Figure 1.
Alignment of protein sequences shows a high level of conservation between E. coli and Yersinia IscR.
(A) The Y. pseudotuberculosis DNA sequence, which displays the unique insertions sites for the two transposon mutants generated from our genetic screen. A space in the DNA sequence and a solid black line indicate the site of insertion for either iscR::Tn1 or iscR::Tn2. (B) Multiple sequence alignment was performed on the IscR protein sequence from E. coli K12-MG1655 and each of the three human pathogenic Yersinia spp., Y. pseudotuberculosis IP 32953 (Y. pstb), Y. enterocolitica 8081 (Y. ent) and Y. pestis CO92 (Y. pestis) using ClustalW [86]. The N-terminal helix-turn-helix DNA-binding motif is indicated by a black box. The three conserved cysteine residues (C92, C98 and C104) responsible for coordinating an Fe-S cluster are in bold and identified by black arrows [33]. Asterisks indicate nucleotides that are conserved across all four species.
Figure 2.
IscR modulates Y. pseudotuberculosis T3SS function.
(A) HEK293T cells expressing an NFκB luciferase reporter gene were infected with either T3SS-positive Y. pseudotuberculosis lacking the six known effector proteins YopHEMOJT (Δyop6), two isogenic iscR transposon mutants (Δyop6/iscR::Tn1 and Δyop6/iscR::Tn2), the iscR deletion mutant (Δyop6/ΔiscR), or a T3SS null mutant (ΔyscNU). At 4 h post-inoculation, T3SS functionality was determined by assessing the ability of the mutants to trigger NFκB activation in host cells by measuring bioluminescence. Shown are the average raw luminescence values of the sample compared to uninfected ± standard error of the mean (SEM) from five independent experiments. *p≤0.05, as determined by one-way ANOVA followed by Bonferroni post hoc test where each indicated group was compared to the appropriate negative (ΔyscNU) and positive (Δyop6) controls. (B) To analyze T3SS-dependent pore formation in macrophages, C57Bl/6 immortalized BMDMs were infected with Δyop6, a T3SS-defective mutant lacking the translocator protein YopB (Δyop6/ΔyopB), the iscR deletion mutant (Δyop6/iscR), or the iscR complemented strain (Δyop6/iscR pIscR), or were left uninfected. At 2 h post-inoculation, pore formation was determined by assessing the number of cells that took up ethidium bromide (EtBr) compared to the total number of Hoechst-stained cells. Shown are the averages ± SEM from three independent experiments. *p≤0.05 relative to both Δyop6 and Δyop6/iscR pIscR, as determined by one-way ANOVA followed by Bonferroni post hoc test where each indicated group was compared to the appropriate negative (Δyop6/ΔyopB) and positive (Δyop6) controls. (C) Y. pseudotuberculosis IP2666 wild type (WT), iscR deletion (ΔiscR), iscR complemented (ΔiscR pIscR), apo-locked IscR (apo-IscR), and apo-IscR complemented (apo-IscR pIscR) strains were grown in 2xYT low calcium media at 37°C to induce type III secretion in the absence of host cells. Proteins in the bacterial culture supernatant were precipitated and visualized alongside a protein molecular weight marker (L) on a polyacrylamide gel using commassie blue. Sample loading was normalized for OD600 of each culture. Results are representative of three independent experiments.
Figure 3.
IscR is required for full virulence of Y. pseudotuberculosis.
Mice were infected with 2×108 CFU of either WT Y. pseudotuberculosis or ΔiscR mutant via orogastric gavage. At 5 days post-inoculation, the Peyer's patches (PP), mesenteric lymph nodes (MLN), spleens and livers were collected, homogenized and CFU determined. Each symbol represents one animal. Unfilled symbols indicate that CFU were below the limit of detection. The data presented are from three independent experiments. *p<0.05, ***p<0.001 as determined by an unpaired Wilcoxon-Mann-Whitney rank sum test. Dashes represent the geometric mean.
Figure 4.
IscR impacts global gene expression in Y. pseudotuberculosis under iron replete conditions.
RNAseq analysis was performed on WT and ΔiscR Y. pseudotuberculosis after growth in M9 at 37°C for 3 h (T3SS-inducing conditions), at which point total RNA was collected and processed. The resulting libraries were sequenced using the HiSeq2500 Illumina sequencing platform for 50 bp single reads and analyzed via the CLC Genomics Workbench application (CLC bio). RPKM expression levels of 225 genes demonstrated a fold change of ≥2, and were deemed significant by Bayseq test with a corrected FDR post hoc test from three independent experiments (p≤0.05). Shown are the functional ontologies of the (A) 133 genes that are up-regulated in the ΔiscR mutant relative to the wild type and (B) 92 that are down-regulated.
Figure 5.
Deletion of IscR leads to increased transcription of Fe-S cluster biogenesis genes and robust transcription of T3SS genes.
Quantitative real-time PCR analysis of WT and ΔiscR Y. pseudotuberculosis was performed (A) for the Fe-S cluster biogenesis genes, iscS and erpA and (B) for the T3SS genes, yscF, yscN and lcrF. Experiments were carried out from cultures grown in M9 at 37°C for 3 h. Shown are the averages ± SEM from three independent experiments. *p<0.05, **p<0.001, ***p<0.0001 as determined by a Student t test.
Table 1.
Genes repressed by IscR, identified by RNAseq analysis.
Table 2.
Genes activated by IscR, identified by RNAseq analysis.
Figure 6.
The apo-IscR mutant strain displays decreased motility and disruption of electrical potential.
(A) Motility was analyzed by spotting 1 µl aliquots of either a nonmotile strain (Δyop6/flhDY.pestis), WT, ΔiscR, or apo-locked IscR Y. pseudotuberculosis onto motility agar plates. The diameters of the colonies were determined one day later and used to calculate percent motility relative to WT, which was set at 100%. Shown is the average percent motility ± SEM and is representative of three independent experiments. ***p≤0.0001 as determined by one-way ANOVA followed by Bonferroni post hoc test where each indicated group was compared to the appropriate negative (Δyop6/flhDY.pestis) and positive (WT) controls. (B) Proton motive force (PMF) was measured using JC-1 dye for Y. pseudotuberculosis IP2666 wild type (WT), iscR deletion mutant (ΔiscR), iscR complemented (ΔiscR pIscR), apo-IscR, and apo-IscR complemented (apo-IscR pIscR) strains grown in M9 at 37°C for 3 hours. The protonophore CCCP was added to a WT sample as a negative control (CCCP). Decreases in PMF were measured as a decrease in red (590 nm) fluorescent cells relative to green (530 nm). The data is presented as total fluorescence intensities at 590 (red) relative to 530 (green) ± SEM and is representative of three independent experiments. *p≤0.05, as determined by one-way ANOVA followed by Bonferroni post hoc test where each indicated group was compared to the appropriate negative (CCCP) and positive (WT) controls.
Figure 7.
Y. pseudotuberculosis lacking a functional IscR display decreased transcription of a number of pYV encoded genes.
Middle and inner rings: heatmap [83] representations of log2-ratios (log2(RPKMmutant/RPKMwt) for each gene on the pYV plasmid for both the ΔiscR (middle ring) and apo-IscR (inner ring) mutants relative to wild type. Outer ring: pYV base coordinate position from Y. pseudotuberculosis IP32953. Known genes are identified and the virA, virB and virC operons highlighted by black arcs. On the interior right side is the color bar legend displaying log2-ratios from −3.5 to 2. Using this scale, orange/red colorations represent genes with decreased transcription in the mutant relative to the wild type strain and blue/green coloring represents increases in gene transcription for the mutant relative to the wild type. Tan/cream denotes no change.
Figure 8.
IscR binds a novel motif 2 site within the lcrF promoter region.
(A) Displayed is the promoter region of the yscW-lcrF operon including −35 and −10 regions, the transcriptional start site (+1) and the ribosome binding site (RBS) [24]. The IscR type 2 DNA-binding site is indicated by a black box. The nine bases previously found to be important for IscR binding are indicated by asterisks [35]. (B) Y. pseudotuberculosis IP2666 wild type (WT), iscR deletion (ΔiscR), ΔiscR complemented with Y. pseudotuberculosis iscR (ΔiscR pIscRY.pstb), and ΔiscR complemented with E. coli iscR (ΔiscR pIscRE.coli) strains were grown in 2xYT low calcium media at 37°C to induce type III secretion in the absence of host cells. Proteins in the bacterial culture supernatant were precipitated and visualized alongside a protein molecular weight marker (Ladder) on a polyacrylamide gel using commassie blue. Sample loading was normalized for OD600 of each culture. These results are representative of three independent experiments. (C) The competitor DNA sequences used for the competition assay and the resulting IC50 concentrations are displayed. Nucleotides in bold and underlined correspond to those that were changed in the mlcrF sequence and have been found to be important for IscR binding in E. coli [33]. (D) Competition assay utilizing 59 nM E. coli apo-locked IscR (IscR-C92A) and 5 nM TAMRA labeled hya DNA [33]. Assay were performed using a range of 8 to 1000 nM unlabeled competitor DNA, including the known E. coli hya site competitor (closed triangles), the in silico identified Y. pseudotuberculosis lcrF site competitor (closed circles), mutated lcrF (mlcrF) site competitor (open circles), and the negative control Y. pseudotuberculosis isc in silico identified motif I site competitor (open triangles). Shown are the averages ± SEM from three independent experiments.
Figure 9.
Regulation of the isc and lcrF operons by IscR.
(A) Model of isc operon transcriptional control in the Y. pseudotuberculosis wild type and apo-locked IscR strains based on previous work on E. coli IscR [32], [34] and on data shown here. In wild type bacteria, the Isc Fe-S cluster biogenesis pathway loads a [2Fe-2S] cluster onto IscR (holo-IscR) [32], which recognizes a type 1 DNA-binding motif in the isc promoter to repress transcription in a negative feedback loop. Expression of the apo-locked IscR allele (***, IscR-C92A/C98A/C104A) results in loss of holo-IscR-mediated repression, thereby increasing transcription of the isc operon relative to wild type, resulting in a 30-fold increase in iscR. (B) Model depicting the mechanism by which IscR controls the Y. pseudotuberculosis Ysc T3SS. Holo- and apo-IscR are predicted to bind a newly identified type 2 DNA-binding site within the yscW-lcrF operon encoding the LcrF T3SS master regulator. Subsequently, LcrF expression leads to transcription of the LcrF regulon, which includes the lcrGVH-yopBD and virC operons and yop genes [17], [20], [22], [53], [54]. These genes encode the majority of T3SS structural, regulatory, and effector proteins. However, through an as yet undefined mechanism, overexpression of apo-locked IscR leads to a decrease in the proton motive force, which is required for type III secretion [50]. As Yop secretion positively regulates yop gene transcription [51], [52], the secretion defect of the apo-locked IscR mutant is predicted to lead to a decrease in effector yop transcription.
Table 3.
Y. pseudotuberculosis strains used in this study.