The prevalence of Salmonella outer protein B (sopB) gene among different serovars of Salmonella enterica isolated from man, birds and animals was investigated by PCR amplification techni[r]
Trang 1Prevalence & phenotypic expression of sopB gene among clinical isolates of Salmonella enterica
H Rahman*
Department of Microbiology, College of Veterinary Sciences, Assam Agricultural University, Guwahati, India
Received December 28, 2004
Background & objectives: Salmonella induced enteritis is a complex phenomenon involving a number
of pathogenic factors Type III secretions play a central role in the development of Salmonella induced
enteritis One such Type III secretion protein is Salmonella outer proteinB (SopB) Reports on the
prevalence of this gene among different serovars of Salmonella of Indian origin appears to be lacking.
The present investigation reports on the prevalence of sopB gene and its phenotypic expression
(SopB protein) among different serovars of Salmonella enterica isolated from man and animals.
Methods: A total of 50 isolates of S enterica belonging to 11 serovars isolated from cases of enteric
infection in man, birds and animals were tested for the presence of sopB gene by polymerase chain
reaction (PCR) using its specific primers The in vitro phenotypic expression of SopB protein was
detected by dot-ELISA using anti-SopB serum.
Results: All the 50 isolates of S enterica belonging to 11 were found to carry sopB gene irrespective
of their serovars like Typhimurium, Enteritidis, Gallinarum, Choleraesuis, Virchow, etc., and source
of isolation Of these, 41 isolates were found to express sopB gene phenotypically as detected by
dot-ELISA using anti-SopB serum Of the different serovars, all but Gallinarum expressed Sop B protein
phenotypically in vitro.
Interpretation & conclusion: Our findings indicated that sopB gene was wildely distributed and
conserved among Salmonella irrespective of their serovars and source of isolation Further work
need to be done to study the factors associated with the phenotypic expression of this gene.
Key words Animals - man - polymerase chain reaction (PCR) - Salmonella - sopB gene - SopB protein - type III secretion
83
Salmonellae are widely distributed in nature and
cause a spectrum of diseases in man and animals
but their virulence factors responsible for the
induction of gastroenteritis and/or systematic infection are still poorly understood Moreover, the
different subspecies and serovars of Salmonella
*Present address: Principal Scientist & Head, Division of Veterinary Public Health, Indian Veterinary Research Institute Izatnagar,
Bareilly 243122 (U.P.), India
Trang 2differ considerably in their virulence for man and
animals1 S enterica serovar Typhi (S Typhi) is
highly pathogenic for humans but never associated
with animals, while S Gallinarum is a pathogen
for poultry but a rare cause of infections in
humans2 Salmonella possesses a dedicated protein
secretion system denoted as type III secretion
system (TTSS) which is involved in the early stage
of Salmonella infection3 This sophisticated system
is found to contribute to the pathogenesis by
directing secretion and translocation of several
virulence-associated proteins (effector proteins)
directly into the cytoplasm of host cells4 In recent
years several translocated effector proteins have
been identified that might be involved in the
pathogenesis of salmonellosis5-9 One such group
of effector proteins of Salmonella is Salmonella
outer proteins (Sop) These proteins are encoded
by sop genes and several polymorphisms of these
genes have been identified (sopA-E)10 Of the
different types of Sop proteins, SopB protein (a
60 kDa), initially identified in the culture filtrate
of S enterica serovar Dublin was found to be
associated with Salmonella induced diarrhoea and
gastroenteritis4 Though reports on the prevalence
of this gene among different serovars of Salmonella
isolated in different countries are available11, such
information on Salmonella from India appears to
be lacking The present investigation was therefore
carried out to report on the prevalence of sopB gene
and its phenotypic expression among different
serovars of S enterica isolated from man and
animals in India
Material & Methods
Bacterial isolates: A total of 50 isolates of S enterica
belonging to 11 different serovars isolated from human patients with gastroenteritis and birds and animals suffering from enteric infection were included in this study (Table) All these were isolated
in the Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India and serotyped at National Salmonella and Escherichia Centre, Central Research
Institute, Kasauli (HP), India A strain of Salmonella Dublin (2229) and a strain of Escherichia coli
(C-600) used as positive and negative controls,
respectively for sopB gene, were obtained from
Dr H Tschape, Director, Robert Koch Institute, Germany The strains were maintained on nutrient agar slants before use
Fig PCR reaction and gel electrophoresis of PCR products (sop B gene, 1348 bp) of Salmonella enterica serovars (1) S Dublin
2229 (+ve control), (2) S.Typhimurium, (3) Template (Target) DNA control (4) S Virchow, (5) S Choleraesuis (6) S Newport, (7)
S Typhimurium, (8) S Richmond, (9) E coli C-600 (-ve control), (M) Molecular standard, (10) S Bareilly, (11) S Agona, (12)
S Typhimurium, (13) S Enteritidis, (14) S Java, (15) S Enteritidis, (16) S Typhimurium.
1348 bp
Trang 3Detection of sopB gene by PCR amplification:
Bacterial cells from overnight cultures were
suspended in sterile distilled water and boiled at
1000C for 10 min After boiling, the cell suspensions
were cooled in ice bath and were immediately tested
for the presence of sopB gene by PCR amplification
technique12
Primers used for PCR reaction were sopB PRSB1
5-CAA CCG TTC TGG GTA AAC AAG AC-3 (upper
primer) and sopB PRSB2 5-AGG ATT GAG CTC
CTC TGG CGA T-3 (lower primer)4 (GENSET,
Singapore) The nucleotide sequence corresponds to
the respective gene from S Dublin (sop B:
AF060858)4 These primers flank a 1348 bp segment
in the sopB gene sequence (Fig.) The PCR mixture
(25 ml) contained 10x PCR buffer (Perkin-Elmer,
USA), 1 µM of each primer, 200 µM each of dATP
dGTP, dCTP and dTTP (Perkin-Elmer, USA), 1 mM
of MgCl2 solution, 0.25 U of Ampli Tag Gold
Polymerase (Perkin-Elmer, USA) and 2.5 µl of template (target) DNA preparation from test organism The PCR incubation was performed in a thermal-cycler (Perkin-Elmer, USA) in 30 cycles of denaturation (940C, 1 min), primer annealing (550C,
1 min) and primer extension (720C, 2 min) This was followed by incubation at 720C for 10 min and storage
at 40C Then a 15 µl aliquot of each PCR product was electrophoretically separated in agar gel (1%) containing 0.5 µl ethidium bromide per ml (Serva, Germany) The separated bands were visualized and analysed under a UV transilluminator (300 nm) and photographed using Gel Doc 2000 documentation system (Bio-Rad, USA)
Table Prevalence of sopB gene and its phenotypic expression among different serovars of Salmonella enterica isolated from human,
birds and animals
Salmonella enterica serovars Source No of isolates tested No of isolates positive in
PCR, Polymerase chain reaction
+, positive; -, negative
Trang 4Isolation of SopB protein secreted by different
strains of Salmonella: Bacteria were grown on Luria
broth (LB) agar overnight at 370C One colony from
agar plate was inoculated in 5 ml of LB broth
containing 0.3 M NaCl and incubated at 370C for 6
h on a rotary shaker (100 rpm) The culture was then
diluted 4 times in fresh LB broth (final volume 20
ml) and incubated at 370C for 18 h on a rotary shaker
(100 rpm) Then the culture was cooled in an
ice-bath for 30 min and centrifuged (20000xg, at 40C
for 1 h) The culture supernatant was collected and
filtered (0.45 mm, Sartorius, Germany) The protein
present in the supernatants was precipitated with
10 per cent trichloroacetic acid (Serva, Germany)
The sediments were dissolved in 0.4 ml of NaOH
(0.1 M) to which 2.0 ml ice cooled acetone (-200C)
was added and incubated at -200C for 20 min The
suspension was centrifuged (20000xg, at 40C for
15 min) The sediments were redissolved in 2.0 ml
of acetone (-200C) and incubated and centrifuged
as above The sediments were dried at room
temperature and dissolved in 0.1 ml of PBS (pH 7.2).
Detection of SopB protein by Dot-ELISA using
anti-SopB serum: Protein prepared from each isolate was
subjected to Dot-ELISA using anti-SopB serum for
the detection of SopB13 Two µl of each protein
preparation and its dilution were dotted on
nitrocellulose (NC) membrane strips (Sigma, USA)
and dried at 370C for 1 h The unsaturated sites were
blocked by immersing the strips in 1 per cent
solution of skimmed milk powder in PBS (0.01 M,
pH 7.2) for 1 h at 370C The strips were washed
three times in PBS-T (0.01 M PBS, pH 7.2 with 0.5%
Tween-20) for five min each The strips were dipped
in the anti-SopB serum diluted 1: 20,000
(predetermined) in Tris-buffer (0.02 M, pH 7.2) and
incubated at 370C for 1 h (antiserum to SopB protein
was obtained through the courtesy of Dr H Tschape,
Director, Robert Koch Institute, Germany) After
incubation, the strips were washed thrice in PBS-T
and incubated with anti-rabbit IgG-horse radish
peroxidase (HRPO) conjugate (Boehringer, Germany) at a dilution of 1:1000 for 1 h at 370C Finally, the strips were washed thrice in PBS-T and immersed in freshly prepared substrate solution (1 chloro-4 naphthol, Sigma, USA) containing 30 per cent H2O2 The enzymatic reaction was stopped by washing the strips in running tap water and a positive reaction was indicated by the presence of deep purple dot against a white background within
10 min
Results & Discussion
The prevalence of Salmonella outer protein B (sopB) gene among different serovars of Salmonella enterica isolated from man, birds and animals was
investigated by PCR amplification technique and its
in vitro phenotypic expression was detected by
dot-ELISA The organisms that gave rise to 1348 bp in
the sopB gene sequence as did by the reference strain S Dublin (2229) were taken as positive for the presence of sopB gene (Fig.) All the 50 isolates
of S enterica belonging to 11 serovars tested for the presence of sopB gene were found to carry sopB
gene irrespective of their serovars like Typhimurium, Enteritidis, Gallinarum, Virchow, Agona, Choleraesuis, Paratyphi B, Bareilly,
Newport, etc., and source of isolation (Table) The data obtained indicated that sopB gene was widely
distributed and conserved among all the serovars
of Salmonella and present in all clinical isolates.
These observations are in agreement with findings
of other workers4,6
The SopB protein secreted by different serovars
of S enterica and the reference strain S Dublin
(2229) was isolated and analyzed by dot-ELISA The isolates that produced 60.0 kDa proteins, which specifically reacted with SopB antibody were taken
as the producer of SopB protein Of the 50 isolates,
41 belonging to serovars Typhimurium, Enteritidis, Virchow, Agona, Choleraesuis, Paratyphi B,
Trang 5Bareilly, Newport, etc., were found to produce SopB
protein (Table) Although all the isolates of serovar
Gallinarum were found to harbour sopB gene, they
did not express it phenotypically in vitro The
missing phenotypic expression among genetically
sopB gene positive strains indicated that some
environmental signal might be necessary for
induction of sopB gene in S Gallinarum Earlier
studies revealed that some signals are required for
in vitro induction of proteins by genes14 The
optimum in vitro expression of Salmonella
enterotoxin (stn) gene was found to be associated
with a number of factors which include
co-cultivation of the organisms with host cell15,
autoinducers like norepinephrine available in the
intestine16, other environmental conditions like
cultivation of organism under shaking condition,
additional growth factors17, etc These factors might
have also been required for induction of sopB gene.
Further work on this regard is envisaged
The SopB protein is one of the important type III
secretions and associated with enteritis10 SopB is an
inositol phosphate phosphatase capable of
hydrolyzing several inositol phosphates resulting in
elevated cellular level of Ins(1,4,5,6)P4 which in turn
induces electrolytes and fluid secretion and
recruitment of polymorphnuclear (PMN) cells in
Salmonella infected intestinal mucosa18,19 Thus, the
SopB is regarded as a novel bacterial enterotoxin6
Further research on the mechanism of phenotypic
expression of this gene would be helpful in the
control of Salmonella induced enteritis.
Acknowledgment
The author thanks to Dr H.Tschape, Director, Robert Koch
Institute, Germany for providing anti-SopB serum.
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Reprint requests: Dr H Rahman, Head, Division of Veterinary Public Health, Indian Veterinary Research Institute
Izatnagar, Bareilly 243122, India e-mail: hr19@rediffmail.com