Total Synthesis of Irciniastatin A (Psymberin)

The total synthesis of (+)-iriciniastatin A (psymberin) is reported in 19 steps and 6% overall yield. Key reactions include a highly convergent enolsilane-oxocarbenium ion union to generate the C8-C25 fragment and a late-stage coupling of a hemiaminal and acid chloride to complete the synthesis

Distinct TLR- and NLR-Mediated Transcriptional Responses to an Intracellular Pathogen

How the innate immune system tailors specific responses to diverse microbial infections is not well understood. Cells use a limited number of host receptors and signaling pathways to both discriminate among extracellular and intracellular microbes, and also to generate responses commensurate to each threat. Here, we have addressed these questions by using DNA microarrays to monitor the macrophage transcriptional response to the intracellular bacterial pathogen Listeria monocytogenes. By utilizing combinations of host and bacterial mutants, we have defined the host transcriptional responses to vacuolar and cytosolic bacteria. These compartment-specific host responses induced significantly different sets of target genes, despite activating similar transcription factors. Vacuolar signaling was entirely MyD88-dependent, and induced the transcription of pro-inflammatory cytokines. The IRF3-dependent cytosolic response induced a distinct set of target genes, including IFNβ. Many of these cytosolic response genes were induced by secreted cytokines, so we further identified those host genes induced independent of secondary signaling. The host response to cytosolic bacteria was reconstituted by the cytosolic delivery of L. monocytogenes genomic DNA, but we observed an amplification of this response by NOD2 signaling in response to MDP. Correspondingly, the induction of IFNβ was reduced in nod2−/− macrophages during infection with either L. monocytogenes or Mycobacterium tuberculosis. Combinatorial control of IFNβ induction by recognition of both DNA and MDP may highlight a mechanism by which the innate immune system integrates the responses to multiple ligands presented in the cytosol by intracellular pathogens

U.S. Natural Resources and Climate Change: Concepts and Approaches for Management Adaptation

Public lands and waters in the United States traditionally have been managed using frameworks and objectives that were established under an implicit assumption of stable climatic conditions. However, projected climatic changes render this assumption invalid. Here, we summarize general principles for management adaptations that have emerged from a major literature review. These general principles cover many topics including: (1) how to assess climate impacts to ecosystem processes that are key to management goals; (2) using management practices to support ecosystem resilience; (3) converting barriers that may inhibit management responses into opportunities for successful implementation; and (4) promoting flexible decision making that takes into account challenges of scale and thresholds. To date, the literature on management adaptations to climate change has mostly focused on strategies for bolstering the resilience of ecosystems to persist in their current states. Yet in the longer term, it is anticipated that climate change will push certain ecosystems and species beyond their capacity to recover. When managing to support resilience becomes infeasible, adaptation may require more than simply changing management practices—it may require changing management goals and managing transitions to new ecosystem states. After transitions have occurred, management will again support resilience—this time for a new ecosystem state. Thus, successful management of natural resources in the context of climate change will require recognition on the part of managers and decisions makers of the need to cycle between “managing for resilience” and “managing for change.

Fis Is Essential for Yersinia pseudotuberculosis Virulence and Protects against Reactive Oxygen Species Produced by Phagocytic Cells during Infection

All three pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3 Secretion System (T3SS) and its associated effector proteins. During mammalian infection, these effectors are injected into innate immune cells, where they block many bactericidal functions, including the production of reactive oxygen species (ROS). However, Y. pseudotuberculosis (Yptb) lacking the T3SS retains the ability to colonize host organs, demonstrating that chromosome-encoded factors are sufficient for growth within mammalian tissue sites. Previously we uncovered more than 30 chromosomal factors that contribute to growth of T3SS-deficient Yptb in livers. Here, a deep sequencing-based approach was used to validate and characterize the phenotype of 18 of these genes during infection by both WT and plasmid-deficient Yptb. Additionally, the fitness of these mutants was evaluated in immunocompromised mice to determine whether any genes contributed to defense against phagocytic cell restriction. Mutants containing deletions of the dusB-fis operon, which encodes the nucleoid associated protein Fis, were markedly attenuated in immunocompetent mice, but were restored for growth in mice lacking neutrophils and inflammatory monocytes, two of the major cell types responsible for restricting Yersinia infection. We determined that Fis was dispensable for secretion of T3SS effectors, but was essential for resisting ROS and regulated the transcription of several ROS-responsive genes. Strikingly, this protection was critical for virulence, as growth of Delta dusB-fis was restored in mice unable to produce ROS. These data support a model in which ROS generated by neutrophils and inflammatory monocytes that have not been translocated with T3SS effectors enter bacterial cells during infection, where their bactericidal effects are resisted in a Fis-dependent manner. This is the first report of the requirement for Fis during Yersinia infection and also highlights a novel mechanism by which Yptb defends against ROS in mammalian tissues

Identification of MrtAB, an ABC Transporter Specifically Required for <em>Yersinia pseudotuberculosis</em> to Colonize the Mesenteric Lymph Nodes

<div><p>A highly conserved virulence plasmid encoding a type III secretion system is shared by the three <em>Yersinia</em> species most pathogenic for mammals. Although factors encoded on this plasmid enhance the ability of <em>Yersinia</em> to thrive in their mammalian hosts, the loss of this virulence plasmid does not eliminate growth or survival in host organs. Most notably, yields of viable plasmid-deficient <em>Yersinia pseudotuberculosis</em> (<em>Yptb</em>) are indistinguishable from wild-type <em>Yptb</em> within mesenteric lymph nodes. To identify chromosomal virulence factors that allow for plasmid-independent survival during systemic infection of mice, we generated transposon insertions in plasmid-deficient <em>Yptb</em>, and screened a library having over 20,000 sequence-identified insertions. Among the previously uncharacterized loci, insertions in <em>mrtAB</em>, an operon encoding an ABC family transporter, had the most profound phenotype in a plasmid-deficient background. The absence of MrtAB, however, had no effect on growth in the liver and spleen of a wild type strain having an intact virulence plasmid, but caused a severe defect in colonization of the mesenteric lymph nodes. Although this result is consistent with lack of expression of the type III secretion system by Wt <em>Yptb</em> in the mesenteric lymph nodes, a reporter for YopE indicated that expression of the system was robust. We demonstrate that the ATPase activity of MrtB is required for growth in mice, indicating that transport activity is required for virulence. Indeed, MrtAB appears to function as an efflux pump, as the ATPase activity enhances resistance to ethidium bromide while increasing sensitivity to pyocyanin, consistent with export across the inner membrane.</p> </div

<i>ΔdusB-fis</i> is not defective for Type 3 Secretion or effector translocation.

<p>A) Stationary phase cultures of WT, <i>ΔyscF</i>, Δ<i>dusB-fis</i>, and Δ<i>fis</i> strains were diluted 1:40 into L broth with 20mM sodium oxalate and 20 mM MgCl₂. Cultures were grown for 2 hours at 26°C and then shifted to 37°C for 2 hours. After growth, proteins from culture supernatants were precipitated, resolved on a polyacrylamide gel alongside a standard protein ladder, and visualized using commaasie blue. B-C) Stationary phase cultures of WT, <i>ΔyscF</i>, and Δ<i>dusB-fis</i> strains containing YopH-TEM (HTEM) or YopE-TEM (ETEM) beta-lactamase reporters were grown as in A. After growth, the culture supernatant was mixed with nitrocefin at a final concentration of 100 μg/mL. After 10 minutes of incubation, the A₄₉₀ of samples was measured using a BioTek Synergy HT plate reader. Statistical significance was calculated using One Way ANOVA analysis with the Dunnett’s multiple comparison test on log₁₀-transformed values. Each bar represents the mean and standard error of 3 biological replicates. D-E) Stationary phase cultures of WT, <i>ΔyopB</i>, and Δ<i>dusB-fis</i> strains fused containing H-TEM or E-TEM beta-lactamase reporters were grown as in A-C. HEp-2 cells were infected at a multiplicity of infection of 10 for 1 hr and then incubated with CCF4 to determine the percentage of cells containing translocated effectors (% blue). Data was log₁₀ transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing each strain to WT. Each bar represents the mean and standard error of 3–6 biological replicates. F-G) C57BL6/J mice were inoculated intravenously with a pool of 10³ bacteria, containing an equal mixture of <i>ΔdusB-fisΔyscF -</i>Kan^R and <i>ΔyscF</i>. 24 hours prior to and post-infection, mice were intraperitoneally injected with RB6-8C5 antibody. Mice were euthanized at 3 days post-infection and livers (F) and spleens (G) were collected, and dilutions of tissue homogenates were plated onto selective and non-selective media to determine the C.I. C.I. values were log₁₀ transformed and statistical significance was calculated using a Mann-Whitney t-test. * indicates p≤0.05, ** indicates p≤0.01, **** indicates p≤0.0001.</p

<i>ΔdusB-fis</i> is sensitive to neutrophils and inflammatory monocytes <i>in vivo</i>.

<p>C57BL6/J mice were inoculated intravenously with a pool of 10³ bacteria, containing an equal mixture of WT and WT<i>-</i>Kan^R, <i>Δfis-</i>Kan^R and WT, <i>ΔdusB-fis</i> and WT<i>-</i>Kan^R, or <i>ΔdusB-fis</i>::<i>dusB-fis</i> and WT<i>-</i>Kan^R. 24 hours prior to and post-infection, some mice were intraperitoneally injected with either the 1A8 or RB6-8C5 antibody. Other mice were intraperitoneally injected with the MC-21 antibody 1 day prior to infection and each day after until completion of the experiment. Mice were euthanized at the indicated times and livers (A) and spleens (B) were collected, and dilutions of tissue homogenates were plated onto selective and non-selective media to determine the C.I. C.I. data was log₁₀ transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing the C.I. values of mutant strains to WT or C.I. values in non-depleted and depleted mice. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.</p

<i>ΔdusB-fis</i> is sensitive to ROS produced by the NADPH oxidase complex during mouse infection.

<p>C57Bl/6 or C57Bl/6 gp91^phox-/- mice were inoculated intravenously with 1x10³ CFU of a 1:1 mixture of WT and <i>ΔdusB-fis-</i>Kan^R. Livers (A,C,E) and spleens (B,D,F) were collected, weighed, homogenized, and plated for CFUs on selective and non-selective agar at 3 days-post-infection. (A-B) The number of bacteria recovered from selective and non-selective plates was used to determine the C.I. of <i>ΔdusB-fis-</i>Kan^R. Each data point represents an individual mouse. C.I. data was log₁₀ transformed and statistical significance was calculated using the Mann-Whitney t-test. (C-D) The CFU/gram was determined by dividing the total number of recovered CFU on non-selective plates by the weight of the tissue. CFU/g data was log₁₀ transformed and statistical significance was calculated using the Mann-Whitney t-test. (E-F) The number of bacteria recovered from selective and non-selective plates was used to determine the quantity of <i>ΔdusB-fis-</i>Kan^R and WT bacteria in each organ sample. CFU/g data was log₁₀ transformed and statistical significance was calculated using One Way ANOVA analysis with the Dunnett’s multiple comparison test. * indicates p≤0.05, ** indicates p≤0.01, *** indicates 00.001, **** indicates p≤0.0001.</p

<i>dusB-fis</i> is required for resistance to oxidative stress.

<p>A) Stationary phase cultures of WT and <i>ΔdusB-fis</i> were diluted 1:100 into L broth and L broth adjusted to pH 5.5 and the OD₆₀₀ of cultures was measured at 1-hour-intervals for 12 hours during growth with aeration. Each symbol represents the mean of 2–4 biological replicates. B) Stationary phase cultures were diluted 1:100 into a well of a 96-well plate containing L broth or L broth + 250 μM 2,2’- Bipyridyl and OD₆₀₀ measurements were recorded at 15-minute intervals during growth with aeration. Lines represent the mean of 3 biological replicates. (C-D) Exponential phase cultures were washed and diluted 1:50 into M9 glucose medium or M9 glucose containing 2.5mM DETA NONOate (C) or M9 glucose containing 1.5mM H₂O₂ (D) for 60 minutes. Survival was calculated by determining the number of CFUs recovered following treatment divided by the number of CFUs recovered from untreated cultures. The mean and standard error of 3 biological replicates for DETA NONOate treatment and 6–10 biological replicates for H₂O₂ treatment are shown. Survival values were log₁₀ transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing each strain to WT. E) <i>Δfis</i> fails to up-regulate ROS-responsive genes after exposure to H₂O₂. Exponential phase cultures were washed and diluted 1:50 into M9 glucose medium or M9 glucose containing 20 μM H₂O₂ and were incubated for 10 minutes with aeration. RNA isolated from treated and untreated samples was used to generate cDNA, and qPCR reactions were performed. Relative expression was determined by normalizing to 16S RNA as well as to expression in untreated samples using the ΔΔCT method. Bars represent the mean and standard error of 8 biological replicates. Unpaired Mann-Whitney t-tests were performed to calculate statistical differences between expression of each gene in WT and <i>Δfis</i>. * indicates p≤0.05, ** indicates p≤0.01, **** indicates p≤0.0001, ns indicates not significant.</p