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.     

Protective immunities induced by porins from mutant strains of Salmonella typhimurium

The protective immunity against Salmonella typhimurium-infection in mice immunized with porins from mutant strains of S. typhimurium was studied. A high level of protection against S. typhimurium infection was achieved in mice immunized with native porins from S. typhimurium LT2 (wild-type strain) but not from S. typhimurium SH6017, SH6260, or SH5551 (mutant strains), which produce 34K, 35K, or 36K porin, respectively. Moreover, when mice were immunized with mixtures of 34K, 35K, and 36K porins (34K + 35K, 35K + 36K, 34K + 36K, or 34K + 35K + 36K porin) or LT2 porin heated at 100 C for 2 min in 2% SDS (heat-denatured LT2 porin), the degree of protective immunities in the mice was very much lower than that in the mice immunized with the native LT2 porin. However, antisera raised against these porins showed no significant differences of the antibody titer against LT2 porin or LT2 whole cells. On the other hand, mice immunized with the native LT2 porin--but not 34K, 35K, 36K, 34K + 35K + 36K, and the heat-denatured LT2 porins--exhibited significant levels of delayed-type hypersensitivity reaction and interleukin-2 production when they were elicited with whole cells of S. typhimurium LT2. These observations suggested that the high level of protection induced by the native LT2 porin immunization was dependent on the induction of cell-mediated immunity.     

The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.     

A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) when grown under anaerobic conditions. All but one of the biosynthetic genes (cob) are located in a single operon which includes genes required for the production of cobinamide and dimethylbenzimidazole, as well as the genes needed to form cobalamin from these precursors. We isolated strains carrying mutations (cobD) which are unlinked to any of the previously described B12 biosynthetic genes. Mutations in cobD are recessive and map at minute 14 of the linkage map, far from the major cluster of B12 genes at minute 41. The cobD mutants appear to be defective in the synthesis of 1-amino-2-propanol, because they can synthesize B12 when this compound is provided exogenously. Labeling studies in other organisms have shown that aminopropanol, derived from threonine, is the precursor of the chain linking dimethylbenzimidazole to the corrinoid ring of B12. Previously, a three-step pathway has been proposed for the synthesis of aminopropanol from threonine, including two enzymatic steps and a spontaneous nonenzymatic decarboxylation. We assayed the two enzymatic steps of the hypothetical pathway; cobD mutants are not defective in either. Furthermore, mutants blocked in one step of the proposed pathway continue to make B12. We conclude that the aminopropanol for B12 synthesis is not made by this pathway. Expression of a lac operon fused to the cobD promoter is unaffected by vitamin B12 or oxygen, both of which are known to repress the main cob operon, suggesting that the cobD gene is not regulated.     

A common virulence region on plasmids from eleven serotypes of Salmonella

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

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.     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.     

Genetics of Sensitivity of Salmonella Species to Colicin M and Bacteriophages T5, T1, and ES18

Nearly all of 62 strains of Salmonella paratyphi B were sensitive to colicin M and phage T5 but resistant to phages T1 and ES18 and to colicin B. All tested S. typhimurium strains were resistant to colicin M and phage T5, and many were sensitive to phage ES18. A rough S. typhimurium LT2 strain given the tonA region of Escherichia coli or S. paratyphi B became sensitive to colicin M and phage T5. We infer that the tonA allele of S. paratyphi B, like that of E. coli, determines an outer membrane protein that adsorbs T5 and colicin M but not phage ES18, whereas the S. typhimurium allele determines a protein able to adsorb only ES18. The partial T1 sensitivity of a rough LT2 strain with a tonA allele from E. coli or S. paratyphi B and also the tonB(+) phentotype of an E. coli B trp-tonB Delta mutant carrying an F' trp of LT2 origin showed that S. typhimurium LT2 has a tonB allele like that of E. coli with respect to determination of sensitivity to colicins and phage T1. Rough S. paratyphi B, although T5 sensitive, remained resistant to T1 even when given F' tonB(+) of E. coli origin. Classes of Salmonella mutants selected as resistant to colicin M included: T5-resistant mutants, probably tonA(-); mutants unchanged except for M resistance, perhaps tolerant; and Exb(+) mutants, producing a colicin inhibitor (presumably enterochelin). Some Exb(+) mutants were resistant to a bacteriocin inactive on E. coli but active on all tested S. paratyphi B and S. typhimurium strains (and on nearly all other tested Salmonella). A survey showed sensitivity to colicin M in several other species of Salmonella.     

Cohabitation within mice of Salmonella typhimurium seqA mutant increases its virulence

The efficacy of a vaccine is conditioned by its capacity to elicit a protective immune response. The principal safety concerns of live vaccine are virulence reversion. The aim of this work was to evaluate the virulence of Salmonella typhimurium seqA mutant after cohabitation with mice. Our results indicated that LD50 of hosted strains were at least twofold lower than those of parental strains. Also, the in vivo competition assays have showed that the development of a systemic infection was most obvious for recovered strains than for control strains. In addition, the number of hosted mutants colonizing spleen and liver was relatively higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. For instance recovered-mutant attached to epithelial cells (KB cells) better than parental strains. According to these results, we report that in vivo adaptation of Salmonella typhimurium seqA mutants can increase their virulence.     

Genetic characteristics and protective properties of neamin-resistant mutant Salmonella typhimurium and S. dublin of the Nea(r) Str(s) and Nea(r) Str(r) 500 classes

The genetic analysis of attenuated mutants, class Nea(r) Str(s), with the use of bacteriophage P 22 has shown that mutation rendering the mutants resistant to neamine is localized in gene nea A. In experiments with the intraperitoneal infection of mice, the appearance of this mutation in S. typhimurium and S. dublin virulent strains has been found to lead to the decrease of virulence in 100% of clones. On the basis of the data obtained in this investigation, region str-spc in S. typhimurium and S. dublin has been mapped. In contrast to mutation spc A, mutations nea A and str A have been shown to inhibit the action of amber suppressor. The investigation has confirmed the regularity, previously established for Shigella flexneri, concerning the relationship between the influence of mutations, occurring in the genes which determine resistance to neamine and streptomycin and control the synthesis of ribosomal proteins S4, S5, S12 and S17, on the virulence of S. typhimurium and S. dublin and the effect of these mutations on the accuracy of the translation of genetic information in the biosynthesis of protein: mutation spc A has been found to produce no changes in the virulence of salmonellae, while mutations nea A and str A cause its loss. Salmonella strains carrying mutations nea A and nea B have shown pronounced protective properties in experiments on mice.     

Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic conditions. Of the 30 cobalamin synthetic genes, 25 are clustered in one operon, cob, and are arranged in three groups, each group encoding enzymes for a biochemically distinct portion of the biosynthetic pathway. We have determined the DNA sequence for the promoter region and the proximal 17.1 kb of the cob operon. This sequence includes 20 translationally coupled genes that encode the enzymes involved in parts I and III of the cobalamin biosynthetic pathway. A comparison of these genes with the cobalamin synthetic genes from Pseudomonas denitrificans allows assignment of likely functions to 12 of the 20 sequenced Salmonella genes. Three additional Salmonella genes encode proteins likely to be involved in the transport of cobalt, a component of vitamin B12. However, not all Salmonella and Pseudomonas cobalamin synthetic genes have apparent homologs in the other species. These differences suggest that the cobalamin biosynthetic pathways differ between the two organisms. The evolution of these genes and their chromosomal positions is discussed.     

Salmonella typhimurium metE operator-constitutive mutations

We used a metE-lacZ fusion phage (lambda Elac) to select for mutants with operator-constitutive mutations in the Salmonella typhimurium metE control region. All of the mutations identified were found to lie within a region containing tandemly-repeating 8-bp palindromes with the consensus sequence 5'-AGACGTCT-3', previously proposed to be the binding region for the metJ-encoded repressor. Lysogens carrying mutant lambda Elac phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. Although repression of metE expression by vitamin B12 is not disrupted in metJ+ lysogens, vitamin B12 repression is disrupted in lysogens lacking an active MetJ repressor. These results suggest that there is an interaction between the metJ-encoded repressor and the vitamin B12 repression system mediated by the metH gene product.     

Mechanism of the protective immunity against murine typhoid: persistence of salmonella L forms in the liver after immunization with live-cell vaccines

Abstract Live-cell vaccines of Salmonella typhimurium , either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd₁ mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunozation. Growth of S. typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization. In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S. schottmülleri 8006 sharing the same O antigenic components with those of S. typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180. However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization. A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti- S. typhimurium antibody responses. Thus, the present data suggest that the long-lived immunity conferred upon live S. typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the brain length of salmonella O-antigens.     

Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium.

When an Escherichia coli K-12 culture was starved for glucose, 50% of the cells lost viability in about 6 days. When a K-12 mutant lacking five distinct peptidase activities, CM89, was starved in the same manner, viability was lost much more rapidly; 50% of the cells lost viability in about 2 days, whereas a parent strain lacking only one peptidase activity lost 50% viability in about 4 days. Compared with the wild-type strain and with its parent strain CM17, CM89 was defective in both protein degradation and protein synthesis during carbon starvation. Similar results were obtained with glucose-starved Salmonella typhimurium LT2 and LT2-derived mutants lacking various peptidase activities. An S. typhimurium mutant lacking four peptidases, TN852, which was deficient in both protein degradation and synthesis during carbon starvation (Yen et al., J. Mol. Biol. 143:21-33, 1980), was roughly one-third as stable as the isogenic wild type. Isogenic S. typhimurium strains that lacked various combinations of three of four peptidases and that displayed protein degradation and synthesis rates intermediate between those of LT2 and TN852 (Yen et al., J. Mol. Biol. 143:21-33, 1980) displayed corresponding stabilities during carbon starvation. These results point to a role for protein degradation in the survival of bacteria during starvation for carbon.