Hour
%LDHrelease
LDH release byhorsebloodmononuclearcells oninfection
with wild type and mutantstrains ofS. Abortusequi(10
bacteria percell)
WT
PC
aroA
htrA
aroA+htrA

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
NumberofbacteriainLog10 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

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.

Characterisation of salmonella abortusequi strains harbouring defined mutations in aro a, htra and phopq genes

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