Rational design of Salmonella recombinant vaccines

Salmonella enterica is an important pathogen of animals and humans causing a variety of infectious diseases. The large number of cases of typhoid fever due to S. enterica serovar Typhi infections gives rise to the continuous need for improved vaccines against this life-threatening infection. However, S. enterica is also an interesting organism to act as a live attenuated carrier for the presentation of recombinant heterologous antigens. Comprehensive experimental studies have been performed and a detailed knowledge of the molecular mechanisms of important virulence factors is available. This allows the rationale design of improved Salmonella carrier strains and the development of novel strategies for the expression and presentation of recombinant antigens. Here, we review recent advances in generation of live attenuated Salmonella vaccines and discuss criteria for expression strategies of heterologous antigens by Salmonella carrier strains.     

Targeting of the actin cytoskeleton during infection by Salmonella strains

Many bacterial pathogens produce virulence factors that alter the host cell cytoskeleton to promote infection. Salmonella strains target cellular actin in a carefully orchestrated series of interactions that promote bacterial uptake into host cells and the subsequent proliferation and intercellular spread of the organisms. The Salmonella Pathogenicity Island 1 (SPI1) locus encodes a type III protein secretion system (TTSS) that translocates effector proteins into epithelial cells to promote bacterial invasion through actin cytoskeletal rearrangements. SPI1 effectors interact directly with actin and also alter the cytoskeleton through activation of the regulatory proteins, Cdc42 and Rac, to produce membrane ruffles that engulf the bacteria. SPI1 also restores normal cellular actin dynamics through the action of another effector, SptP. A second TTSS, Salmonella Pathogenecity Island 2 (SPI2), translocates effectors that promote intracellular survival and growth, accompanied by focal actin polymerization around the Salmonella-containing vacuole (SCV). A number of Salmonella strains also carry the spv virulence locus, encoding an ADP-ribosyl transferase, the SpvB protein, which acts later during intracellular infection to depolymerize the actin cytoskeleton. SpvB produces a cytotoxic effect on infected host cells leading to apoptosis. The SpvB effect appears to promote intracellular infection and may facilitate cell-to-cell spread of the organism, thereby enhancing virulence.     

The frequency of fim genes among Salmonella serovars

Salmonella serovars were examined for the presence of fim gene sequences using specific DNA probes. All strains, regardless of their ability to express surface-associated fimbriae, retain a considerable amount of DNA homologous to the gene probes used. The phenotypically non-fimbriate FIRN and non-FIRN strains of S. typhimurium retain detectable amounts of fim gene sequences and, therefore, may not be genotypically non-fimbriate. The MS adhesin can be expressed by type 2 fimbriate bacteria when they are transformed with discrete regions of the fim gene cluster. However, this conversion to a hemagglutinating phenotype is not associated with a small region of DNA. Therefore, the inability of type 2 fimbria-producing strains of Salmonella to mediate hemagglutination does not appear to be due to a small deletion in a single fim gene.     

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.     

Maintenance of the Salmonella-containing vacuole in the juxtanuclear area: a role for intermediate filaments

Until recently, intermediate filaments (IF) were thought to be only involved in resistance to physical stress and mechanical integrity of cells and tissues. Recent data indicate that IF play a much more important role in cellular physiology including organelle structure and positioning within the cell. Here, we show that Salmonella enterica serovar Typhimurium (S. typhimurium) induces in epithelial cells and macrophages the formation of an aggresome-like structure with a dramatic remodelling of cytoplasmic IF (vimentin and cytokeratin) networks and the adaptor proteins 14-3-3 which are recruited around intracellular S. typhimurium microcolonies. These rearrangements are not necessary for bacterial replication. Depletion of vimentin and cytokeratin by siRNA indicates that IF remodelling is required to maintain Salmonella microcolonies in the juxtanuclear area.     

Heteroduplex analysis of Salmonella virulence plasmids and their prevalence in isolates of defined sources.

Strains of the Salmonella serovars S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis harbour large plasmids which are required for extraintestinal colonization after oral infection of mice. Electron microscopic heteroduplex analysis showed that these virulence plasmids share large regions of homology. Nine hundred and eighty-six isolates of different origins were analysed for the presence of these plasmids by using a cloned fragment of a S. choleraesuis virulence plasmid as a gene probe. Virulence plasmids were detected in nearly 100% of strains isolated from animal organs or human blood. Frequencies of detection ranged from 48 to 87% in strains of faecal, food or environmental origin. These results suggest that Salmonella virulence plasmids are required for systemic infections in humans and livestock.     

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD₅₀ value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.     

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichia coli that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Certain DNA probes derived from accessory genes of cloned K88 and F41 determinants hybridize with Escherichia coli strains that express K88 or F41 and with certain other E. coli strains that do not express these antigens. We found that these probes hybridized with human enteroinvasive E. coli, and with bovine E. coli isolates which produced a fatal septicemia in experimentally infected piglets. These strains did not hybridize with probes derived from the structural subunit genes encoding the K88 and F41 antigens. E. coli strains isolated from turkeys with septicemia, Shigella and Salmonella strains did not hybridize to the K88 and F41 accessory gene probes. The K88 and F41 accessory gene probes hybridized with a 200 kb plasmid which is required for invasion by human enteroinvasive E. coli. The K88 and F41 accessory gene homology in the bovine isolates was located on a 150 kb transmissible plasmid but was unrelated to plasmids encoding aerobactin, Vir, or colicin V, which are suspected virulence factors in septicemic E. coli. A common plasmid-encoded antigen was associated with bovine isolates that hybridized with the K88 and F41 accessory gene probes. This included strains which express CS31A, a surface antigen associated with bovine septicemic E. coli, which also hybridized with the K88 and F4 accessory gene probes. The results suggest that the K88 and F41 accessory gene probes hybridized with sequences that may be associated with a common mechanism of pilus expression in distinct groups of E. coli pathogens.     

Virulence and vaccine potential of phoP mutants of Salmonella typhimurium

We have constructed Salmonella typhimurium phoP mutants and found them to be avirulent and able to induce a protective immune response. BALB/c mice survived challenge with phoP derivatives of the highly virulent S. typhimurium strains SR-11 and SL1344 when inoculated intraperitoneally and per oral with doses equivalent to 10⁴ 50% lethal doses (LD₅₀) of the parent virulent strains. The avirulent mutants were able to establish an infection of the Peyer's patches of orally infected animals for up to 10 days after inoculation but were very inefficient at reaching the spleens. Despite the low level of infectivity of these mutants, immunized animals developed a delayed-type hypersensitivity (DTH) response to Salmonella antigens and resisted challenge with up to 10⁴ LD₅₀ of the virulent parent strain 30 days after immunization.     

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10³ cfu mL⁻¹ for Campylobacter jejuni, 10⁴ cfu mL⁻¹ for Salmonella, and 10⁵ cfu mL⁻¹ for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples (n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.     

Supplement 1995 (no. 39) to the Kauffmann-White scheme

This supplement reports the characterization of 23 new Salmonella serovars recognized in 1995 by the WHO Collaborating Centre for Reference and Research on Salmonella: 11 were assigned to S. enterica subsp. enterica, 5 to subspecies salamae, 2 to subspecies diarizonae, 3 to subspecies houtenae, 1 to subspecies indica, and 1 to S. bongori.     

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio – Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n = 20), Typhimurium (n = 4), Zanzibar (n = 2), Manhattan (n = 1), Bovismorbificans (n = 1), Amsterdam (n = 1), Saint Paul (n = 1), Kentucky (n = 1) and Muenster (n = 1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1–5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.     

Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.     

Supplément 1996 (no. 40) to the Kauffmann-White scheme

This supplement reports the characterization of 13 new Salmonella serovars recognized in 1996 by the WHO Collaborating Centre for Reference and Research on Salmonella: 8 were assigned to S. enterica subsp. enterica, 3 to subspecies salamae and 2 to subspecies diarizonae.     

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45 + 26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.     

Supplement 1989 (n° 33) to the Kauffmann-White scheme

This supplement reports the characters of 18 new Salmonella serovars recognized in 1989 by the WHO Collaborating Centre for Reference and Research on Salmonella: 11 were assigned to S. enterica subsp. enterica, 3 to subspecies salamae, 1 to subspecies arizonae, 2 to subspecies houtenae and 1 to S. bongori.     

Type IV(B) pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CFTR is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the ?F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells.     

Induction of TNF-α mRNA in murine macrophages by virulent and avirulent strains ofSalmonella choleraesuisserovar Typhimurium and serovar Choleraesuis

TNF-αmRNA induction in murine macrophages by virulent and avirulentSalmonellastrains was measuredin vitroby RT-PCR method. Virulence plasmid-cured strains ofS. choleraesuisserovar Typhimurium and serovar Choleraesuis, andrpoS-defective mutant ofS. choleraesuisserovar Typhimurium induced significantly higher level of TNF-αmRNA than their parent (virulent) strains in macrophages of C3H/HeN mice. When macrophages of LPS-low responder (C3H/HeJ) mice were used, the difference of the induction level was not observed, indicating that LPS was involved in the enhanced level of TNF-αmRNA induction by avirulentSalmonellastrains. LPSs from virulent and avirulent strains were analysed, but no difference was found for cytokine-inducing activity, and chemical properties. Those results suggested that avirulentSalmonellastrains were damaged more easily, and released more LPS in macrophages to enhance TNF-αinduction.     

Extra-intestinal infections with multiply drug-resistantSalmonella typhimurium in hospitalized patients in jordan

During the 12-year period from 1978 to 1989,Salmonella typhimurium was the most frequently isolated serotype (592/1,500; 39.5 %) among all clinicalSalmonella isolates at Jordan University Hospital. Extra-intestinal infections due toSalmonella typhimurium accounted for 68 (11.5 %) isolates. A high percentage ofSalmonella typhimurium strains (52–90 %) were resistant to commonly used drugs in Jordan. Most of the antibiotic-resistant strains ofSalmonella typhimurium (10/12) examined which were from extra-intestinal sources contained a large plasmid (55 MDa) in addition to two to four small plasmids. These strains were also able to transfer most or part of their drug resistance in vitro. It is concluded that the invasive potential ofSalmonella typhimurium isolates is probably associated with the presence of a large virulence plasmid and multiple antibiotic resistance.     

Interactions of Yersinia enterocolitica with polarized human intestinal Caco-2 cells

The in vitro interactions of Yersinia enterocolitica, Salmonella typhimurium and Escherichia coli with polarized human colonie carcinoma (Caco-2) cells are described. Invasion of a confluent Caco-2 cell monolayer by Yersinia and Salmonella took place within 4 h after contact, which was in marked contrast to E. coli which did not invade Caco-2 cells. Cytoplasmic extrusions developed on the apical membrane and indicated the site of entrance of bacteria into the Caco-2 cells. Intracellular Yersinia and Salmonella were surrounded by a vacuolar membrane. Single as well as multiple bacteria were enclosed within a single vacuole. At 6 h after contact some of the intracellular yersiniae were found free in the cytoplasm. Furthermore, morphological signs of degeneration of Caco-2 cells such as vacuolization and autophagy were observed. Caco-2 cells infected with Salmonella also showed degenerative changes but the salmonellae resided within membranebound vacuoles in contrast to Yersinia. These observations are in contrast to those described for the invasion of other cell lines (not derived from intestinal epithelium) by Yersinia and may reflect more closely the interactions between Yersinia and the intestinal epithelium during gastrointestinal infection.