Characterization of the pigmentation locus in Yersinia pestis pathogenesis of pneumonic plague

Title from PDF of title page (University of Missouri--Columbia, viewed on June 5, 2012).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Dissertation advisor: Dr. Deborah Mae AndersonVita.Includes bibliographical references.Ph. D. University of Missouri--Columbia, 2011."December 2011"[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The extreme virulence of Yersinia pestis in all three forms of plague disease is attributed to its multiple virulence factors. Y. pestis pathogenesis research often focuses on characterization of these factors to better understand their regulation and mechanisms, in hopes of identifying potential targets useful for development of therapeutic and preventative options. Concern regarding a potential outbreak of Y. pestis disease in the form of pneumonic plague has led to heightened focus on elucidating the pathogenesis of this specific form of disease. In the studies presented here, we describe our discovery of the pigmentation (pgm) locus as containing one or multiple virulence factors necessary for the development of pneumonic plague. Pgm-deficient strains are commonly used for plague pathogenesis research due to its exclusion from select agent restrictions. However, our results have demonstrated its inapplicability as a model for pneumonic plague research as pgm-deficient strains are unable to cause respiratory disease. Further characterization of the siderophore-producing yersiniabactin (Ybt) system located in the pgm locus identified the Ybt siderophore as playing an essential role in bacterial growth within the lungs as well as potential immunomodulation of the host response. Additional studies to better understand the exact mechanism behind the effects of Ybt are needed to determine whether knowledge of this virulence factor can be used to our advantage in treatment and prevention

Absence of Inflammation and Pneumonia during Infection with Nonpigmented Yersinia pestis Reveals a New Role for the pgm Locus in Pathogenesis▿

Yersinia pestis causes primary pneumonic plague in many mammalian species, including humans, mice, and rats. Virulent Y. pestis strains undergo frequent spontaneous deletion of a 102-kb chromosomal DNA fragment, known as the pigmentation (pgm) locus, when grown in laboratory media, yet this locus is present in every virulent isolate. The pgm locus encodes, within a high-pathogenicity island, siderophore biosynthesis genes that are required for growth in the mammalian host when inoculated by peripheral routes. Recently, higher challenge doses of nonpigmented Y. pestis were reported to cause fatal pneumonic plague in mice, suggesting a useful model for studies of virulence and immunity. In this work, we show that intranasal infection of BALB/c mice with nonpigmented Yersinia pestis does not result in pneumonic plague. Despite persistent bacterial colonization of the lungs and the eventual death of infected mice, pulmonary inflammation was generally absent, and there was no disease pathology characteristic of pneumonic plague. Iron given to mice at the time of challenge, previously shown to enhance the virulence of pgm-deficient strains, resulted in an accelerated disease course, with less time to bacteremia and lethality, but lung inflammation and pneumonia were still absent. We examined other rodent models and found differences in lung inflammatory responses, some of which led to clearance and survival even when high challenge doses were used. Together, the results suggest that the Y. pestis pgm locus encodes previously unappreciated virulence factors required for the induction of pneumonic plague that are independent of iron scavenging from the mammalian host

Opposing Roles for Interferon Regulatory Factor-3 (IRF-3) and Type I Interferon Signaling during Plague

<div><p>Type I interferons (IFN-I) broadly control innate immunity and are typically transcriptionally induced by Interferon Regulatory Factors (IRFs) following stimulation of pattern recognition receptors within the cytosol of host cells. For bacterial infection, IFN-I signaling can result in widely variant responses, in some cases contributing to the pathogenesis of disease while in others contributing to host defense. In this work, we addressed the role of type I IFN during <em>Yersinia pestis</em> infection in a murine model of septicemic plague. Transcription of IFN-β was induced <em>in vitro</em> and <em>in vivo</em> and contributed to pathogenesis. Mice lacking the IFN-I receptor, <em>Ifnar</em>, were less sensitive to disease and harbored more neutrophils in the later stage of infection which correlated with protection from lethality. In contrast, IRF-3, a transcription factor commonly involved in inducing IFN-β following bacterial infection, was not necessary for IFN production but instead contributed to host defense. <em>In vitro</em>, phagocytosis of <em>Y. pestis</em> by macrophages and neutrophils was more effective in the presence of IRF-3 and was not affected by IFN-β signaling. This activity correlated with limited bacterial growth <em>in vivo</em> in the presence of IRF-3. Together the data demonstrate that IRF-3 is able to activate pathways of innate immunity against bacterial infection that extend beyond regulation of IFN-β production.</p> </div

IFNAR dependent depletion of neutrophils correlates with disease.

<p>Wild type C57BL/6 and <i>Ifnar^−/−</i> mice were challenged by intranasal infection of <i>Y. pestis</i> KIM D27. On day 5 post-infection, 6 mice per group were euthanized and tissues processed for (A) bacterial load, (B) serum cytokines and (C) histology. Samples for all assays were obtained for each mouse; open shapes are wild type, closed shaped are <i>Ifnar^−/−</i> mice; representative data are shown; n = 12 mice per strain assayed in 2 independent trials. All values for titers and cytokines were not statistically significant (P>0.9) between wild type and <i>Ifnar^−/−</i> mice as determined by Kruskal Wallis rank sum test. (C) H&E stains of <i>Ifnar^−/−</i> liver and spleen (top panels, left to right) and wild type liver and spleen (bottom panels, left to right). Images shown are representative of the mice with positive bacterial titers. Scale bar indicates 100 µm.</p

IFNAR-dependent depletion of Ly6G⁺ cells in the bone marrow.

<p>Bone marrow cells of wild type C57BL/6 and <i>Ifnar^−/−</i> mice that were not infected (NI) (n = 3 mice per group assayed in 2 independent trials) or on day 5 post-challenge by intranasal infection with <i>Y. pestis</i> KIM D27 (n = 13 mice per group assayed in 3 independent trials) and stained with anti-Ly6G/6C-FITC followed by flow cytometry analysis. Data was gated on high stained cells as indicated in the scatter plot (A) and are shown for mice with the greatest percentage neutrophils in each group from a single trial. (B) Mean percent neutrophils from representative trial, error bars indicate the standard deviation from the mean. White bars indicate wild type mice, black bars indicate <i>Ifnar^−/−</i> mice; *P<0.05 between wild type and <i>Ifnar^−/−</i> mice determined by two-tailed Kruskal Wallis test.</p

<i>Irf3^−/−</i> mice develop septicemic plague at an accelerated rate.

<p>Groups of 3 wild type C57BL/6 and <i>Irf3^−/−</i> mice were challenged by intranasal infection of <i>Y. pestis</i> KIM D27. (A) Bacterial load for lungs, liver and spleen of wild type and <i>Irf3^−/−</i> mice on days 2 and 3 post-infection; open symbols indicate wild type, closed symbols indicate <i>Irf3^−/−</i>; *P<0.05 for all three tissues compared to wild type as determined by Kruskal Wallis rank sum test. (B) Mean severity scores for histopathology of lungs, liver and spleen on day 3; error bars indicate the standard deviation from the mean scores. (C–G) Wild type tissues are the left panels, <i>Irf3^−/−</i> tissues are the right panels; Day 2 (C), 3 (D–E), and moribund (F–G) hematoxylin and eosin (H&E) stain for liver (C–D,F) or spleen (E, G). Scale bar indicates 50 µm. Data shown are representative from 2 independent experiments (n = 6 mice per strain).</p

Increase in sensitivity of <i>Irf3^−/−</i> mice is dependent on the absence of the <i>Y. pestis</i> pigmentation locus.

<p>Groups of five male and female wild type C57BL/6 and <i>Irf3^−/−</i> mice were challenged by intranasal infection of 1×10⁴ CFU <i>Y. pestis</i> KIM5⁺ and monitored over 14 days for development of disease. Data shown were collected in 3 independent experiments, n = 5 mice per group in each experiment; 15 mice total per strain. P = 0.7295 between wild type and <i>Irf3^−/−</i> mice as determined by Mantel Cox log rank test.</p

Type I interferon is produced following infection of C57BL/6 mice.

<p>(A–B) Wild type female C57BL/6 mice were challenged by intranasal infection with <i>Y. pestis</i> KIM D27. Groups of 3 mice were analyzed for bacterial load and gene expression on days 2, 4 and 7 post-infection. (A) Bacterial titer recovered in the lungs, liver and spleen. Bars depict the median titer (n = 6 total per group, 2 independent trials); *P<0.05 as determined by Kruskal Wallis rank sum test. (B) mRNA expression in the lungs. Gene expression values were normalized to the housekeeping gene <i>ywhaz</i> and are presented as a ratio compared to age-matched female C57BL/6 mice that were not infected. Data shown are a subset of a total of 17 analyzed from two independent experiments (n = 6 mice total per time point); error bars depict the standard deviation from the mean. (C) RAW 264.7 cells were infected by <i>Y. pestis</i> KIM D27, mRNA isolated and probed for expression of the indicated genes by real time PCR. Data were normalized to housekeeping gene <i>ywhaz</i>. Data shown is representative of two independent trials; *P<0.05 compared to not infected as determined by Wilcoxan matched pairs rank test.</p

<i>Irf3^−/−</i> mice are more sensitive to pulmonary infection by <i>Y. pestis</i>.

<p>(A) Groups of 5–7 male and female wild type C57BL/6, <i>Irf3^−/−</i>, and <i>Irf7^−/−</i> mice were challenged by intranasal infection of <i>Y. pestis</i> KIM D27 and monitored for 14 days; *P<0.05 compared to wild type as determined by Gehan-Wilcoxan test; data shown are combined from three independent experiments for each strain (n = 20 for wild type, n = 13 for <i>Irf3^−/−</i> mice, n = 11 for <i>Irf7^−/−</i> mice). (B) Groups of 3 wild type C57BL/6, <i>Irf3^−/−</i> and <i>Irf7^−/−</i> mice were challenged by intranasal infection with <i>Y. pestis</i> KIM D27. On days 2 and 4 post-infection, mice were euthanized by CO₂ asphyxiation, lungs harvested and processed for RNA isolation and real time PCR. mRNA expression values were normalized to housekeeping gene <i>ywhaz </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002817#ppat.1002817-Joyce1" target="_blank">[19]</a>; y-axis represents fold change compared to mice that were not infected. Error bars depict the standard deviation from the mean. Data shown were collected in a single trial (n = 3 for all day 2 samples; only 2 <i>Irf3^−/−</i> and <i>Irf7^−/−</i> mice survived until day 4, n = 3 for wild type C57BL/6 on day 4). *P<0.05, ***P<0.005 as determined by one way ANOVA followed by Bonferroni's multiple comparison test.</p