Véronique Robbe-Saule

VIRULENCE AND VACCINE POTENTIAL OF SALMONELLA TYPHIMURIUM MUTANTS DEFICIENT IN THE EXPRESSION OF THE RPOS (SIGMA S) REGULON

The alternative sigma factor RpoS (σs) is required for Salmonella virulence in mice. We report the immunizing capacity of Salmonella typhimurlum rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S. typhimurium. When administered orally or intraperitoneally, rpoS derivatives of the mouse‐virulent S. typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single‐dose live vaccines. rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis. Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design. Consistent with this, Salmonella typhi Ty2, a ‘wild‐type’ strain used widely for the development of human live‐vaccine candidates against typhoid fever, was shown to be defective for rpoS. In addition, our results demonstrate that rpoS not only controls the growth and persistence of S. typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.

Identification of a non-haem catalase in Salmonella and its regulation by RpoS (sigmaS).

We report the identification and functional analysis of katN, a gene encoding a non‐haem catalase of Salmonella enterica serotype Typhimurium. katN, which is not present in Escherichia coli, is located between the yciGFE and yciD E. coli homologues in the Salmonella genome. Its predicted protein product has a molecular weight of 31 826 Da and is similar to the Mn‐catalases of Lactobacillus plantarum and Thermus spp. Its product, KatN, was visualized as a 37 kDa protein in E. coli maxicells. A KatN recombinant protein, containing six histidine residues at its C‐terminus, was purified, and its catalase activity was observed on a non‐denaturing polyacrylamide gel. KatN was also visualized by catalase activity gel staining of bacterial cell extracts. Its expression was shown to be regulated by growth phase and rpoS. Northern blotting indicated that kat forms an operon with the upstream yciGFE genes. A putative rpoS‐regulated promoter was identified upstream of yciG. Southern blotting revealed that katN is conserved within Salmonella serovars. katN homologues were found in Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens. A katN mutation did not appear to affect the hydrogen peroxide (H2O2) response of Salmonella. However, the expression of katN increased the H2O2 resistance of unadapted cells in the exponential phase and of rpoS mutants in stationary phase. Thus, KatN may contribute to hydrogen peroxide resistance in Salmonella in certain environmental conditions.

Crl activates transcription initiation of RpoS-regulated genes involved in the multicellular behavior of Salmonella enterica serovar typhimurium

In Salmonella enterica serovar Typhimurium, the stationary-phase sigma factor sigma(S) (RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein of Escherichia coli interacts with sigma(S) and activates expression of sigma(S)-regulated genes, such as the csgBAC operon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein of Salmonella serovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of the csgD, csgB, adrA, and bcsA genes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His(6)-Crl showed that Crl directly activated sigma(S)-dependent transcription initiation at the csgD and adrA promoters. We observed no effect of Crl on sigma(70)-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28 degrees C. We obtained complementation of the crl mutation by increasing sigma(S) levels. This suggests that Crl has a major physiological impact at low concentrations of sigma(S).

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

ABSTRACT The stationary-phase-inducible sigma factor, σS (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σS protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σS, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.

RELATIONSHIPS BETWEEN H-NS, SIGMA S, SPVR AND GROWTH PHASE IN THE CONTROL OF SPVR, THE REGULATORY GENE OF THE SALMONELLA PLASMID VIRULENCE OPERON

Abstract The σS-regulated gene spvR of Salmonella typhimurium encodes an autoregulatory protein required for transcriptional activation of the virulence operon spvABCD. A mutation in the histone-like protein H-NS, which negatively controls the σS level, has been reported to increase spv gene expression in S. typhimurium strain LT2. In agreement with this, we found that transcription of spvR and spvABCD was derepressed in hns strains of Escherichia coli and S. typhimurium. Moreover, levels of spv gene expression in hns rpoS double mutants were higher than expression levels in mutants deficient in rpoS alone, and were close to those measured in wild-type strains. This demonstrates that H-NS contributes to spv gene regulation independently of its function in controlling the σS level. Since the same start site was used for spvR gene transcription in wild-type as in hns and hns rpoS mutant strains, it is likely that the spvR promoter, spvRp1, can be recognized efficiently by an RNA polymerase containing σ70. The spvR promoter region shows an intrinsic DNA curvature that might be a determinant in H-NS- and/or σS-mediated control. A single amino acid substitution, Leu to Pro at position 265, abolished the regulatory function of SpvR in E. coli and Salmonella, implicating the C-terminal domain of SpvR in its structure and/or regulatory function. The spvR265 allele is not transcribed at detectable levels in hns or hns rpoS strains, suggesting that activation of spvRp1 in these strains remains dependent on SpvR. Thus, we propose a model for spvR gene regulation in which SpvR acts as a co-regulator of an RNA polymerase containing either σ70 (in the absence of H-NS) or σS, to induce transcriptional initiation at spvRp1. Moreover, growth-phase regulation of spv gene expression was maintained in hns and hns rpoS strains, indicating that an additional element, besides σS, is involved in the growth-phase regulation in rich medium.

Growth phase and SpvR regulation of transcription of Salmonella typhimurium spvABC virulence genes

The 90 kb virulence plasmid of Salmonella typhimurium is required for bacterial growth beyond the small intestine to deeper tissues such as the spleen and liver of orally inoculated mice. We constructed transcriptional lacZ fusions within the cloned plasmid-borne virulence genes spvA, spvB and spvC of S. typhimurium to demonstrate that spvR encodes a trans-acting positive regulator for the transcription of spvA, spvB and spvC. Data suggesting that the activation of spvABC transcription is dependent on the growth phase of both S. typhimurium and Escherichia coli grown in Luria Broth (LB) are also presented. Complementation experiments for virulence in mice confirmed that at least spvR and spvC are virulence genes and further suggested that the spvRABC gene cluster consists of at least three transcriptional units containing spvR, spvC and spvABC, respectively. Reinitiation of transcription at spvC was confirmed in vitro, using a lacZ fusion, and was shown to be independent of SpvR-mediated control in LB.

Physiological Effects of Crl in Salmonella Are Modulated by σS Level and Promoter Specificity

The small regulatory protein Crl activates sigma(S) (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind sigma(S) in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the sigma(S)-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other sigma(S)-dependent phenotypes and genes in a Deltacrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on sigma(S) levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS(LT2) allele that shows reduced expression of sigma(S); the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS(LT2) mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by sigma(S), a finding consistent with a role for Crl in sigma factor competition via the facilitation of sigma(S) binding to core RNA polymerase.

Binding of the Unorthodox Transcription Activator, Crl, to the Components of the Transcription Machinery

The small regulatory protein Crl binds to σS, the RNA polymerase stationary phase σ factor. Crl facilitates the formation of the σS-associated holoenzyme (EσS) and thereby activates σS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with σS, which results in an increase of the association rate of σS to core RNA polymerase without any effect on the dissociation rate of EσS. Crl is also able to associate with preformed EσS with a higher affinity than with σS alone. Furthermore, even at saturating σS concentrations, Crl significantly increases EσS association with the katN promoter and the productive isomerization of the EσS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to σ70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the σ70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-σS holoenzymes.

Effect of Growth Temperature on Crl-Dependent Regulation of σS Activity in Salmonella enterica Serovar Typhimurium

The small regulatory protein Crl favors association of the stationary-phase sigma factor sigma(S) (RpoS) with the core enzyme polymerase and thereby increases sigma(S) activity. Crl has a major physiological impact at low levels of sigma(S). Here, we report that the Crl effects on sigma(S)-dependent gene expression, the H(2)O(2) resistance of Salmonella enterica serovar Typhimurium, and the resistance of this organism to acidic pH are greater at 28 degrees C than at 37 degrees C. Immunoblot experiments revealed a negative correlation between sigma(S) and Crl levels; the production of Crl was slightly greater at 28 degrees C than at 37 degrees C, whereas the sigma(S) levels were about twofold lower at 28 degrees C than at 37 degrees C. At both temperatures, Crl was present in excess of sigma(S), and increasing the Crl level further did not increase the H(2)O(2) resistance level of Salmonella and the expression of the sigma(S)-dependent gene katE encoding the stationary-phase catalase. In contrast, increasing the sigma(S) level rendered Salmonella more resistant to H(2)O(2) at 28 degrees C, increased the expression of katE, and reduced the magnitude of Crl activation. In addition, the effect of Crl on katE transcription in vitro was not dependent on temperature. These results suggest that the effect of temperature on Crl-dependent regulation of the katE gene and H(2)O(2) resistance are mediated mainly via an effect on sigma(S) levels. In addition, our results revealed that sigma(S) exerts a negative effect on the production of Crl in stationary phase when the cells contain high levels of sigma(S).

IDENTIFICATION OF CIS-ACTING DNA SEQUENCES INVOLVED IN THE TRANSCRIPTION OF THE VIRULENCE REGULATORY GENE SPVR IN SALMONELLA TYPHIMURIUM

The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of thespvABCD virulence operon ofSalmonella typhimurium. An alternative sigma factor, σS (RpoS), in conjunction with SpvR, controls the transcription of thespvR gene. In this study, we used a combination of primer extension experiments and deletion/fusion analyses of thespvR gene to identify sequences involved inspvR transcription inS. typhimurium. When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription ofspvR inS. typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of thespvR start codon. The level ofspvR transcription originating atspvRp1 was 20-fold higher in the wild-type strain than in therpoS mutant. In both strains, however, transcription atspvRp1 requires the SpvR protein. 5′ Deletions up to position −86, relative to thespvR start codon, did not inhibit inducibility by σS and/or SpvR. In contrast, 5′ deletion up to −75 abolished the activation ofspvRp1 by SpvR in both the wild-type strain andrpoS mutant. Within the 11-bp sequence lying between position −86 and position −75, a 10-bp consensus motif TNTNTGCANA, present in both thespvR andspvA promoter regions, was identified and may contain the DNA recognition site for SpvR. In addition, we detected initiation of transcription within thespvR coding region. This finding may have implications for comparative studies of regulation withspvR gene fusions.