Indian Journal of Experimental Biology Vol. 43, July 2005, pp. 631-634

Occurrence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals

H Rahman l*, W D Hardt2, H V Murugkar3 & D K Bhattacharyya

Department of Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati 781 022, India

Received 15 September 2004; revised 23 March 2005

Salmonella pathogenesis is a complex phenomenon and a Type III secretion system plays a central role in the development of Salmonella-induced enteritis. One such Type III secretion protein is Salmonella outer protein E (SopE). Prevalence 'of sopE gene and its phenotypic expression (SopE protein) among different serovars of Salmonella enterica isolated from man and animals were investigated. Of 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE, 130 strains belonging to three serovars viz., Enteritidis, Gallinarum and Virchow were found to carry sopE gene irrespective of their source of isolation when tested by PCR amplification technique using its specific primers. Of these 130 strains, 112 strains were found to express SopE protein phenotypically as detected by Dot-ELISA using SopE antibody. Among the different serovars tested only serovars Gallinarum, Enteritidis and Virchow expressed SopE protein phenotypically in vitro. Role of SopE protein in pathogenesis of salmonellosis has been discussed.

Keywords: PCR, Salmonella. SopE. Type III secretion

Salmonellae are widely distributed in nature and cause a spectrum of diseases in man and animals but their virulence factors responsible for induction of gastroenteritis and/or systematic infection are still poorly understood. Moreover, the different subspecies and serovars of Salmonella differ considerably in their virulence for man and animals l

. S. enterica serovar Typhi (S. Typhi) is highly pathogenic for humans, but nearly never associated with animals. While S. Gallinarum is a pathogen for poultry, but a rare cause of infection in humans2

. Like many other Gram negative bacteria, Salmonella possesses a dedicated protein secretion system denoted 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 virulence­associated proteins (effector proteins) directly into the cytoplasm of host cells4. In recent years, several translocated effector proteins have been identified that

Present address: - 1.3Division of Animal Health, ICAR Research Complex for NEH Region, Umroi Road, Umiam, Meghalaya 793103, India 2Max von Pattenkofer Institute, Pattenkofer Stresse 9a, 80336, Munchen, Germany *Correspondent author- Phone: (0364) 2570071 (0); 2570033 (R); Fax: (0364) 2570071 Email: hrl9@rediffmail.com

might b~ involved . in the pathogenesis of salmonellosis5

-7

• Invasion of Salmonella into the epithelial cells is considered to be an essential feature of pathogenesis. Of the different types TTSS of

. Salmonella, Salmonella outer proteins (Sop) are found to contribute to the pathogenesis by directing secretion and translocation of several bacterial effector proteins into the cytoplasm of host cells5

.

These proteins are encoded by sop genes8• Several

polymorphisms of sop genes have been identified (sopA-E)9. SopE protein contributes to the expression of Salmonella invasion by stimulating membrane ruffling6

• SopE protein (29 kDa) was initially identified in the culture medium of Salmonella enterica serovar Dublin4. The sopE gene is located outside SPI-l (Salmonella Pathogenicity Island) and is found to be encoded within a temperate phage7

.

Although the reports on the prevalence of this gene among different serovars of Salmonella isolated in different countries are available5

•9

, such information on Salmonella of Indian origin appears to be meagerlO. The present investigation reports on the prevalence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica isolated from man and animals in India.

Materials and Methods Bacterial strains - A total of 305 strains of

Salmonella enterica belonging to 11 different

632 INDIAN J EXP BIOL, JULY 2005

serovars isolated from human patients with gastroententls and animals suffering from salmonellosis were available for this study (Table 1). All these strains were isolated at 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) were used as positive and negative controls, respectively for sopE gene. These isolates were maintained on nutrient agar slants before use.

Detection of sopE gene by peR amplification -Bacterial cells from overnight cultures were suspended in sterile distilled water and boiled at 100°C for 10 min. After boiling, the cell suspensions were cooled in ice bath and were immediately tested for the presence of sopE gene by PCR amplification technique. Primers used for PCR reaction were sopE­P4 5'-ACA CAC TTT CCA CGA GGA AGC G-3' (upper primer) and sopE-M2 5'-GGA TGC CTT CTG ATG TTG ACT GG-3' (lower primert These primers flank a segment (398-bp) in the sopE gene sequence (Fig. 1). The PCR mixture (25 J.ll) contained lOx PCR buffer (Perkin-Elmer, USA), IJ.lM of each primer, 200 J.lM each of dATP, dGTP, dCTP and dTTP (Perkin-Elmer, USA), ImM of MgCh solution, 0.25 U of AmpliTaq Gold Polymerase (Perkin-Elmer, USA) and 2.5 J.ll 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 (94°C, 1 min), primer annealing (55°C, 1 min) and primer extension (nOC, 2 min). This was followed by incubation at n oc for 10 min and storage at 4°C. Then aliquot (I5 J.ll) of each PCR product was electrophoretically separated through agar gel (2%) containing 0.5 J.lI ethidium bromide per ml (Pharmacia, USA) and was visualized and photographed by Gel Doc System (Pharmacia, USA).

Isolation of SopE protein secreted by different strains of Salmonella - Bacteria were grown on LB agar overnight at 37°C. One colony from agar plate was inoculated in 5 m] of LB broth containing NaCl (0.3 M) and incubated at 37°C for 6 hr on a rotary shaker (l00 rpm). The culture was then diluted 4 times in fresh LB broth (final volume 20 ml) and incubated at 37°C for 18 hr on a rotary shaker

(100 rpm). Then the culture was cooled in an ice-bath for 30 min and centrifuged (20,000 g, at 4°C for 1 hr) . The culture supernatant was collected and filtered (0.45 J.lm, Sartorius, Germany). The protein present in the supernatants was precipitated with 10% (v/v) of 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 (-20°C) was added and incubated at -20°C for 20 min. The suspension was centrifuged (20,000 g, at 4°C for 15 min) . The sediments were redissolved in 2.0 ml of acetone (-20°C) and incubated and centrifuged as above. The sediments were dried at room temperature and dissolved in PBS (PH 7.2) and subjected to Dot­ELISA.

Detection of SopE protein by Dot-ELISA llsing anti-SopE serum - Protein prepared from each strain was subjected to Dot-ELISA using anti-SopE serum obtained through the courtesy of Dr W 0 Hardt, Max von Pattenkofer Institute, Munchen, Germany for the detection of SopE protein II. Two J.ll of each protein preparation and its dilution were dotted on nitrocellulose (NC) membrane strips (Sigma, USA) and dried at 37°C for 1 hr. The unsaturated sites were blocked by immersing the strips in I % solution of skimmed milk powder in PBS (0.01 M, pH 7.2) for 1 hr at 37°C. The strips were washed three times in PBS-T (O.OIM PBS, pH 7.2 with 0.5% Tween-20) for 5 min each. The strips were dipped in the anti -SopE serum diluted 1 :60,000 (predetermined) in Tris-buffer (0.02 M, pH 7.2) and incubated at 37°C for 1 hr. After incubation, the strips were washed thrice in PBS-T and incubated with anti-rabbit IgG-horse radish peroxidase (HRPO) conjugate (Boehrin&er, Germany) at a dilution of I: 1000 for 1 hr at 37°C. Finally, the strips were washed thrice in PBS-T and immersed in freshly prepared substrate solution (1 chloro-4 naphthol, Sigma, USA) containing 30% H20 2 (Sigma, USA). 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. The antibody was used at a dilution of 1:60,000.

Results and Discussion Prevalence of Salmonella outer protein E (sopE)

gene and expression of the protein among different serovars of Salmonella enterica are presented in Table 1 and Fig. 1. The organisms that gave rise to PCR

RAHMAN el al.: OCCURRENCE OF SOPE GENE AND ITE PHENOTYPIC EXPRESSION 633

products of 398 bp using sopE gene primers as did by the reference strain S. Dublin (2229) were taken as positive for the presence of sopE gene (Fig. 1), Of the 305 strains of S. enterica belonging to 11 serovars tested for the presence of sopE gene, 130 strains belonging to three serovars namely Entertidis (117), Gallinarum (7) and Virchow (6) were found to carry sopE gene irrespective of their source of isolation (Table 1). Of the 130 positive strains for sopE gene with PCR only 112 strains belonging to three serovars viz., Enteritidis (103), Gallinarum (5) and Virchow (4) produced positive results by SopE dot-ELISA. The sopE gene or the product could not be detected either by PCR or with dot-ELISA in the other serovars like Typhimurium, Choleraesuis, Java (Paratyphi B), Bareilly, Newport, etc. tested in this study (Table I). As expected, results indicated that sopE gene appeared to be distributed and conserved among only

Tablel- Prevalence of sopE gene and its phenotypic expression among different serovars of Salmonella enterica

isolated from man and animals

Salmonella ellterica serovar

Typhimurium

Enteritidis

Weltevreden

Choleraesuis

Paralyphi B var Java

Virchow

Paratyphi C

Bareilly

Bovismorbi ficans

Gallinarum

Newport

Total

Source No. of No. of strains

Human

Pigs

Birds

Calves

Human

Pigs

Birds

Calves

Pigs

Birds

Pigs

Human

Pigs

Birds

Birds

Calves

Pigs

Pigs

Calves

Calves

Birds

Birds

strains positive in tested -P-C-R~-D-o-t--E-L-IS-;-\

27

51

42

7

37

35

41

4

6

5

II

2

3

6

4

2

6

2

2

4

7

I

305

o o o o 37

35

41

4

o o o o o o 4

2

o o o o 7

o 130

o o o o 33

31

35

4

2

2

5

112

a few serovars of Salmonella irrespective of their source of isolation which was in corroboration with the observations of earlier workers'J·IO. Although SopE protein is one of the important type III secretions and associated with Salmonella invasion of epithelial cells by stimulating membrane ruffling which is an essential feature of Salmonella pathogenesis8

, its location outside SPI-I and is encoded by a temperate bacteriophage, sopE gene may not be present in all strains of SalmonellaS , This suggests that there may be some other proteins with functions similar to those of SopE exist in Salmonella. A recent study identified such a protein termed as SopE2 in Salmonella 12 which was found to be present in all strains of Salmonella and encoded by a chromosomal gene8

. It is interesting to note that sopE gene is present in each and every clinical isolate of S. Enteritidis, S. Gallinarum and S. Virchow, while it was found to be absent in all other serovars tested including S. Typhimurium (Table I) . This observation is in accordance with earlier studies where sopE gene has also been found to be absent in all isolates of S. Typhimurium of Indian origin 10. The other reason for absence of sopE gene may be presence of sopE gene only in a few phage types of

bp

bp

Fig. 1 - Distribution of sopE gene in Salnumella isolates (Lane I : S. Dublin 2229, +ve control; Lane 2: E. coli C-600, -ve control ; Lane 3,4: S. Bovismorbificans; Lane 5, 6: S. Typhimurium; Lane 7,21 : DNA Ladder; Lane 8: S. Choleraesuis; Lane 9-14,26: S. Enteritidis; Lane '15-18: S. Virchow; Lane 19: S. Paratyphi B; Lane 20, 22-24: S. Gallinarum; Lane 25: S. Weltevreden)

INDIAN J EXP BIOL, JULY 2005

S. Typhimurium (DT68 , DTl04, DTl75, DT204)9. Although phage types of S. Typhimurium in the present study were not known, earlier studies on S. Typhimurium of Indian ongm have reported prevalence of DT003, DT096, DTl93, DT004 phage types 13.

Although the sopE gene was detected in 130 strains, only 112 strains expressed SopE protein phenotypically indicated that sopE gene was not fully expressed in vitro (Table 1). In vitro expression of a gene is associated with a number of factors like suitable environmental conditions'4, contact with host ceil's, suitable autoinducers'6, etc. which might have not been adequately present in the methods used for

-the detection of SopE protein in the present study.

Acknowledgement The authors are thankful to Department of

Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi for providing financial help for the present study and the Director, N:ttional Salmonella and Escherichia Centre, Central Research Institute, Kasauli (HP), India for serotyping the Salmonella isolates.

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