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.     

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.     

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.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.     

PhoQ基因重组鼠伤寒沙门氏菌株的构建及毒力研究

应用PCR技术从鼠伤寒沙门氏菌基因组DNA中克隆phoQ基因片段,构建原核表达pUC18重组质粒,测定序列(GenBank登录号为DQ787014),并转入鼠伤寒沙门氏菌,经异丙基硫代半乳糖苷(IPTG)诱导,进行高效表达。对重组菌株、野生菌株进行毒力检测对比实验,通过口腔注入45日龄健康无菌KM小鼠,测定其半数致死量(LD50)。结果发现：重组菌株与野生菌株的毒力存在显著差异,其半致死量分别为3.981×107 cf u/ mL and 5.012×102 cf u/ mL,PhoQ基因重组菌株的毒力远远低于非重组菌株。说明phoQ基因是调节鼠伤寒沙门氏菌致病机制中一个重要的调节因子。     

Nucleotide sequence of mkaD, a virulence-associated gene of Salmonella typhimurium containing variable and constant regions

We have identified the nucleotide (nt) sequence of mkaD, a virulence-associated gene of the Salmonella typhimurium virulence plasmid, pEX102. The gene shows 98% homology on nt sequence level to mkfA, a corresponding gene of the S. typhimurium virulence plasmid pIP1350. The few nt changes, however, caused more extensive changes on the amino-acid level. The differences between mkaD and mkfA were clustered in distinct variable regions rather than being randomly scattered along the sequence. A third salmonellar virulence plasmid, pLT2, contained an mkaD gene identical to that of pEX102. Our observation suggests that the conserved virulence determinant on the plasmids of Salmonellae may contain different alleles of the same gene.     

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.     

Induction of rpoS in Salmonella typhimurium by nutrient-poor and depleted media is slower than that achieved by a competitive microflora

The presence of a viable competitive microflora, at greater than 10(8) cfu ml-1, is known to advance the induction of RpoS-mediated gene expression in a sub-population of Salmonella typhimurium. As starvation is known to induce RpoS, one action of the competitive microflora could be to cause depletion of essential nutrients. The aim of the current experiments was to determine whether this was the case by examining RpoS induction in Salm. typhimurium in reduced nutrient media. RpoS-mediated gene expression in Salm. typhimurium was not advanced so significantly in 'conditioned' or diluted medium as it was in the presence of competitors, which indicates that nutrient depletion was not the responsible mechanism. The effect of a competitive microflora has implications for models of bacterial survival during food processing, as RpoS ultimately regulates both stress resistance and virulence.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

FlgM is a primary regulator of sigmaD activity, and its absence restores motility to a sinR mutant.

We have used mini-Tn1O mutagenesis to identify negative regulators of sigmaD activity. Nine independent insertions were mapped to five genes: flgM, flgK, fliD, fliS, and fliT, suggesting that FlgM export is regulated similarly in Bacillus subtilis and Salmonella typhimurium. We show that a deletion of flgM can restore sigmaD activity to a sinR null mutant of B. subtilis, although fla/che operon expression is affected by neither SinR nor FlgM.     

Genomic Subtraction Identifies Salmonella typhimurium Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon, stf, Which Are Absent in Salmonella typhi

Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2

Two large virulence loci encoding type III secretion systems are present on the chromosome of Salmonella typhimurium. Salmonella pathogenicity island 2 (SPI2) is important for the survival of S. typhimurium in host organs and forms an insertion of about 40 kb at the tRNA(Val) gene. However, several indications suggested that SPI2 was not the result of a single event of horizontal gene transfer. We characterized the portion of SPI2 towards the 30 cs boundary and performed mutational analysis to investigate the contribution of this region to S. enterica virulence. This analysis indicates that SPI2 may be composed of at least two different genetic elements. About 15 kb of the 40 kb of SPI2 contain genes without a significant contribution to systemic infections in the model of murine salmonellosis. Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure.     

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.