Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Senthilkumar B, Anbarasu K, Senbagam D, Rajasekarapandian M

1Centre for Biotechnology, Muthayammal College of Arts & Science, Rasipuram-637 408, Tamil Nadu, India

2Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India

3Department of Biotechnology, Vivekanandha College of Engineering for Women, Tiruchengode-637 205. Tamil Nadu, India

4Department of Zoology, Arignar Anna Government Arts College, Namakkal-637 001, Tamil Nadu, India

Induction of deletion mutation on ompR gene of Salmonella enterica serovar Typhi isolates from asymptomatic typhoid carriers to evolve attenuated strains for vaccine development

Senthilkumar B1*, Anbarasu K2, Senbagam D3, Rajasekarapandian M4

1Centre for Biotechnology, Muthayammal College of Arts & Science, Rasipuram-637 408, Tamil Nadu, India

2Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India

3Department of Biotechnology, Vivekanandha College of Engineering for Women, Tiruchengode-637 205. Tamil Nadu, India

4Department of Zoology, Arignar Anna Government Arts College, Namakkal-637 001, Tamil Nadu, India

Objective:To develop attenuated strains of Salmonella enterica serovar Typhi (S. typhi) for the candidate vaccine by osmolar stress.Methods:S. typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Namakkal, Tamil Nadu, India. Both strains were grown in LB (Luria Bertani) medium supplemented with various concentration of NaCl (0.1- 0.7M) respectively. The effect of osmolar stress was determined at molecular level by PCR using MGR 06 and MGR 07 primers corresponding to ompR with chromosomal DNA of S. typhi SS3 and SS5 strains. Attenuation by osmolar stress results in deletion mutation of the S. typhi strains was determined by agglutination assays, precipitation method, SDS PAGE analysis and by animal models.

Results:The 799 bp amplified ompR gene product from wild type S. typhi SS3 and SS5 illustrate the presence of virulent gene. Interestingly, there was only a 282 bp amplified product from S. typhi SS3 and SS5 grown in the presence of 0.5, 0.6 and 0.7 M NaCl. This illustrates the occurrence of deletion mutation in ompR gene at high concentration of NaCl. Furthermore, both the wildtype and mutant S. typhi outer membrane SDS-PAGE profile reveals the differences in the expression of ompF, ompC and ompA proteins. In mice, wild type and mutant strains lethal dose (LD50) were determined. The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU.mL-1. When the mice were injected with the mutants in same dosage, no clinical symptoms were observed; whereas the serum antibody titre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response. These results suggest that S. typhi SS3 and SS5 may be used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S. typhi strains were attenuated and could be good vaccine candidates in future.

ARTICLE INFO

Article history:

Received 24 September 2014

Received in revised form 10 October 2014

Accepted 15 November 2014

Available online 20 December 2014

Attenuated live vaccine

1. Introduction

Salmonella enterica serovar Typhi(S. typhi) are Gramnegative, motile, non-lactose fermenting, facultative, intracellular pathogenic bacteria causing typhoid fever exclusively in humans, responsible for both morbidity and mortality in worldwide. It has been well documented that typhoid fever is a systemic infection characterized by the presence ofS. typhiin the liver, spleen, and bone marrow, invades and survives within macrophages and tissues of the reticuloendothelial system[1].S. typhicolonizes gall bladder and survive in the infected individual after the symptoms disappeared, serving as a reservoir for the spreading of the typhoid fever[2]. In developing countries,Salmonellaeinfection poses a greatest health concern in the water, foodindustry[3] and continues to be a worldwide health problem. It has been reported that the morbidity of typhoid fever is highest in Asia with 93% of the global level (WHO). Number of studies has been describing the emergence of multidrug resistantS. typhiwhich renders concern on use of antibiotics in the treatment process[4-8]. Given the fact, currently use of vaccines to prevent the Salmonellae infections holds great promise in clinical research as an alternative approach to antibiotics as well to reduce the disease burden. It has been reported thatS. typhilike other enteric pathogens has to respond quickly to the changing host environments encounteredin vivo, which will exert different demands and stressesie. osmolarity, pH, oxygen tension and nutrient starvation on the bacterial cell[1]. Bacteria possess systems for sensing these external environments, responding by co-ordinately controlling the expression of genes whose products are employed to assist survival under different condition[9].

Traditional approaches to the development of vaccines for bacterial diseases include parenteral injection of purified components of live vaccines or killed whole cell organisms. In contrast live oral vaccines have several advantages over parenteral vaccine such as low cost, easy to preparation and administration, safe to the organism as well as to the administrator. But the development of live vaccines has limitations such as understanding of the pathogenesis of the disease at molecular level. It is prerequisite that the candidate live vaccine strains must be non- revertable genetic alterations that affect the virulence of the organism, but not its induction of an immune response. Attenuation of virulentSalmonellastrains has been demonstrated by evaluatingSalmonella typhimurium mutantsin the murine typhoid model[10].

Several classes and combinations of mutant ofSalmonella typhimuriumattenuation have already been reported, including those with mutations in genes encoding key enzymes in the aromatic biosynthetic pathway (aromutants) [11] and those with mutations in controlling the expression of genes in response to environmental stimuli (ompRmutants) [12]. It has been well documented that attenuated strains ofS. typhiin humans can stimulate both humoral and cellmediated immune responses. It is proven that cell-mediated immune responses are very important mediators of the protection conferred byTy21aand other attenuatedS. typhilive oral vaccine strains[13].

CVD 909 live attenuatedS. typhiinduces cell mediated immune response, however second dose affects ability due to constitutive expression of Vi Ag[14].S. typhiTy2derivative TSB7 harbouring deletion mutation in ssaVandaroCdemonstrated to elicite immune response[15].

The importance ofompR-envZgenes is well characterized inS. typhiin response to osmolar stress and its virulence in human beings[10]. Moreover, the mutation inompRcan attenuate the virulence characteristics also reported[1]. Considering the importance of NaCl concentration in the expression ofompRgene in the present study, we propose that a high osmolar concentration can be a strong stress and used for induction of deletion mutations inompRgene to attain attenuated strains which is not revert in animal models to develop candidate vaccines.

2. Materials and methods

2.1. Sample collection

Early morning stool samples were collected from the asymptomatic typhoid carriers in Namakkal District and transported to the laboratory using screw-capped tubes with Cary-Blair medium.

2.2. Identification of S. typhi strains

The preliminary morphological and biochemical tests[16] were performed for the identification ofS. typhiincluding Gram’s staining, motility test, catalase test, oxidase test, sugar fermentation, indole test, methyl red test, vogesproskauer test, citrate utilization test, triple sugar iron test and urease test.S. typhiwas also identified based on the growth pattern on enrichment medium (Selenite-F broth), Differential medium (Mac Conkey Agar), and Selective medium (Bismuth Sulphite Agar, Xylose, Lysine and Deoxycholate Agar).

2.3. Confirmatory test for identification of S. typhi strains

The bacterial agglutination test (high titre serum agglutination test) was adopted to identifyS. typhi[17]. A drop of saline was placed in clean microscopic slide and a small amount of culture ofS. typhifrom the solid medium was emulsified using inoculation loop followed by a drop of antiserum (SPAN) ofS. typhiwas added and mixed with sterile stick. The slide was observed for agglutination within 2 min.

2.4. Induction of mutation on SS3 and SS5 strains

LB broth was prepared and various concentrations of sodium chloride ranging from 0.1 to 0.7 M were supplemented.S. typhiSS3 and SS5 strains were inoculated into the LB broth and incubated for two weeks of period with repeated subculture. From these cultures, DNA was isolated[18] and subjected to amplification ofompRgene using MGR 06 and MGR 07 primers[1].

2.5. Polymerase chain reaction (PCR)

Total genomic DNA ofS. typhiisolates grown on LB broth was prepared by phenol/chloroform/isoamyl alcohol extraction (25:24:1) and spooling from ethanol, as described by Sambrookeet al[19]. RNA contamination in DNA was eliminated using RNase A (BioBasic INC, Canada) PCR was performed in a final volume of 50 μL containing 1 μL of genomic DNA (100 μg) as template, 0.5 μL of each primer, (ompRgene forward primer 5’AGG GGC GTT TTC ATC TCG-3’ (MGR 06) and reverse primer 5’-ACC AGG CTG ACG AAC AG-3’ (MGR 07) (First Base, Singapore) (20 μm), 48 μL of Master Mix (Promega) to make a final concentration of 15 mM MgCl2, 100 mM Tris-Hydrochloride, 4 μL of 2.5 mM dNTPs mix (dATP, dCTP, dGTP, dTTP) and 2.5 Units of Thermo stable DNA polymerase (Promega). Subsequent amplifications were then performed in a Thermal Cycler (Techgene, UK), according to the following profile: 35 cycles of 2 min at 94 ℃, 30 sec at 94 ℃, 1 min at 49 ℃, 1 min at 72℃ for extension, and 7 min at 72 ℃ for the final extension. Amplified products were purified using Eppendrof perfectprep gel cleanup kit and analyzed by electrophoresis in 1% (w/v) agarose (Sigma) gel. DNA sequence was done by automated sequencer at MWG Biotech Private Limited, Bangalore, India. DNA sequence data reported here was deposited in GenBank under the accession number of EU834745 and EU849617.

2.6. Detection of Vi antigen in SS3 and SS5 strains

S. typhistrains were placed on a clean glass slide, heated at 60 ℃ for 30 min and cool. Equal amount of Vi antiserum (Acme Progen Biotech India Pvt Ltd, India) was placed and mixed gently for agglutination test[1].

2.7. Detection of intra cellular accumulation of Vi polysaccharide

S. typhistrains were grown overnight on LB agar (Hi Media) plates containing aromatic compounds at 37 ℃. Cells were harvested, suspended in PBS and the optical density was adjusted to 0.8-1.0 at 650 nm. Then the cells were disrupted by sonication in 30 sec bursts for a total of 2.5 min with an interval of 30 sec on ice. Counter current immunoelctrophoresis was done[20] to detect intracellular accumulation of Vi polysaccharide.

2.8. Outer membrane protein isolation from SS3 and SS5 wild and mutant strains

To begin with total cell, envelopes were removed by sonication of aerobically grown LB broth cultures. The sonication was carried out with 5 mL aliquots of cells (OD at 650 nm of 0.8-1.0) suspended in 10 mM sodium phosphate buffer (pH 7.2). The resulting sonicated material was subjected to centrifugation to remove the cell debris at 100 000gfor 10 min. Eventually, the inner membrane was solubilized by 1% sodium lauryl sarcosinate treatment, and outer membrane enriched fraction was collected by a final centrifugation at 100 000gfor 30 min. The pellets were resuspended in phosphate buffer and then analyzed in 10% SDS-PAGE[21].

2.9. Assessment of virulence in mice

Six mice per group for SS3 and SS5 wild type strains and mutants were used. Female mice aged 6-8 weeks were used to determine LD50of the wild strains with 0.5 mL doses of log phase culture of attenuated SS3 and SS5 strains of having 3 log CFU.mL-1. Second injection was being given on 8th day with the same dose. After the 14th day mice was bleed by retero orbital plexus. The antiserum was separated and stored in aliquots with 0.1% sodium azide (Merck) at -20 ℃[22]. Mice were injected intraperitoneally with various log phase culture (12 h of LB broth culture again inoculated in the LB broth and incubated for 3 h) of 10 fold dilutions of SS3 and SS5 wild isolates were used. The animals were monitored for morbidity and mortality[23,24].

3. Results

3.1. Identification of S. typhi strains

Based on biochemical tests and specific morphological growth characteristics in the respective medium such as the production of red colour deposits in the Selenite F broth, jet-black colour colonies on BSA, red smooth colonies on XLD and colourless colonies on Mac Conkey agar, the isolates were identified asS. typhi. A clear agglutination of emulsified colony confirms the isolates wereS. typhi, with antiserum on the glass slide (bacterial agglutination test).

3.2. Amplification of ompR gene encoding the virulence region SS3 and SS5 strains

PCR primers MGR 06 (F) and MGR 07(R) corresponding to the region ofompRused to amplify the chromosomal DNA fromS. typhiisolates. The PCR products ofompRgenes were analyzed by agarose gel electrophoresis. The amplified product was also compared with standard DNA ladder (Sigma).S. typhiSS3 and SS5 strains were give rise to 799 bp forompR(Figure 1). In case of the mutants, 282 bpompRgene products (Figure 2) were observed using same MGR 06 (F) and MGR 07 (R) primers, and 517 bp was found to be deleted due to osmolar stress at 0.5, 0.6 and 0.7 M NaCl(Table 1).

Figure 1. Amplified ompR gene of S. typhi wild strains.Lane1-1 kb DNA ladder (Sigma), Lane 2- S. typhi SS3, Lane 3- S. typhi SS5.

Figure 2. Amplified ompR gene of S. typhi mutant strains.Lane1: 1 kb DNA ladder (Sigma), Lane 2: S. typhi SS3, Lane 3: S. typhi SS5.

Table 1NaCl concentration to induce mutation on S. typhi SS 3 and SS 5 strains.

3.3. Intra cellular accumulation of Vi polysaccharide

The lack of agglutination ofompRmutants with antiserum was due to a defect in the expression of Vi polysaccharide. Counter current immunoelctreophoresis was performed on the bacterial sonicates with Vi antiserum to test the presence of Vi polysaccharide. The sonicates of mutant strains failed to produce any line of precipitation indicated that Vi polysaccharide was not being accumulated intracellularly inompRmutants, whereas wild strains were produced the precipitation band (Figure 3).

Figure 3. Immunodiffusion pattern for sonicated S. typhi strain reacted with Vi antiserum.

Figure 4. SDS-PAGE (10%) analysis of OMPs from S. typhi upon treatment with various concentrations of NaCl.Lane 1-Protein molecular weight marker (GeNei, India), Lane 2-0.1 M NaCl osmolar induced S. typhi, Lane 3- 0.2 M NaCl osmolar induced S. typhi, Lane 4- 0.3 M NaCl osmolar induced S. typhi, Lane 5- 0.4 M NaCl osmolar induced S. typhi, Lane 6-0.5 M NaCl osmolar induced S. typhi, Lane 7- 0.6 M NaCl osmolar induced S. typhi.

3.4. Expression and regulation of the porins ompC and ompF in the SS3 and SS5 strains

BecauseompRis known to regulate expression of porin channel, in the present study we probe into the effect of theompRmutation on the expression ofompCandompFby comparing with outer membrane proteins of the mutant and wild type strains ofS. typhi. The expression ofompC,ompF, andompAin the mutants as well as in the wild type strains were analyzed by SDS-PAGE. We observedompF,ompCandompAwere found to be 36 kDa, 35 kDa and 33 kDa respectively in the wild type strain, whereas the mutant isolates showing onlyompAwhich do not have porin forming properties. Thus, it was found that expression ofompCandompFwas down regulated in mutants (Figure 4).

3.5. Phenotypic characterization of SS3 and SS5 mutant strains

Phenotypic test was carried out to identify the mutantstrains which were no longer agglutinated with Vi antiserum (Table 2). These strains were also screened for the presence ofompRdeletions. From these results, it is suggested that theompRcomponent systems were involved in the regulation of Vi synthesis inS. typhi.

Table 2 Agglutination test of S. typhi mutants grown on LB agar with different osmolarity.

3.6. Assessment of virulence in mice

LD50was found to be 3 log CFU.mL-1for SS3 and SS5 strains in this study. The experimental animals died within 72 h after inoculation intraperitoneally indicating these strains were virulent. However, the mutant strain injected mice group showed no mortality or any clinical symptoms. Serum was collected from the mutant mice injected with the attenuated strains of SS3 and SS5. The collected serum was added with broth culture of SS3 and SS5 wild-type strains on a glass slide. Agglutination within 2 min was observed and the results indicated antibody was raised against the mutant isolates without any symptoms. The immunized animal’s serum antibody titre was found to be 1:60. This study obviously revealed that the mice immunized with attenuated strains were found to be protected with elicited humoral immune response.

4. Discussion

Typhoid fever continues to be an important infection in endemic countries and among travellers to these areas. Typhoid fever has been declining in many middle-income countries, whereas the bulk of cases occur in the Indian subcontinent and in south-east Asia[25].

A total of 138 enteric fever cases were found in United Kingdom during 2005-2010. Of these 65% and 35% cases were caused byS. typhiandSalmonella paratyphirespectively. These individuals were acquired the infection form abroad in the age group 16-40 years and under 16 years. Indian, Pakisthani, Banhgladesh patients have been made the majority (87%) of the typhoid cases. New entrant and foreign visitors accounted for 10% of the cases, 92% cases had a history of recent foreign travel; 57%patients had travelled to visit friends and relatives and 23% patients were new entrants / foreign visitors from the Indian subcontinent[26].

Typhoid bacilli has become a major threat to the public due to the severity of the disease , recurrence of disease through asymptomatic carrier state[27] emergence of multi drug resistance[4,28] and its use as a potential candidate in bioterrorism[29], thereby it is necessary to develop potential candidate vaccine for typhoid fever[30].

Attenuated strain development for bacterial vaccines by virulent gene knock out is one of the key research areas at present against infectious bacterial diseases prevention. In this study, we described a modified approach for the generation of mutantSalmonellastrains to be attenuated against Salmonellosis in mice model study. Development of attenuatedS. typhistrains and their ability to elicit protective immunity in animal models have already been reported[31,32]. It is proved that live attenuated vaccines elicit potent cell mediated responses. It has also been proved that the family of two component regulatory system genes responses to various environmental stimuli in bacteria and its involvement in bacterial virulence. Towards that end it has been studied that alterations in porin channel by demanding bacterial growth with the presence of sugars[33]. On such alteration in theompCporin, R74C substitution (ompCR74C orompC1 cys) has been reported. TheompCandompFporin genes were transcriptionally regulated by a classical two component signal transduction regulatory system consistent of theompR-envZproteins[34,35]. Furthermore,Salmonellastrains harbouring mutations in two component regulatory system ofphoP-phoQ, could be attenuated to developSalmonellavaccines[36].

In vivo down regulation of Vi Ag provides proof of principle that it is possible to generate a live attenuated vaccine that induces specific antibodies after single oral administration[37].

The WHO has been recommended for the programmatic use of new-generation typhoid vaccines in high-risk areas of countries where typhoid fever is still endemic. Past and current typhoid vaccination programs that have taken place in Thailand (using the old whole-cell vaccine) and in China, Vietnam and India (using the new-generation injectable Vi polysaccharide vaccine)[38]. Vi polysaccharide vaccine will not protect the patients ifS. typhistrains are negative for Vi polysaccharide. The recent data of the community vaccination in high incidence areas of Kolkata, Karachi, and north Jakarta showed the cost effectives of Vi polysaccharide vaccine in children[39].

It is well documented thatompRgene ofS. typhiis involved in the regulation of the synthesis of Vi capsularpolysaccharide in response to osmolority. Considering all these facts we used high osmolar conditions to study the effect ofompRgene regulation in theSalmonellaisolates from asymptomatic typhoid carriers. Remarkably, in agreement with, Pickardet al[1], we found that Vi expression is very sensitive with osmolarity of the growth medium. Interestingly, our results showed that 0.5, 0.6 and 0.7 M of NaCl yielded mutant attenuated strains which leads to 282 bp PCR amplified products. We speculate that resulting mutant strains due to deletion of 517 bp inompRgene. Moreover, as expected[22,40] in this study, the SDS-PAGE results showed that in the wild type isolates the outer membrane proteinsviz.ompC,ompFandompAwere found to be 36, 35 and 33 kDa respectively. In contrast, the mutant strains only expressedomp Aat 33 kDa, other two proteins ofompCandFwere found to be down regulated.

In mice challenging study, the animals were died within 72 h of intraperitoneal injection of wild type strains SS3 and SS5 indicating that the strains were virulent, whereas, no clinical symptoms were observed when the mice were intraperitoneally injected with the high concentration NaCl osmolar stress inducedompRmutant strains alternatively, they produce antibody titre after 15 days.

In 2007, a live attenuated vaccine against typhoid fever DV-STM-07 has been shown to be potent in murine model of salmonellosis[41]. The vaccine developed through independent chemical conjugation of Vi polysaccharide ofS. typhiand o polysaccharide of S. paratyphi A to CRM197, a non toxic mutant of diphtheria toxin. The vaccine was found to be inducing humoral response than unconjugated Vi polysaccharide[42].

With these findings, we believe that the NaCl osmolar stress induced mutants may be used for vaccine development. In conclusion, this is the first time, we report thatS. typhiisolated from asymptomatic typhoid carriers with its down regulated expression of Vi polysaccharide upon NaCl osmolar stress induced deletion mutation inompRgene would be a rational object against which to elicit antibody for a candidate of typhoid vaccine.

Conflict of interest statement

We declare that we have no conflict of interest.

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ment heading

10.1016/S1995-7645(14)60165-6

*Corresponding author: Balakrishnan Senthilkumar, Centre for Biotechnology, Muthayammal College of Arts & Science, Rasipuram-637 408, Tamil Nadu, India. Tel: +91-9443286292

Fax: +91+91-4287 220227

E-mail: senthilmicro@yahoo.co.in

Salmonella enterica serovar Typhi Mutants