Analysis of Virulence-Associated Petrobactin Reacquisition in Bacillus anthracis.

In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. FpuA has high homology to the receptors associated with ATP-binding Cassette (ABC) transporter complexes, indicating that the mechanism of petrobactin reacquisition requires an ABC transport system. ABC transporters are used by bacteria for cross-membrane transport of many small molecules. Recently, the additional components of petrobactin reacquisition have been identified. We have shown that either of two distinct permeases, FpuB or FatCD, are required for iron acquisition and play redundant roles in petrobactin transport. Additionally, three ATPase proteins are sufficient to provide the energy required for petrobactin reacquisition. These results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis. Furthermore, these ABC-transport proteins are essential in cell viability, virulence and pathogenicity. The ABC ATPases share conserved sequences across all bacterial species, and only requires ATP as a substrate, making these proteins prime candidates for a high-throughput inhibitor search for small molecules that can potentially block siderophore import and thus the necessary act of iron acquisition by B. anthracis and other dangerous pathogens. Sixteen top ATPase inhibitor hits were identified in a pilot high-throughput screen at the University of Michigan Center for Chemical Genomics. Eight of which were reconfirmed at the bench-top to reveal three compounds as promising leads in the development of therapeutics or tools to probe ATPase activity.PhDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97997/1/shandeed_1.pd

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways

Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins

Intoxication Mediated by CdtA and CdtC Subunits.

<p>Jurkat, HeLa, or CHO-A745 cells were seeded in clear-bottom 384-well plates, incubated overnight, then challenged with the indicated toxin concentrations. Holotoxin, black circles; CdtAB, red squares; CdtBC, blue triangles. Intoxication was allowed to proceed for 48 h (Jurkat) or 72 h (HeLa and CHO-A745). Cell viability was measured by ATPlite reagent (Perkin Elmer), and normalized to ATPlite signal from unintoxicated controls. Data represent average values from three independent experiments, each performed in triplicate, +/- standard deviation. Lines represent nonlinear curve fit calculated using Prism 5 (GraphPad).</p

Holotoxin Assembly Method Affects Sensitivity to EGA.

<p>CHO-A745 cells were intoxicated as above in the presence or absence of 12.5 μM EGA. Additionally, cells were challenged with a combination of purified CdtA, CdtB, and CdtC subunits that were combined at the time of intoxication without further purification of assembled holotoxin (Ec-ABC). Cell viability was measured by ATPlite and normalized as above. Data represent average values from three independent experiments, each performed in triplicate.</p

Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Ec-Cdt Dimers and Trimers.</p

Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.

<p>Average values and standard deviation (+/-) were determined from at least three biological replicates, each performed in triplicate. NT, not tested; ND, value not determined due to lack of cytotoxicity.</p><p>Tissue Culture LD₅₀ Values for Hd-Cdt Dimers and Trimers.</p

Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

<div><p>Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by <i>Haemophilus ducreyi</i> (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.</p></div

The interaction of Derl2 and p97 is not required for CDT intoxication.

<p>(a) Derl2-GFP fails to bind p97, similar to Derl2ΔC. 293 cells were transfected with vectors encoding S-tagged versions of the indicated forms of Derl2. After 3 days, the cells were lysed and western blot was performed on S-protein precipitates with anti-p97 and anti-S-tag antibodies (b) Overexpression of Derl2-GFP does not affect Hd-CDT intoxication of parental A745TKR cells. Parental A745TKR cells expressing empty vector, Derl2 or Derl2-GFP were intoxicated with Hd-CDT, similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">Fig. 1</a>. (c, d) Derl2-GFP and Derl2ΔC complement sensitivity to Hd-CDT in CHO-CDT^RC1. CHO-CDT^RC1 cells expressing empty vector, Derl2, (c) Derl2-GFP or (d) Derl2ΔC were intoxicated similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">Fig. 1</a>. (e) Dominant negative p97 reduces sensitivity of 293 cells to Hd-CDT. 293 cells stably expressing TCRαGFP were transfected with plasmids encoding CD4 and either dominant negative (R586A) or control (R700A) p97, followed by intoxication with Hd-CDT for 48 hours and staining with Hoechst and anti-CD4 antibodies. Flow cytometry was performed to obtain geometric mean fluorescence values for TCRαGFP (GFP) in CD4+ cells and cell cycle profile of CD4 negative (grey shaded; untransfected control) and CD4 positive cells (black lines). (f) The Derl2 WR motif is not required for intoxication by Hd-CDT. CHO-CDT^RC1 cells expressing empty vector, wildtype Derl2, Derl2 Q53A, Derl2 W55A or Derl2 T59A were intoxicated similar to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004295#ppat-1004295-g001" target="_blank">figure 1</a>. (g–i) Retrograde trafficking of Hd-CDT in p97 deficient cells is blocked at the endoplasmic reticulum. (g) Following transfection with pH2B-GFP (blue) and either dominant negative or control p97, wildtype and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with anti-Hd-CdtB (green) antibody and anti-calreticulin antibody (red). White scale bars indicate 5 µm. pH2B-GFP pseudo-colored blue; Hd-CdtB pseudo-colored green and calreticulin pseudo-colored red (h, i) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER. Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments, percent viability is normalized to unintoxicated controls and error bars indicate standard error.</p

CdtC Mediates Cholesterol Dependency of Ec-CDT.

<p>CHO-A745 cells were seeded at 8 x 10³ cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH₂AX by laser scanning cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143977#pone.0143977.g002" target="_blank">Fig 2B</a>. Data were normalized against pH₂AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.</p