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 17.1pir

• 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 pir.

• 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 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 .


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

Log1

0Recovery of wild type & mutant S. Abortusequi from

livers of IP inoculated mice

3 DPI

7 DPI

14 DPI

24 DPI

30 DPI


2

3

4

5

6

7

8


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


2

3

4

5

6


Log1


kidneys of IP inoculated mice 3 DPI

7 DPI

14 DPI

24 DPI

30 DPI


2

2.5

3

3.5

4


Log1

0Recovery of wild type and mutants of S. Abortusequi

from liver of orally inoculated mice

7 DPI

14 DPI

24 DPI


2

2.1

2.2

2.3

2.4

2.5

2.6

2.7


Log


spleens of orally inoculated mice

7 DPI

14 DPI

24 DPI


-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


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


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


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


-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


-5.0

0.0

5.0

10.0

15.0

20.0


% 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


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


0.00

1.00

2.00

3.00

4.00

5.00

6.00


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


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


05

1015202530354045


% 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


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


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


0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00


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


-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


-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


2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0


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


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.


Health & Medicine

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