Identification and characterization of Salmonella Typhimurium peptidyl-prolyl cis-trans isomerase encoded by Ppi gene
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Date
2016
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Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS)
Abstract
species can cause wide-ranging disease from mild to moderate
food-poisoning enteritisto a systemic, sometimes fatal typhoid infections. Intraphagosomal
survival of S. Typhimurium, at least in part, depends upon detoxifying
phagocyte generated ROS/RNS. Primary anti-oxidant enzymes of Salmonella like
catalases and peroxidases playan important role to cope up oxidative stress by
catalytically degrading ROS/RNS. Proteins and peptides are the primary target of
oxidative damage. Among several changes in amino acids, conversion of cis to trans
is an important modification occurs during oxidative stress leading to protein
inactivation. Peptidyl-prolyl cis-trans isomerases (PPIase) catalyse the cis-trans
isomerization of the peptidyl- prolyl peptide bond in oligopeptides there by maintain
the proteins activity.
The aim of study was to identify and characterise peptidyl-prolyl cis-trans
isomerases (PPIases) from the bacteria Salmonella Typhimurium, the causative agent
of the disease Salmonellosis. The long term goal was to assess their potential as
vaccine candidates.
In current study, expression and purification of S. Typhimurium PpiA, PpiB
and PpiC (PPiase encoding genes) in recombinant form were performed. Then, cloned
PpiA, PpiB and PpiC were sequenced. There was 100 % homology observed with S.
Typhimurium LT2 serover.
PpiA, PpiB and PpiC were expressed in T7 lys E. coli cells. The expressed
recombinant PpiA, PpiB and PpiC proteins were purified by Ni-NTA affinity
chromatography. The MALDI-TOF MS and MS-MS were used to identify and
conform these proteins. The recombinant form PpiA, PpiB and PpiC proteins were
shown to have characteristic PPIase activity increasing with oxidative stress
conditions.
Further, to evaluate the role of the PpiA, PpiB and PpiC in S. Typhimurium
(ST) virulence, deletion mutant strains of a ppiA, ppiB and ppiC were generated
(ΔppiA, ΔppiB and ΔppiC). The ppiA, ppiB and ppiC gene deletion strains were found
to be stable and it did not show any growth defect in in vitro culture. The
susceptibility of ST compared with ΔppiA, ΔppiB and ΔppiC against oxidants such as
H2O2, HOCl and phagocytic cells. All there mutants (ΔppiA, ΔppiB and ΔppiC) were
shown more sensitive towards H2O2 and HOCl as compared to ST. It was observed
these mutant strains sensitive towards stationary-phase and heat survival. Further,
ΔppiA, ΔppiB and ΔppiC were also found to be more susceptible towards phagocytes
tested i.e. macrophage and monocytes derived macrophages cells. In conclusion, we
found that ST PpiA, PpiB and PpiC contributes resistance against oxidants and aids in
intra phagocytic survival in S. Typhimurium.
Description
Thesis on "Identification and characterization of Salmonella Typhimurium peptidyl-prolyl cis-trans isomerase encoded by Ppi gene" submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Manoj Kumawat.