A trans-acting leader RNA from a Salmonella virulence gene.

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress.

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

tRNAPro -mediated downregulation of elongation factor P is required for mgtCBR expression during Salmonella infection.

Bacterial ribosome requires elongation factor P to translate fragments harbouring consecutive proline codons. Given the abundance of ORFs with potential EF-P regulated sites, EF-P was assumed to be constitutively expressed. Here, we report that the intracellular pathogen Salmonella enterica serovar Typhimurium decreases efp mRNA levels during course of infection. We determined that the decrease in efp mRNA is triggered by low levels of charged tRNAPro , a condition that Salmonella experiences when inside a macrophage phagosome. Surprisingly, downregulation of EF-P selectively promotes expression of the virulence mgtC gene and contributes to Salmonella's ability to survive inside macrophages. The decrease in EF-P levels induces ribosome stalling at the consecutive proline codons of the mgtP open reading frame in the mgtCBR leader RNA, and thus allows formation of a stem-loop structure promoting transcription of the mgtC gene. The substitution of proline codons in the mgtP gene eliminates EF-P-mediated mgtC expression and thus Salmonella's survival inside macrophages. Our findings indicate that Salmonella benefits virulence genes by decreasing EF-P levels and inducing the stringent response inside host.

The iron-sensing fur regulator controls expression timing and levels of salmonella pathogenicity island 2 genes in the course of environmental acidification.

In order to survive inside macrophages, Salmonella produces a series of proteins encoded by genes within Salmonella pathogenicity island 2 (SPI-2). In the present study, we report that Fur, a central regulator of iron utilization, negatively controls the expression of SPI-2 genes. Time course analysis of SPI-2 expression after the entry of Salmonella into macrophages revealed that SPI-2 genes are induced earlier and at higher levels in the absence of the Fur regulator. It was hypothesized that Fur repressed the SPI-2 expression that was activated during acidification of the phagosome. Indeed, as pH was lowered from pH 7.0 to pH 5.5, the lack of Fur enabled SPI-2 gene expression to be induced at higher pH and to be expressed at higher levels. Fur controlled SPI-2 genes via repression of the SsrB response regulator, a primary activator of SPI-2 expression. Fur repressed ssrB expression both inside macrophages and under acidic conditions, which we ascribe to the direct binding of Fur to the ssrB promoter. Our study suggests that Salmonella could employ iron inside the phagosome to precisely control the timing and levels of SPI-2 expression inside macrophages.

The Salmonella virulence protein MgtC promotes phosphate uptake inside macrophages.

The MgtC virulence protein from the intracellular pathogen Salmonella enterica is required for its intramacrophage survival and virulence in mice and this requirement of MgtC is conserved in several intracellular pathogens including Mycobacterium tuberculosis. Despite its critical role in survival within macrophages, only a few molecular targets of the MgtC protein have been identified. Here, we report that MgtC targets PhoR histidine kinase and activates phosphate transport independently of the available phosphate concentration. A single amino acid substitution in PhoR prevents its binding to MgtC, thus abrogating MgtC-mediated phosphate transport. Surprisingly, the removal of MgtC's effect on the ability to transport phosphate renders Salmonella hypervirulent and decreases a non-replicating population inside macrophages, indicating that MgtC-mediated phosphate transport is required for normal Salmonella pathogenesis. This provides an example of a virulence protein directly activating a pathogen's phosphate transport inside host.

The mgtCBR mRNA Leader Secures Growth of Salmonella in Both Host and Non-host Environments.

Upon intracellular cues, bacterial mRNA leaders often form secondary structures that determine expression of a downstream protein-coding region(s), thereby providing bacteria with a mechanism to control the amounts of necessary proteins in the right locales. Here we describe a polycistronic mRNA leader that secures bacterial growth by preventing dysregulated expression of the protein-coding regions. In Salmonella, the mgtCBR mRNA encodes the virulence protein MgtC and the Mg2+ transporter MgtB. A mutant designed to produce leaderless mgtCBR mRNA induced MgtC and MgtB in conditions that promote mgtC transcription. The dysregulated expression of MgtC and MgtB impaired bacterial growth under all such non-host environments. While MgtC, but not MgtB, normally reduces ATP levels in a process requiring the F1F0 ATP synthase, dysregulated MgtC and MgtB reduced ATP levels independently of the F1F0 ATP synthase, which correlated with the mutant's growth defect. The mutant showed dysregulated MgtC expression and attenuated survival inside macrophages. While MgtB normally does not affect the phenotype, MgtB impaired intramacrophage survival of the mutant in the presence of MgtC. We provide an example showing that a polycistronic mRNA leader prevents the dysregulated function of protein-coding regions to allow bacteria to proliferate across complex niches.

Elongation factor P controls translation of the mgtA gene encoding a Mg2+ transporter during Salmonella infection.

Ribosome often stalls on mRNA sequences harboring consecutive proline codons. Elongation factor P (EF-P) is required for the stalled ribosome to continue translation and thus the absence of EF-P affects translation of the associated open reading frame. Here we report that EF-P controls translation of the mgtA gene encoding a Mg2+ -transporting ATPase from the intracellualr pathogen Salmonella enterica serovar Typhimurium. EF-P's effect on mgtA translation is dependent on the 550th and 551st proline codons in the coding region and thus substitution of those proline codons eliminates EF-P-mediated control of MgtA protein without affecting the Mg2+ -transporting activity of the mgtA gene. The Pro550 and Pro551-substituted mgtA gene promotes Salmonella's intramacrophage survival and mouse virulence, suggesting that EF-P-mediated translational control of the mgtA gene is required for Salmonella pathogenesis.

Elongation factor P restricts Salmonella's growth by controlling translation of a Mg2+ transporter gene during infection.

When a ribosome translates mRNA sequences, the ribosome often stalls at certain codons because it is hard to translate. Consecutive proline codons are such examples that induce ribosome stalling and elongation factor P (EF-P) is required for the stalled ribosome to continue translation at those consecutive proline codons. We found that EF-P is required for translation of the mgtB gene encoding a Mg2+ transporter in the mgtCBR virulence operon from the intracellular pathogen Salmonella enterica serovar Typhimurium. Salmonella lacking EF-P decreases MgtB protein levels in a manner dependent on consecutive proline codons located in the mgtB coding region despite increasing transcription of the mgtCBR operon via the mgtP open reading frame in the leader RNA, resulting in an altered ratio between MgtC and MgtB proteins within the operon. Substitution of the consecutive proline codons to alanine codons eliminates EF-P-mediated control of the mgtB gene during infection and thus contributes to Salmonella's survival inside macrophages where Salmonella experiences low levels of EF-P. This suggests that this pathogen utilizes a strategy to coordinate expression of virulence genes by an evolutionarily conserved translation factor.

Loss of vancomycin resistance not completely dependent on the Tn1546 element in Enterococcus faecium isolates.

We investigated characteristics of 3 Enterococcus faecium strains (SHY-1, SHY-2, and SHY-3) isolated successively from 1 patient. In vitro susceptibility testing was performed using broth microdilution method. Change of vancomycin MIC was monitored during incubation with vancomycin for SHY-3 strain. Genetic backgrounds were determined both by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In addition, the genetic variations among Tn1546 element were investigated by polymerase chain reaction (PCR) assay and sequencing. vanA and vanX expression of 3 strains was evaluated using quantitative real-time (qRT)-PCR method. Although all the strains possessed the vanA gene, SHY-3 was susceptible to glycopeptides, while SHY-1 and SHY-2 were resistant to glycopeptides. Judged by MLST and PFGE, 3 strains have the same genetic background. The vancomycin resistance of SHY-3 was not recovered after exposure to vancomycin. The vanA and vanX genes were expressed in strains SHY-1 and SHY-2 but not in strain SHY-3, although the SHY-2 and SHY-3 strains shared the same arrangement of the van gene cluster, a common 88-bp deletion in vanS gene. Our results indicate that vancomycin resistance might not be completely dependent on the Tn1546 element.

The RstB sensor acts on the PhoQ sensor to control expression of PhoP-regulated genes.

The PhoP response regulator and PhoQ sensor, which are encoded by the phoPQ operon, constitute the PhoP/PhoQ two-component system. Genome-wide transcription analysis revealed that heterologous expression of the RstB protein, a sensor of the RstA/RstB two-component system, leads to enhanced transcription of PhoP-activated genes in wild-type Salmonella. We determined that RstB-induction increases the levels of phoP mRNA as well as PhoP protein, while lack of the phoPQ genes abolishes RstB-promoted transcription of the PhoP-regulated genes. This regulatory function of RstB did not require its enzymatic activities, and thus the truncated RstB protein containing only periplasmic and transmembrane regions was able to promote PhoP-activated transcription. The RstB protein appeared to target the PhoQ sensor rather than the PhoP response regulator because RstB-induction failed to enhance transcription of the PhoP-regulated genes in a strain maintaining the normal PhoP function, even without PhoQ.