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.

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 tRNA , 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.

bIn 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.

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 virulence operon in the intracellular pathogen serovar Typhimurium binds to the coding region of mRNAs in the 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 mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the flagellin gene that prevents the leader RNA-mediated down-regulation increases the-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the nucleotide substitution renders hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a-acting leader RNA from the same virulence 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.

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 Mg 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 Mg 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 Mg 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 Mg levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

No abstract

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.