Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri.

This paper shows that compounds in defined growth media strongly influence the expression of the effectors of virulence in the human invasive pathogen Shigella flexneri. Ornithine in conjunction with uracil reduces the haemolytic ability of wild-type cultures more than 20-fold and the expression of the type III secretion system more than 8-fold, as monitored by an mxiC : : lacZ transcriptional reporter. mxiC gene expression is further decreased by the presence of methionine or branched-chain amino acids (15-fold or 25-fold at least, respectively). Lysine and a few other aminated metabolites (cadaverine, homoserine and diaminopimelate) counteract the ornithine-mediated inhibition of haemolytic activity and of the expression of a transcriptional activator virF reporter. The complete abolition of invasion of HeLa cells by wild-type bacteria by ornithine, uracil, methionine or branched-chain amino acids establishes that these metabolites are powerful effectors of virulence. These findings provide a direct connection between metabolism and virulence in S. flexneri. The inhibitory potential exhibited by the nutritional environment is stronger than temperature, the classical environmental effector of virulence. The implications and practical application of this finding in prophylaxis and treatment of shigellosis are discussed.

Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting.

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.

Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification-deficient mutant of Shigella flexneri: a possible role in adaptation of virulence.

The wild-type strain YSH6000 of Shigella flexneri growing in minimal medium contains the modified nucleoside epoxy-Q (oQ) in a subset of tRNAs. This nucleoside is lacking in tRNA from a tgt mutant of this bacterium. When these bacteria are growing in minimal medium, the expression of virulence genes is 10-fold lower in the tgt mutant than in the wild type, although only a twofold reduction in the expression of these virulence factors is observed in broth. Such a strong media-dependant expression of virulence genes was not observed in the wild type. Accordingly, the level of the positive regulator of virulence, VirF, is much lower in the mutant than in the wild type. However, the transcription of the virF gene in minimal medium is the same in the wild type and in the tgt mutant. As the undermodification of tRNA is not affected by the quality of the growth medium, we conclude that such an environmental change in growth conditions partly restores virulence gene expression by counteracting poor translation of the virF mRNA mediated by an oQ-deficient tRNA. Virulence gene expression is partly restored in the tgt mutant by the addition of a mixture of arginine and methionine. Addition of the polyamine putrescine, synthesis of which is metabolically related to that of arginine and methionine, has a comparable stimulatory effect on virulence gene expression. These results not only suggest a role for amino acids and polyamines in the environmental regulation of virulence gene expression in S. flexneri, but also demonstrate a strong and specific involvement of tRNA modifications, and especially oQ, in the adaptation of virulence gene expression to the nutritional quality of the growth medium.

Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri.

The modified nucleosides 2'-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m(5)U54), and pseudouridine, present at position 55 (Psi55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli. The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m(5)U54), and truB (Psi55). The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-DL-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Psi55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Psi55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes.