Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis

An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy. Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S. typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age. A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S. typhimurium or S. enteritidis showed that delta cya delta crp S. typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella. The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated. S. enteritidis and S. typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella. S. typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S. enteritidis showed no pathological lesion in the visceral and reproductive organs. Vaccination with chi 3985 prevented transmission of S. typhimurium or S. enteritidis into eggs laid by vaccinated layers with no effect on egg production. To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers.     

Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.     

Site-directed mutational analysis of the osmotically regulated proU promoter of Salmonella typhimurium

We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control. We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect. However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.     

Inoculum composition and Salmonella pathogenicity island 1 regulate M-cell invasion and epithelial destruction by Salmonella typhimurium

In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S. typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S. typhimurium. Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S. typhimurium. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems.     

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, alphaS239 to alphaT244, in the alpha subunit M1-M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between alphaG240 and alphaT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues alphaG240, alphaE241, alphaK242, and alphaT244 line a narrow part of the channel, in which this gate is located.     

Isolation of a cytotoxin from L-form Salmonella typhimurium

A cytotoxic protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 micrograms/ml, with a complete lysis obtained at 5 micrograms/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1-0.5 micrograms/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor alpha. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 micrograms/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium

The dependence of hormonal responses to exercise on sexual maturation was tested in three-year longitudinal experiment on 34 girls (11-12 years old at the beginning of the study). Sexual maturation of the girls was evaluated using Tanner scale. Girls were divided into three groups: maturation stages 1-2, 2-4 and 4-5. Children performed a 20-min cycle exercise at 60% of maximal oxygen uptake (VO max) once a year. Cortisol, insulin, somatotropin, beta-estradiol, progesterone and testosterone were determined in venous blood by RIA procedures. High basal levels of beta-estradiol and somatotropin appeared in stages 2-4 (387 +/- 92 pmol.l-1) and 12.9 +/- 2.85 ng.ml-1, respectively) and 4-5 (358 +/- 54 pmol.l-1) and 14.3 +/- 1.53 ng.ml-1, respectively). The basal progesterone level increased with maturation, testosterone appeared in the blood in stages 2-4 and 4-5. The exercise resulted in increased levels of cortisol and somatotropin, and a drop in insulin in all girls. The cortisol response was most pronounced in stage 1-2. Postexercise insulin concentration was the highest in stage 4-5. beta-estradiol level increased by 23% in stages 1-2 and 4-5, while the response was insignificant in stages 2-4. Exercise-induced progesterone increase was significant in stage 4-5. In conclusion, sexual maturation associates with several quantitative changes in exercise-induced hormonal responses.     

NAD-dependent DNA-binding activity of the bifunctional NadR regulator of Salmonella typhimurium

NadR is a 45-kDa bifunctional regulator protein. In vivo genetic studies indicate that NadR represses three genes involved in the biosynthesis of NAD. It also participates with an integral membrane protein (PnuC) in the import of nicotinamide mononucleotide, an NAD precursor. NadR was overexpressed and purified as a His-tagged fusion in order to study its DNA-binding properties. The protein bound to DNA fragments containing NAD box consensus sequences. NAD proved to be the relevant in vivo corepressor, but full NAD dependence of repressor activity required nucleotide triphosphates. DNA footprint analysis and gel shift assays suggest that NadR binds as a multimer to adjacent NAD boxes. The DNA-repressor complex would sequester a potential RNA polymerase binding site and thereby decrease expression of the nad regulon.     

Elimination of Salmonella typhimurium infection by the strategic movement of pigs

Three field investigations were carried out to assess the feasibility of raising salmonella-free finishers from pigs born in infected herds, by moving the pigs to clean and disinfected facilities before their expected exposure to the bacteria from the environment. Three herds with persistently high levels of subclinical infection with S typhimurium in the finishing pigs were used. They practised all-in all-out management in the nurseries and in the grower units. A total of 844 pigs were moved, either at weaning, from the nursery, or from the grower unit to newly built or rigorously cleaned and disinfected finishing units with no known history of salmonella infection. No detectable infection was observed at slaughter either serologically or bacteriologically by random testing of the pigs which had been moved, whereas a proportion of the pigs raised at the same time in the continuous systems on the farms were found to be infected.     

Supramolecular structure of the Salmonella typhimurium type III protein secretion system

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.     

Low pH adaptation and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium periodically confronts acid environments during its life. These situations arise in chemically compromised ponds, soil, degradative cellular organelles, host digestive systems, and may even result from byproducts of their own metabolism. The levels of acid that are encountered range from mild to extreme. As a neutralophile, S. typhimurium prefers to grown in pH environments above pH 5.5. They can survive down to pH 4 for extended periods of time. However, the limits of endurance can be stretched if the organisms are first adapted to a moderate acid pH before exposing them to acidity below pH 4.0. This adaptation, called the acid-tolerance response (ATR), includes several log phase and stationary phase systems. Some of these systems are dependent on an alternate sigma factor for RNA polymerase called sigma s, whereas other systems are sigma s-independent. A key to the ATR is the synthesis of a series of acid shock inducible proteins (ASPs), 51 for log phase ATR and 15 for stationary phase ATR. Some of these ASPs require sigma s for their synthesis; others require the participation of the ferric uptake regulator protein Fur. Effective acid tolerance involves RecA-independent DNA repair systems, iron, and facets of fatty acid metabolism. Aspects of medium composition and carbon metabolism are also known to influence the nature of acid tolerance in this organism. In addition to aiding survival in the natural non-host environment, aspects of acid tolerance are also tied to virulence, as evidenced by the involvement of the mouse virulence locus mviA and the fact that acid-sensitive strains of S. typhimurium exhibit reduced virulence. This review summarizes these aspects of acid adaptation and includes a discussion of acid-regulated gene expression.     

Neutrophils prevent extracellular colonization of the liver microvasculature by Salmonella typhimurium

The early course of hepatic infection with the facultative intracellular bacterial pathogen Salmonella typhimurium was examined in control mice and in mice selectively depleted of neutrophils by treatment with a granulocyte-specific monoclonal antibody. The results show that >200-fold more salmonellae were recovered in livers of the latter group of mice than in livers of the former group by 24 h of parenterally initiated infection. Comparative histological examination of the livers from both groups of mice indicated that neutrophils participate in early anti-Salmonella defense in the liver in part by aborting infection in permissive hepatocytes and by inhibiting extracellular bacterial colonization of the hepatic microvasculature. It is shown in addition that systemic salmonellosis was also severely exacerbated in neutropenic mice infected intragastrically with the pathogen.     

Phosphate groups in lipopolysaccharides of Salmonella typhimurium rfaP mutants

Lipopolysaccharides (LPS) of Salmonella typhimurium rfaP mutants and of a galE strain as a control were subjected to analysis by 31P-NMR in order to assess the location of phosphate groups. This was done to obtain direct proof for our earlier finding by chemical analysis that phosphate was lacking in the core oligosaccharide part of the mutant LPS, whereas the core oligosaccharide normally contains several phosphate groups. Such phosphate deficiency has been associated with the increased susceptibility of the rfaP mutants to hydrophobic antibiotics and detergents. Analysis of the de-O-acylated LPS derivatives of S. typhimurium rfaP strains SH7770, SH8551, and SH8572 by 31P-NMR revealed an almost total lack of phosphate groups in the core oligosaccharide part, the LPS phosphates being largely accounted for by the two monophosphate monoesters of lipid A, linked to positions C-1 and C-4' of the lipid A backbone. Core oligosaccharide-linked phosphates were detected in minor proportions only, indicating the presence of some normally phosphorylated core oligosaccharide, due to the inherently leaky nature of the mutation.     

The subunit structure of alpha-acetohydroxyacid isomeroreductase from Salmonella typhimurium

Alpha-Acetohydroxyacid isomeroreductase from Salmonella typhimurium has a native molecular weight of 220,000. The constituent polypeptide chains exhibit anomalous but unimodal electrophoretic migration on sodium dodecyl sulfate-urea polyacrylamide gels. The subunit molecular weight, determined by sedimentation equilibrium in 6 M guanidine hydrochloride, is 57,000. The apparent tetrameric nature of the native enzyme was confirmed by determining the types of oligomers formed upon cross-linking with dimethylsebacimidate. Analysis of tryptic peptides suggests that the polypeptide chains have an identical amino acid sequence. Carbohydrate analysis, ultraviolet absorption spectrum, and atomic absorption spectrum are consistent with the lack of cobalamine and cobalt. The Michaelis constants are as follows: alpha-acetolactate, 2.9 x 10-4 M; alpha-aceto-alpha-hydroxybutyrate, 7.8 x 10-4 M; NADPH, 1.5 x 10-5 M; Mg2+, 7.7 x 10-4 M. The catalytic constants (molecules of substrate catalyzed per min per molecules of enzyme) for alpha-acetolactate and alpha-aceto-alpha-hydroxybutyrate are 1,100 and 4,700, respectively. Comparative tryptic peptide analysis and immunological analysis show that alpha-acetohydroxyacid isomero-reductase and biosynthetic L-threonine deaminase bear no structural relationship and therefore rule out a "shared structure" hypothesis for the putative involvement of L-threonine deaminase in the synthesis of alpha-acetohydroxyacid isomeroreductase.