Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid

The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.     

A new alpha-helical coiled coil protein encoded by the Salmonella typhimurium virulence plasmid.

A new protein of Salmonella typhimurium was identified and characterized. The gene (tlpA) encoding this protein (TlpA) was isolated from the large virulence-associated plasmid of S. typhimurium and sequenced in order to predict the primary structure of TlpA. tlpA encodes a 371-amino acid soluble protein with a calculated M(r) of 41600 and pI of 4.63. Secondary structure predictions and sequence statistics of TlpA indicated a predominant alpha-helical configuration and presence of heptapeptide repeat motifs characteristic of coiled coil proteins. Purified TlpA was shown to have biochemical properties similar to those of coiled coil proteins, including adoption of an alpha-helical configuration and a tendency to form homodimers. Furthermore, TlpA possessed heat resistance, evidence for a chain register and altered mobility in urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels which are characteristics of tropomyosins. TlpA shows 32% overall sequence similarity with rat cardiac myosin and 36% similarity with horse platelet beta-tropomyosin over 226 residues, whereas selected regions possessed significant sequence identities with myosins, tropomyosins, and alpha-helical surface proteins of Streptococcus pyogenes. Our results indicate that TlpA represents a new member of prokaryotic coiled coil proteins.     

Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to human salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.     

Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid

The ability of intracellular growth is plasmid-dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96-kb plasmid pEX102 and transformed into minicell-producing E. coli. Plasmid-directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96-kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC).     

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.     

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype.     

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.     

The plasmid of Salmonella typhimurium LT2

Summary Methods of clonal analysis were applied to the study of heterogeneity of the progeny after crosses of 4 donor strains (Hfr H, Hfr C, KL 16 and KL 99) with 3 recipient strains (PC 0212, AB 712 and ECK 022). Three markers were used in each cross. The distal one was the selective marker. The inheritance of two additional proximal markers characterized the heterogeneity of clones originating from particular zygotes. In most crosses the percentage of heterogeneity exceeded 30. One of the recipient strains, obtained by conjugation of the conventional strain PC 0212 with the donor Hfr H revealed unusual properties in respect to heterogeneity. Exconjugants derived from this recipient (ECK 022) and donor Hfr H and Hfr C had a heterogeneity index of about 5%. It is shown that this unusual behavior reflects a very fast process of segregation of recombinants.In crosses with the donors KL 16 and KL 99 the same recipient revealed normal indices of heterogeneity. All these data are explained assuming that there exists a specific genetic marker which determines the process of decay of merozygotes. Tentatively it is called het. Its approximate localization was deduced from specifically designed experiments, in which the heterogeneity of the progeny was found very different, when the donor KL 16 transmitted different parts of its chromosome to the recipient ECK 022.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.     

Lack of evidence of an association between the carriage of virulence plasmid and the bacteremia of Salmonella typhimurium in humans

The involvement of the virulence plasmid (pSTV) of Salmonella typhimurium in human salmonellosis was examined. Most of the 224 clinical strains isolated from the blood (53) and nonblood samples (171) contained a 90 kb or larger plasmid, most of which were pSTV. The rates of pSTV carriage in the isolates showed no statistically significant difference between those derived from the blood and those from other sources (87% vs. 83%; chi2=0.49, 0.1相似文献   

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe

M.D. HAMPTON, E.J. THRELFALL, J.A. FROST, L.R. WARD AND B. ROWE. 1995. Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991–92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.     

Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium

Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts. Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro. Many virulence genes are specifically induced at particular sites during infection or in cultured host cells. The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host. In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo. Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.     

Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium

Salmonella typhimurium is an intracellular pathogen that can survive and replicate in macrophages. One of the host defense mechanisms that S. typhimurium encounters upon infection is superoxide produced by the phagocytes' NADPH-oxidase. Salmonella has evolved numerous ways of coping with superoxide in the extracellular environment. In addition, Salmonella has to defend itself against superoxide produced as a by-product of aerobic respiration. Over the last decade, research on bacterial mutants has led to the identification of Salmonella strains that differ from their parental strain in susceptibility to superoxide in vitro. However, the consequences of such mutations for bacterial virulence are highly variable, indicating that superoxide sensitivity per se is not a characteristic that renders Salmonella less virulent. By discussing various bacterial mutants classified according to their in vitro sensitivity to superoxide, we will exemplify the complex mechanisms that Salmonella has evolved to cope with superoxide stress.