Fig 1.
Schematic overview of YopN-regulated Yop secretion in Yersinia.
The secretion of effector proteins (Yops) that modulate the host immune system are controlled by the YopN-TyeA-SycN-YscB complex. YopN and TyeA block secretion in concert with the chaperone complex SycN/YscB [20,22]. Dissociation of the plug is assumed to occur through depletion of calcium and after host cell contact resulting in the SycN/YscB-controlled secretion of YopN itself [23,26,27]. HM: host membrane; OM: outer membrane; IM: inner membrane.
Fig 2.
Expression and RNA structures of 5’-UTRs of yopN.
(A) RNA-seq results of the yopN locus at 37°C and identification of two transcriptional start sites from [32] and [38] visualized by the Artemis genome browser (s: short transcript; l: long transcript). (B) Comparison of the relative transcript levels of both yopN 5’-UTRs under non-secretion (+ Ca2+) and secretion (- Ca2+) conditions at 25 and 37°C. Samples of Y. pseudotuberculosis YPIII were taken during the early exponential phase at an OD600 of 0.5 followed by RNA isolation and qRT-PCR. Transcript levels were normalized to the amount of yopN short at 25°C under non-secretion conditions and to the reference genes nuoB and gyrB. The mean transcript amounts and standard deviations comprise the results of three biological replicates. (C,D) PARS-derived RNA secondary structures of the short and long 5’-UTRs of yopN at 37°C including the first 30 nucleotides of the coding region [38]. The putative SD region is highlighted in gray and the start codon in red. Nucleotide exchanges in the Y. enterocolitica sequence are indicated in (C).
Fig 3.
Schematic representation and functional characterization of the short yopN 5’-UTR and mutated variants.
(A) PARS-derived stem-loop RNA structure of the short yopN 5’-UTR (nucleotides 1 to 43) [38] and predicted stabilized (R1–3) and destabilized (D1–2) variants. The putative SD region is highlighted in gray, the start codon in red and mutated nucleotides are highlighted in blue. (B) Plasmid-based translational fusions of 5’-UTRs of interest and bgaB encoding a heat-stable β-galactosidase to test RNA thermometer (RNAT) functionality. Expression of the fusion products is controlled by the arabinose-inducible promotor PBAD. The RNAT of lcrF served as a positive control [34]. (C,D) The β-galactosidase assays of lcrF, the short and long yopN 5’-UTR and the corresponding mutated variants were conducted at 25 and 37°C. Y. pseudotuberculosis YPIII cells carrying plasmids of the fusion constructs were grown to an OD600 of 0.5 at 25°C. Subsequently, transcription of the reporter gene was induced by 0.1% (w/v) L-arabinose and the cultures were split to flasks at 25 and prewarmed flasks at 37°C and incubated for further 30 minutes. Samples were then taken for the β-galactosidase assay. The mean activities in Miller Units and the mean standard deviations were calculated from nine biological replicates. The representative Western blot displays the amount of BgaB-His produced. Protein amounts were adjusted to an optical density of 0.5 and detected by Ponceau S staining after blotting onto a nitrocellulose membrane.
Fig 4.
Temperature-controlled expression of gfp by the short yopN RNAT.
(A) Plasmid-based translational fusions of yopN:gfp were cloned to test RNAT functionality at RNA and protein levels. Transcription of the fusion product is controlled by the arabinose inducible promotor PBAD. (B) Determination of transcript and protein levels of yopN:gfp (WT-gfp) and mutated yopN variants (R1 and D1) by Northern and Western blot analyses, respectively. The RNAT of lcrF served as a positive control [8]. Y. pseudotuberculosis YPIII cells carrying plasmids of the fusion constructs were grown to an OD600 of 0.5 at 25°C. Transcription was induced by 0.1% (w/v) L-arabinose and the cultures were split to flasks at 25°C and prewarmed flasks at 37°C and incubated for further 30 minutes. Samples were then taken for Northern and Western blot analyses. The blots shown represent one of three biological replicates. To ensure equal amounts of RNA, a total of 10 μg of RNA was loaded per sample. Ethidium bromide stained 23S rRNA served as loading control. Protein amounts were adjusted to an optical density of 0.5 and detected by Ponceau S staining after blotting onto a nitrocellulose membrane.
Fig 5.
Temperature-dependent melting of the SD sequence and start codon-containing stem-loop structure of the yopN RNAT.
(A) Enzymatic structure probing of the yopN RNAT (WT) and the stable variant (R1) at 25, 37 and 42°C. Radioactively labeled RNA was treated with single-strand specific RNases T1 (0.016 U) and T2 (0.025 U) at the different temperatures. LOH: alkaline Ladder; LT1: T1 treated RNA at 37°C; C: water control. (B) PARS-derived RNA secondary structure of the yopN RNAT [38]. White arrows indicate T1 and T2 cleavages at all temperatures while black arrows indicate cleavages with increasing temperature. The putative SD region is highlighted in gray and the start codon in red. (C) Quantification of band intensities at 25, 37 and 42°C of selected guanines and adenines. Pixel counting was performed using AlphaEaseFC software and values were normalized to intensities at 25°C. The enzymatic structure probing shown here represents one of two experiments performed.
Fig 6.
Temperature-dependent binding of the 30S ribosomal subunit to the yopN RNAT.
Primer extension inhibition of the yopN RNAT and the stable variant R1 was performed at 25, 37 and 42°C in presence (+) and absence (-) of the 30S ribosomal subunit. A DNA sequencing ladder (ATGC) of the yopN RNAT serves as orientation. Full-length transcripts, termination and the characteristic toeprint signals (+12–14 nt) after ribosome binding are indicated. The primer extension inhibition experiment shown here represents one of two experiments performed.
Fig 7.
The closed RNAT variant R1 is unable to rescue the temperature-sensitive phenotype of the ΔyopN mutant.
(A) Growth experiment of Y. pseudotuberculosis YPIII wild type (WT) and ΔyopN with the empty vector pGM930 (EV) and ΔyopN with vectors containing arabinose-inducible constructs of yopN with the wild type RNAT, the stable variant R1 or the open variant D2. The growth experiment was performed in triplicate. The strains were incubated in secretion-induced (-Ca2+) and secretion-noninduced (+Ca2+) LB medium at 25 and 37°C. Growth was measured by optical density at 600 nm. (B) Visualization of secreted effector proteins by SDS-PAGE using Coomassie blue staining and Western blotting using total-anti-Yop serum. Samples were taken after five hours at 37°C (black arrows) and secreted proteins were TCA-precipitated from filtered supernatants. (C) Production of YopN-Strep was visualized by Western blotting using Strep-tag antibody. Samples were taken as described for (B) and protein levels were adjusted to an optical density of 0.5.
Fig 8.
Visualization of YopE translocation into HEp-2 cells.
YopE translocation assay of Y. pseudotuberculosis YPIII wild type (WT) and ΔyopN with the empty vector pGM930 (EV) and ΔyopN with vectors containing arabinose-inducible complementation constructs of yopN with the wild type RNAT, the stable variant R1 or the open variant D2. All strains carry plasmid pMK-bla coding for a yopE-blaTEM fusion. HEp-2 cells were infected with bacterial strains at an MOI of 50, labeled with CCF4-AM and analyzed by fluorescence microscopy. Blue fluorescence signals of HEp-2 cells indicate efficient YopE-TEM translocation. scale bars: 50 μm.
Fig 9.
Overview of temperature-dependent regulatory pathways regarding the assembly and functionality of the T3SS.
At ambient temperatures (25°C), Yersinia downregulates virulence-associated pathways while the flagellar synthesis is induced [56]. The global regulator YmoA represses the expression of the main virulence regulator lcrF at 25°C [53,57–59]. Besides, RNA thermometers (RNATs) also contribute to repression of specific genes like lcrF itself or the secretion regulator yopN [34]. At virulence-relevant temperatures (37°C), YmoA is degraded by proteases which leads to the derepression of lcrF [59]. Furthermore, melting of the lcrF RNAT increases the expression induction and synthesis of the virulence regulator and thus induce the expression of T3SS genes and effector protein genes (Yop genes) [34]. The transcript of yopN posseses an RNAT that additionally induces its expression at 37°C. In calcium-containing environments, YopN together with TyeA and the chaperone complex SycN/YscB prevents the secretion of Yops by blocking the T3SS channel in the cytosol. In contrast, the complex dissociates under calcium deficiency allowing first the secretion of YopN and subsequently the secretion of further Yops into the surrounding medium [20,22,23,26,27]. Blue box: closed RNAT; red box: open RNAT; red circles: Yops; blue circles: YopN.