The Genomic Blueprint of Salmonella enterica subspecies enterica serovar Typhi P-stx-12

Salmonella enterica subspecies enterica serovar Typhi is a rod-shaped, Gram-negative, facultatively anaerobic bacterium. It belongs to the family Enterobacteriaceae in the class Gammaproteobacteria, and has the capability of residing in the human gallbladder by forming a biofilm and hence causing the person to become a typhoid carrier. Here we present the complete genome of Salmonella enterica subspecies enterica serotype Typhi strain P-stx-12, which was isolated from a chronic carrier in Varanasi, India. The complete genome comprises a 4,768,352 bp chromosome with a total of 98 RNA genes, 4,691 protein-coding genes and a 181,431 bp plasmid. Genome analysis revealed that the organism is closely related to Salmonella enterica serovar Typhi strain Ty2 and Salmonella enterica serovar Typhi strain CT18, although their genome structure is slightly different.     

Protection against experimental salmonellosis by recombinant 49 kDa OMP of Salmonella enterica serovar Typhi: biochemical aspects

Typhoid fever is systemic illness caused by Salmonella enterica serovar Typhi (S. Typhi) in humans. Increasing multidrug resistant strains of S. Typhi and limited effect of available vaccines has necessitated exploring of new immunogens for protection against it. Earlier studies have shown that a crude preparation of outer membrane proteins (OMPs) of S. Typhi evokes strong immune response and induces a protective immunity against infection caused by diverse Gram-negative bacteria. In the present study we have evaluated the protective effect of a purified recombinant 49 kDa (r49kDa) OMP of S. Typhi alone or along with alum or complete Freund’s adjuvant, against a challenge by S. Typhi (0.4 × 50% lethal dose) by biochemical estimation of serum enzymes and oxidative stress enzymes in Swiss albino mice. There was a decrease in activity of alanine aminotransferase by 14.28%, 38.09%, 23.80%; aspartate aminotransferase by 6.25%, 25%, 16.25%; lipid peroxidation by 4.34%, 18.84%, 11.59%; and catalase by 8%, 14%, 10%, respectively, whereas increase in activity of reduced glutathione by 33.33%, 61.11%, 44.44%; glutathione peroxidase by 7%, 16%, 10%; and glutathione reductase by 8%, 20%, 12%, respectively, as compared to control animals challenged with bacteria without pre-immunization. The results indicated that immunization of mice with r49kDa OMP alone or in combination with adjuvants protected and normalized the liver. It reduces the development of oxidative stress in mice against Salmonella infection and the risk of getting typhoid. These results represent an additional supplement to our earlier reported data on protective immunity evoked by this protein.     

Identification of novel vaccine candidate against Salmonella enterica serovar Typhi by reverse vaccinology method and evaluation of its immunization

Salmonella enterica serovar Typhi (S. Typhi) is an essential enteric fever causing bacterium worldwide. Due to the emergence of multidrug-resistant strains, urgently attention is needed to prevent the global spread of them. Vaccination is an alternative approach to control these kinds of infections. Currently available commercial vaccines have significant limitations such as non-recommendation for children below six years of age and poor long-term efficacy. Thus, the development of a new vaccine overcoming these limitations is immediately required. Reverse Vaccinology (RV) is one of the most robust approaches for direct screening of genome sequence assemblies to identify new protein-based vaccines. The present study aimed to identify potential vaccine candidates against S. Typhi by using the RV approach. Immunogenicity of the best candidate against S. Typhi was further investigated. The proteome of S. Typhi strain Ty2 was analyzed to identify the most immunogenic, conserved, and protective surface proteins. Among the predicted vaccine candidates, steD (fimbrial subunit) was the best for qualifying all the applied criteria. The synthetic steD gene was expressed in E.coli, and the mice were immunized with purified recombinant steD protein and then challenged with a lethal dose of S. Typhi. Immunized animals generated high protein-specific antibody titers and demonstrated 70% survival following lethal dose challenge with S. Typhi. Pretreatment of the S. Typhi cells with immunized mice antisera significantly decreased their adhesion to Caco-2 cells. Altogether, steD as a protective antigen could induce a robust and long term and protective immunity in immunized mice against S. Typhi challenge.     

利用间接ELISA定量分析伤寒沙门菌表面呈现表达的流感病毒抗原

目的:建立定量检测伤寒沙门菌表面呈现表达的流感病毒抗原的间接ELISA方法。方法:ELISA板以2.5%的戊二醛溶液预处理,将呈现表达M2e等流感病毒抗原表位的伤寒沙门菌的全细胞抗原在ELISA板上进行干燥包被,通过间接ELISA确立全菌抗原的最佳包被浓度;分别采用化学合成多肽M2e和GST-M2e融合蛋白干燥包被ELISA板,绘制标准曲线,对沙门菌表面呈现表达的抗原进行定量分析;对多肽和融合蛋白干燥包被进行比较,同时确立用于定量表面展示量的回归方程。结果:用多肽包被测定的呈现表达的M2e分子数为9.8×104,以GST-M2e包被测定的呈现表达的M2e分子数为1.3×105。结论:利用全菌干燥包被ELISA板可以对伤寒沙门菌表面呈现表达的抗原进行很好的定量分析。     

Identification of in vivo induced protein antigens of Salmonella enterica serovar Typhi during human infection

During infectious disease episodes, pathogens express distinct subsets of virulence factors which allow them to adapt to different environments. Hence, genes that are expressed or upregulated in vivo are implicated in pathogenesis. We used in vivo induced antigen technology (IVIAT) to identify antigens which are expressed during infection with Salmonella enterica serovar Typhi. We identified 7 in vivo induced (IVI) antigens, which included BcfD (a fimbrial structural subunit), GrxC (a glutaredoxin 3), SapB (an ABC-type transport system), T3663 (an ABC-type uncharacterized transport system), T3816 (a putative rhodanese-related sulfurtransferase), T1497 (a probable TonB-dependent receptor) and T3689 (unknown function). Of the 7 identified antigens, 5 antigens had no cross-immunoreactivity in adsorbed control sera from healthy subjects. These 5 included BcfD, GrxC, SapB, T3663 and T3689. Antigens identified in this study are potential targets for drug and vaccine development and may be utilized as diagnostic agents.     

伤寒沙门菌非编码RNA617的分子鉴定及其对生物膜形成的影响研究

本研究旨在探讨伤寒沙门菌(Salmonella enterica serovar Typhi, S. Typhi)中非编码RNA617(non-coding RNA617,ncRNA617)的分子特性,并研究其对生物膜形成的影响及作用机制。采用Northern blot方法检测ncRNA617的表达,通过cDNA 5’末端快速扩增技术(5’-rapid amplification of cDNA end,5’RACE)和逆转录-聚合酶链式反应(reverse transcriotion-polymerase chain reaction,3’RT-PCR)实验分析ncRNA617可能的转录起始位点和终止位点;构建ncRNA617缺陷菌株、回补菌株和过表达菌株等相关菌株,通过生物膜形成实验,观察ncRNA617对伤寒沙门菌生物膜形成的影响,并用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)分析生物膜形成相关基因表达水平的变化,综合运用生物信息学方法预测ncRNA617和差异基因的结合区域,初步分析ncRNA617发挥调控作用的机制。结果显示,伤寒沙门菌确有ncRNA617的表达,长度约300 nt,其转录起始位点位于mig-14终止密码子下游967 nt处,终止位点位于t2681起始密码子上游 2 378～2 560 nt处。与野生对照菌株相比,ncRNA617缺陷菌株生物膜形成能力增强(P<0.05),回补菌株的生物膜形成能力恢复至野生菌株水平,过表达菌株的生物膜形成能力有所下降(P<0.05)。qPCR结果表明,ncRNA617可负向调控多个生物膜形成相关基因的转录表达水平(P<0.05)。经生物信息学方法预测发现,ncRNA617与差异基因有不同的结合区域。本研究结果提示,ncRNA617在伤寒沙门菌中存在,其长度约270～452 nt。ncRNA617可能通过靶向结合生物膜形成相关基因下调基因表达,从而负向调控伤寒沙门菌生物膜的生成。     

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

Macrophage cell death due to Salmonella enterica serovar Typhi and its acid stress protein has features of apoptosis

Salmonella spp. have been shown to cause apoptosis of various host cell types as a part of their infection process. However, the induction of apoptosis remains to be looked into under the different host environments including acidic stress experienced by the pathogen. In order to simulate the in vivo acidic conditions, we studied the potential of S. typhi and its protein expressed under in vitro acidic conditions to induce apoptosis in macrophages. Murine macrophages were isolated and interacted with serovar Typhi and its acid stress protein for different time periods. The assessment of nucleosomal DNA, and nuclear staining with H-33342 dye and flow cytometry indicated the occurrence of characteristic features of apoptosis. Analysis of data revealed that S. typhi caused apoptotic cell death in 61% of macrophages whereas stress-induced protein alone accounted for apoptotic cell death in 45% of macrophages. The present study, for the first time demonstrates the potential of stress-induced outermembrane component of S. typhi to induce apoptosis. Identification of such factors may offer new insights for understanding the pathophysiology of the disease during the host-pathogen interactions.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.     

The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi EnvZ are determinants of autophosphorylation and OmpR phosphorylation

An initial biochemical characterization of the Salmonella enterica serovar Typhi ( S . Typhi) EnvZ sensor protein and several mutant derivatives was performed. Autophosphorylation levels were higher for Escherichia coli EnvZ, intermediate for S. enterica serovar Typhimurium EnvZ and very low for S . Typhi EnvZ, in spite of their high amino acid sequence identity. Consequently, OmpR phosphorylation was related to EnvZ autophosphorylation. Among the mutant derivatives, a C354G mutation in S . Typhi EnvZ resulted in a substantial increase in autophosphorylation, while mutation of its other cysteine residue at position 277 to L or S decreased the EnvZ autophosphorylation level. Upon heterodimerization, the S . Typhi C354G mutant complemented the wild type in vitro , increasing the EnvZ-P yield of both monomers, in accordance with the model where EnvZ autophosphorylation occurs in trans , indicating that dimer formation is a dynamic process. Hence, the C354 and the C277 residues are fundamental in determining the particular intrinsic biochemical characteristics of EnvZ.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Coordinated expression and immunogenicity of an outer membrane protein from Salmonella enterica serovar Typhi under iron limitation, oxidative stress and anaerobic conditions

Summary Successful pathogens overcome the environmental stresses by the coordinated expression of various genes and eventually proteins. Since, the surface of the microbe is likely to come in contact with the host initially, an attempt was made to identify the outer membrane proteins (OMPs), if any, which may get expressed under more than one environmental conditions simulating the in vivo ones. In the present study, Salmonella enterica serovar Typhi was grown under iron-limited, oxidative stress as well as anaerobic conditions and the OMP profiles were compared. A 69 kDa OMP was found to express with enhanced intensity under the selected stress conditions in comparison to normal conditions. The phenotypic similarity among the proteins was assessed on the basis of their molecular weight, cross reactivity and HPLC. The protein expressed under oxidative stress and anaerobic conditions reacted with the antibodies raised against iron-regulated outer membrane protein (IROMP), indicating the sharing of at least some of the epitopes. A single peak observed after subjecting the pooled 69 kDa protein sample and appearance of a single band on SDS-PAGE thereafter, confirmed the purity and phenotypic similarity of the 69 kDa OMP. Reactivity of pooled 69 kDa protein with 85% of sera from typhoid patients revealed the in vivo expression of this protein. The results of this study indicate the coordination of this phenotype under iron stress, oxidative stress and anaerobic conditions. In view of the expression of the 69 kDa protein under the selected stress conditions and their in vivo immunogenicity, these findings may be relevant for the better understanding of the host–microbe interactions and for the further development of diagnostic and preventive strategies.     

Effects of yeast probiotic formulation on viability, revival and protection against infection with Salmonella enterica ssp. enterica serovar Typhimurium in mice

Aims:  To compare the effects of five yeast probiotic formulations on viability, revival and washout kinetic in the digestive tract of mice, and the protection against an experimental infection with Salmonella enterica serovar Typhimurium.
Methods and Results:  The number of viable cells in five commercial probiotic products codified as A, B, C and D ( Saccharomyces boulardii – lyophilized) and E ( Saccharomyces cerevisiae – aqueous suspension) was determined, as well as revival and washout kinetic in mouse intestine. Protective capacity was evaluated by survival rate and histopathology of liver and intestine of mice treated with each product and then challenged with Salm . Typhimurium.
Conclusions:  Product A contained the highest number of viable cells and, fed to mice, gave the highest counts of viable yeasts and the longest persistence in faeces. Probably as a consequence, the highest survival and protection of intestinal and hepatic tissues were observed when product A was used for mouse treatment. Product E showed low counts in the formulation and was not recovered from mouse intestine.
Significance and Impact of the Study:  Formulation (lyophilization or aqueous suspension) is an important factor for revival and survival of a probiotic product in vivo and consequently for its protective properties.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Multiple-locus variable-number tandem repeat analysis of Salmonella enterica subsp. enterica serovar Typhimurium: comparison of isolates from pigs, poultry and cases of human gastroenteritis

AIMS: Pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) profiles of 195 epidemiologically unrelated Salmonella Typhimurium strains isolated in 1997-2004 from pigs were analysed and the results compared to establish the discriminatory ability of each method. In order to investigate the epidemiology of S. Typhimurium from different populations, the VNTR profiles from pigs were compared with those obtained from 190 S. Typhimurium strains isolated from poultry and 186 strains isolated from human cases of gastroenteritis. METHODS AND RESULTS: A total of 195 strains of S. Typhimurium were tested by PFGE and VNTR. For PFGE, the restriction enzyme XbaI was used, and for VNTR, the number of repeats at five loci (STTR 9, 5, 6, 10pl and 3) were counted and assigned an allele number based on an established VNTR scheme. The results obtained showed improved discrimination of VNTR when compared with PFGE with 34 PFGE profiles identified compared with 96 different VNTR profiles for the pig isolates and 56 different VNTR types within the most common PFGE type. Within the three different populations, VNTR showed distinct subpopulations of VNTR type related not only to source, but also demonstrated common VNTR types within samples obtained from humans, poultry and pigs, especially in strains of phage type DT104. CONCLUSIONS: VNTR has taken the discrimination to a further level than that obtained through PFGE, and demonstrated an overlap in the genetic diversity of isolates tested across the three different populations, confirming previous suggestions that animals have an involvement in the dissemination of S. Typhimurium through the food chain. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Typhimurium remains an important concern as a food-borne zoonotic agent. The VNTR strategy described provides an accurate method of tracing strain dissemination, and adds a further level of discrimination to the PFGE type, providing potential benefits to epidemiological studies and the possibility of deciphering source attribution of cases.