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.     

Genetic Mapping of Is200 Copies in SALMONELLA TYPHIMURIM Strain Lt2

The wild-type Salmonella typhimurium strain LT2 contains six copies of the insertion sequence element IS200 which is unique to Salmonella. We have determined the chromosomal locations of all six copies of IS200 in strain LT2. This was done by mapping the positions of Tn10 elements inserted near each copy of IS200. Such Tn10 insertions were detected by Southern hybridization as IS200-containing restriction fragments with altered electrophoretic mobility. The copies are located at quite evenly spaced sites in the chromosome. Some are found in regions with many known genes; others are in regions with few known functions. There is no indication of a possible function for IS200. The method described here should be applicable to the mapping of IS elements in general.     

Genomic cleavage map of Salmonella typhi Ty2.

The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively. The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, and labelling with 32P, and reelectrophoresis and named in alphabetical order. Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S. typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10. All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S. typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S. typhimurium LT2 to AGCEFDB in S. typhi. In addition, there is a 500-kb inversion which covers the terminus region. Comparisons of lengths of segments between genes showed that S. typhi has segments which differ in size from those in S. typhimurium. The viaB locus, for synthesis of the Vi antigen of S. typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

The susceptibility of Salmonella typhimurium LT2 and S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Divergent effects of tumor necrosis factor-alpha and lymphotoxin-alpha on lethal endotoxemia and infection with live Salmonella typhimurium in mice

During septic shock with Gram-negative microorganisms, mortality is determined by two independent factors: high concentrations of circulating proinflammatory cytokines and multiplication of the microorganisms in the organs of the host. We studied the role of endogenous tumor necrosis factor-alpha (TNF) and lymphotoxin-alpha (LT) in the pathogenesis of lethal endotoxemia and infection with viable Salmonella typhimurium. Compared to wild-type control mice, TNF-/-LT-/- knock-out mice were more resistant (100% versus 25% mortality) to a lethal challenge with LPS, due to a significantly decreased production of the proinflammatory cytokines TNF, IL-1alpha and IL-1beta. In contrast, TNF-/-LT-/- mice were highly susceptible to infection with viable S. typhimurium as compared to wild-type mice (100% versus 0% mortality), and this was accompanied by a 100-fold greater bacterial load in their organs. The effect of endogenous TNF and LT during infection was mediated by a defective recruitment of neutrophils at the site of infection, as well as a reduced intracellular killing of S. typhimurium by these cells. These results show that TNF and LT have crucial, yet opposite effects on lethal endotoxemia induced by S. typhimurium LPS and on the infection of mice with live Salmonella microorganisms, and suggest caution when extrapolating results obtained in the lethal endotoxemia model to bacteremia in patients.     

Accessory replicons of species of Salmonella and Shigella.

Shigella and Salmonella strains isolated from clinical samples were examined. Out of 42 Shigella strains tested, 17 (40%) were found to be colicinogenic and another 3 were lysogenic. All three lysogens yielded a phage antigenically homologous to coliphage P2. Out of 30 strains tested, only 1 was found to be resistant to both neomycin and sulfamethoxazole. Out of 48 strains of Salmonella tested for drug resistance, only 2 showed multiple drug resistance. In contrast to Shigella isolates, the Salmonella isolates were infrequently (approximately 5%) bacteriocinogenic. The frequency of lysogeny in Salmonella strains was found to be 6% when tested on Salmonella typhimurium LT2, but by using a set of five indicators belonging to species Salmonella potsdam, Salmonella mbadanka, Salmonella dublin, Salmonella london, and Salmonella wandsworth, 50% of the strains were shown to be lysogenic. Salmonella phages related to P22 were recoverable from Salmonella saintpaul, Salmonella indiana, and Salmonella heidelberg. Some isolates of S. typhimurium yielded a temperature-sensitive and P22-heterologous phage which was found to be a more efficient transducer of bacterial genetic markers than P22. EcoRI-generated fragments of the DNA of some phages permitted the establishment of a clonal descent for some of the wild-type lysogenic bacterial strains. This last observation points out the potential usefulness of prophages as epidemiological markers.     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

Spontaneous mutators of salmonella typhimurium LT2 generated by insertion of transposable elements.

Spontaneous mutators of Salmonella typhimurium LT2 were generated by inserting the transposable element Tn5 or Tn10 into the bacterial chromosome. Two mutators mapped at the position of the mutH and mutL loci of S. typhimurium, and two other mutators mapped at positions corresponding to the mutS and uvrD loci of Escherichia coli. A fifth mutator, mutB, did not map at a position corresponding to any of the known mutators of S. typhimurium or E. coli. The mutH,L,S and uvrD alleles increased the frequency of both spontaneous base substitution and frameshift mutations, whereas the mutB allele increased the frequency only of spontaneous base substitution mutations. The increased frequency of base substitution mutations was recA+ independent in the mutH, mutL, and uvrD strains and partially recA+ independent in the mutS strain. The uvrD mutation decreased the resistance of the cells to killing by ultraviolet irradiation. The mutH,L,S and uvrD strains showed an increased sensitivity to mutagenesis by the alkylating agents methyl methane sulfonate and ethyl methane sulfonate, but not to mutagenesis by 4-nitroquinoline-1-oxide.     

Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence.

We isolated spontaneous mutations (pmrA) in the smooth strain Salmonella typhimurium LT2 that show increased resistance to the cationic antibacterial proteins of human neutrophils and to the drug polymyxin B. The mutation in one strain, JKS5, maps to 93 min on the S. typhimurium chromosome, near the proP gene and the melAB operon. The mutation, designated pmrA505, confers a 1,000-fold increase in resistance to polymyxin B and a 2- to 4-fold increase in resistance to neutrophil proteins. We cloned both the pmrA505 and pmrA+ alleles and found that the pmrA+ gene is partially dominant over pmrA505. DNA sequence analysis of the pmrA505 clone revealed three open reading frames (ORFs). The deduced amino acid sequences indicated that ORF1 encodes a 548-amino-acid (aa) protein with a putative membrane-spanning domain and no significant homology to any known protein. ORF2 and ORF3, which encode 222- and 356-aa proteins, respectively, show strong homology with the OmpR-EnvZ family of two-component regulatory systems. ORF2 showed homology with a number of response regulators, including OmpR and PhoP, while ORF3 showed homology to histidine kinase-sensor proteins EnvZ and PhoR. Genetic analysis of the cloned genes suggested that ORF2 contained the pmrA505 mutation. Comparison of the pmrA505 and pmrA+ ORF2 DNA sequences revealed a single G-A transition, which would result in a His-to-Arg substitution at position 81 in the ORF2 mutant protein. We therefore designate ORF2 PmrA and ORF3 PmrB. The function of ORF1 is unknown.     

Bacterial lipoprotein induces resistance to Gram-negative sepsis in TLR4-deficient mice via enhanced bacterial clearance

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 x 10(6) CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcgammaIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.     

Stabilization of glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by peptidase-deficient mutants

Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.     

The ends of Tn10 are not IS3.

By heteroduplex and hybridization analysis we showed that the inverted repetition (here called IS10) at the ends of the translocatable tetracycline resistance element Tn10 is not IS3, as had previously been reported by Ptashne and Cohen (J. Bacteriol. 122:776--781, 1975). Further analysis confirmed the homology between IS3 and the alpha beta sequence of F and demonstrated that IS10 was not present in the genomes of Salmonella typhimurium LT2 or Escherichia coli K-12.     

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.     

Identification and characterization of the mutL and mutS gene products of Salmonella typhimurium LT2.

The gene products of the mutL and mutS loci play essential roles in the dam-directed mismatch repair in both Salmonella typhimurium LT2 and Escherichia coli K-12. Mutations in these genes result in a spontaneous mutator phenotype. We have cloned the mutL and mutS genes from S. typhimurium by generating mutL- and mutS-specific probes from an S. typhimurium mutL::Tn10 and an mutS::Tn10 strain and using these to screen an S. typhimurium library. Both the mutL and mutS genes from S. typhimurium were able to complement E. coli mutL and mutS strains, respectively. By a combination of Tn1000 insertion mutagenesis and the maxicell technique, the products of the mutL and mutS genes were shown to have molecular weights of 70,000 and 98,000 respectively. A phi (mutL'-lacZ+) gene fusion was constructed; no change in the expression of the fusion could be detected by treatment with DNA-damaging agents. In crude extracts, the MutS protein binds single-stranded DNA, but not double-stranded DNA, with high affinity.     

The chromosome of Salmonella paratyphi A is inverted by recombination between rrnH and rrnG.

Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.     

Integration of transposon Tn10 into phage L (Salmonella typhimurium)

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.     

Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype.

Salmonella typhimurium ST39 exhibits reduced virulence in mice and decreased survival in mouse macrophages compared with the parent strain SL3201. Strain ST39 is nonmotile, carries an indeterminate deletion in and near the flgB operon, and is defective in the mviS (mouse virulence Salmonella) locus. In flagellum-defective strains, the flgM gene product of S. typhimurium negatively regulates flagellar genes by inhibiting the activity of FliA, the flagellin-specific sigma factor. In this study, flgM of wild-type S. typhimurium LT2 was found to complement the mviS defect in ST39 for virulence in mice and for enhanced survival in macrophages. Transduction of flgM::Tn10dCm into the parent strain SL3201 resulted in attenuation of mouse virulence and decreased survival in macrophages. However, a flgM-fliA double mutant was fully virulent in mice and survived in macrophages at wild-type levels. Thus, the absolute level of FliA activity appears to affect the virulence of S. typhimurium SL3201 in mice. DNA hybridization studies showed that flgM-related sequences were present in species other than Salmonella typhimurium and that sequences related to that of fliA were common among members of the family Enterobacteriaceae. Our results demonstrate that flgM and fliA, two genes previously shown to regulate flagellar operons, are also involved in the regulation of expression of virulence of S. typhimurium and that this system may not be unique to the genus Salmonella.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella paratyphi B.

The genomic cleavage map of Salmonella paratyphi B was determined through digestion with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 19 XbaI sites, 10 BlnI sites, and 7 CeuI sites. The fragments were arranged in order through excision of fragments from the gel, redigestion with a second enzyme, end labelling with 32P, and reelectrophoresis. Tn10 transposons inserted in 61 different genes of S. typhimurium LT2 were transduced by use of bacteriophage P22 into S. paratyphi B. The locations of Tn10 insertions on the chromosome of S. paratyphi B were determined by use of XbaI and BlnI sites in Tn10, revealing the positions of genes with Tn10 insertions in S. paratyphi B. All seven CeuI sites (in rrl genes for 23S rRNA) and most of the XbaI and BlnI sites in rrn genes for Glt-tRNA are conserved, but only about half of the XbaI and BlnI sites outside rrn genes are conserved. Gene order is identical in the 68 genes that we could compare between S. paratyphi B and S. typhimurium LT2, and the lengths of intervals between the genes are often the same, but there are several instances of differences in interval lengths, indicating that insertions or deletions of DNA have occurred during the evolutionary divergence of these bacteria.