Characterization of the transcription factor IscR in Yersinia pseudotuberculosis

Regulation of the Ysc type three secretion system (T3SS) of the human gut pathogen Yersinia pseudotuberculosis has been a widely explored field. Located on the Yersinia virulence plasmid, the Ysc T3SS has been shown to be regulated by environmental cues such as calcium, temperature, and host cell contact, and by bacterial factor such as AraC-like transcriptional regulators and histone-like proteins. These mechanisms allow for Yersinia to respond to environmental changes as well as entry into the host. Through a forward genetic screen to identify novel factors that regulate the T3SS, we discovered IscR, a global transcriptional regulator that coordinates an iron sulfur (Fe-S) cluster. No reports on Yersinia pseudotuberculosis IscR have been published, though Escherichia coli IscR is well studied. Here we describe the identification and initial characterization of this novel Yersinia transcriptional regulator. We constructed an in-frame IscR deletion mutant in Y. pseudotuberculosis (∆iscR) as well as a mutant expressing an iscR allele unable to coordinate an Fe-S cluster (apo-IscR). Interestingly, both the Y. pseudotuberculosis ∆iscR and apo-IscR mutants lacked the ability to secrete effector proteins and target macrophages through the T3SS. In contrast, the ∆iscR mutant displayed normal flagellar motility while the apo-IscR mutant had a severe motility defect. The flagellar basal body is a T3SS itself, indicating that the defect in the Ysc T3SS displayed by the ∆iscR mutant is not a result of gross abnormalities in secretion systems. Accordingly, the ∆iscR mutant showed normal growth in both rich and minimal media, while the apo-IscR mutant displayed a selective growth defect only in rich media. This suggests that the ratio between Fe-S-bound holo-IscR and apo-IscR may be important for balanced growth. Lastly, preliminary data suggest that the ΔiscR mutant has increased resistance to hydrogen peroxide in comparison to the parental strain, while apo-IscR was susceptible. Our findings suggest that Y. pseudotuberculosis IscR is an important regulator of the Ysc T3SS and plays a role in controlling resistance to ROS, motility, and balanced growth in vitro. As the iscR gene is almost identical in Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis, we speculate that IscR may be important for the virulence of all three human pathogenic Yersinia

Impact of host membrane pore formation by the Yersinia pseudotuberculosis type III secretion system on the macrophage innate immune response.

Type III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenic Yersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize the Yersinia T3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1β (IL-1β), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and host cell death. The known Yersinia T3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how the Yersinia T3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed a Yersinia pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopD(ΔTM)) and lacking the T3SS cargo proteins YopHEMOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1β secretion, or TLR-independent Egr1 and TNF-α expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopD(ΔTM) Y. pseudotuberculosis mutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells

Yersinia pseudotuberculosis YopD mutants that genetically separate effector protein translocation from host membrane disruption.

The Yersinia type III secretion system (T3SS) translocates Yop effector proteins into host cells to manipulate immune defenses such as phagocytosis and reactive oxygen species (ROS) production. The T3SS translocator proteins YopB and YopD form pores in host membranes, facilitating Yop translocation. While the YopD amino and carboxy termini participate in pore formation, the role of the YopD central region between amino acids 150-227 remains unknown. We assessed the contribution of this region by generating Y. pseudotuberculosis yopD(Δ150-170) and yopD(Δ207-227) mutants and analyzing their T3SS functions. These strains exhibited wild-type levels of Yop secretion in vitro and enabled robust pore formation in macrophages. However, the yopDΔ150-170 and yopD(Δ207-227) mutants were defective in Yop translocation into CHO cells and splenocyte-derived neutrophils and macrophages. These data suggest that YopD-mediated host membrane disruption and effector Yop translocation are genetically separable activities requiring distinct protein domains. Importantly, the yopD(Δ150-170) and yopD(Δ207-227) mutants were defective in Yop-mediated inhibition of macrophage cell death and ROS production in neutrophil-like cells, and were attenuated in disseminated Yersinia infection. Therefore, the ability of the YopD central region to facilitate optimal effector protein delivery into phagocytes, and therefore robust effector Yop function, is important for Yersinia virulence

IscR is essential for yersinia pseudotuberculosis type III secretion and virulence.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF

IscR impacts global gene expression in <i>Y. pseudotuberculosis</i> under iron replete conditions.

<p>RNAseq analysis was performed on WT and Δ<i>iscR Y. pseudotuberculosis</i> 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<u>≤</u>0.05). Shown are the functional ontologies of the (<b>A</b>) 133 genes that are up-regulated in the Δ<i>iscR</i> mutant relative to the wild type and (<b>B</b>) 92 that are down-regulated.</p

<i>Y. pseudotuberculosis</i> lacking a functional IscR display decreased transcription of a number of pYV encoded genes.

<p>Middle and inner rings: heatmap <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004194#ppat.1004194-Krzywinski1" target="_blank">[83]</a> representations of log₂-ratios (log₂(RPKM_mutant/RPKM_wt) for each gene on the pYV plasmid for both the Δ<i>iscR</i> (middle ring) and apo-IscR (inner ring) mutants relative to wild type. Outer ring: pYV base coordinate position from <i>Y. pseudotuberculosis</i> IP32953. Known genes are identified and the <i>virA</i>, <i>virB</i> and <i>virC</i> 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.</p

IscR binds a novel motif 2 site within the <i>lcrF</i> promoter region.

<p>(<b>A</b>) Displayed is the promoter region of the <i>yscW-lcrF</i> operon including −35 and −10 regions, the transcriptional start site (+1) and the ribosome binding site (RBS) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004194#ppat.1004194-Bohme1" target="_blank">[24]</a>. 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004194#ppat.1004194-Lee1" target="_blank">[35]</a>. (<b>B</b>) <i>Y. pseudotuberculosis</i> IP2666 wild type (WT), <i>iscR</i> deletion (Δ<i>iscR</i>), Δ<i>iscR</i> complemented with <i>Y. pseudotuberculosis iscR</i> (Δ<i>iscR</i> pIscR_Y.pstb), and Δ<i>iscR</i> complemented with <i>E. coli iscR</i> (Δ<i>iscR</i> pIscR_E.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. (<b>C</b>) 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 <i>mlcrF</i> sequence and have been found to be important for IscR binding in <i>E. coli </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004194#ppat.1004194-Nesbit1" target="_blank">[33]</a>. (<b>D</b>) Competition assay utilizing 59 nM <i>E. coli</i> apo-locked IscR (IscR-C92A) and 5 nM TAMRA labeled <i>hya</i> DNA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004194#ppat.1004194-Nesbit1" target="_blank">[33]</a>. Assay were performed using a range of 8 to 1000 nM unlabeled competitor DNA, including the known <i>E. coli hya</i> site competitor (closed triangles), the <i>in silico</i> identified <i>Y. pseudotuberculosis lcrF</i> site competitor (closed circles), mutated <i>lcrF</i> (<i>mlcrF</i>) site competitor (open circles), and the negative control <i>Y. pseudotuberculosis isc in silico</i> identified motif I site competitor (open triangles). Shown are the averages ± SEM from three independent experiments.</p