Shandee Dixon | Oregon Health and Science University (original) (raw)

Papers by Shandee Dixon

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse ar... more 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.

PLoS Pathogens, 2014

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular de... more 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 Gramnegative 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 Nterminal 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 Derl2dependent pathway exploited by retrograde trafficking toxins.

Proceedings of the National Academy of Sciences, 2013

Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results:... more Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results: Purified AsbA-E proteins reconstituted petrobactin assembly in vitro. The crystal structure and enzymatic studies of AsbB highlight its function and role in the siderophore pathway. Conclusion: AsbB characterization demonstrated reaction flexibility and substrate positions in the binding pocket. Significance: Siderophore synthetases represent promising antimicrobial targets, and characterization of these versatile enzymes enables creation of novel compounds.

PLoS Pathogens, 2014

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular de... more 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 Gramnegative 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 Nterminal 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 Derl2dependent pathway exploited by retrograde trafficking toxins.

Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in suscep... more Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock-and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock-or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.

Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbe... more Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766, respectively) in Bacillus anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The ΔfatB strain was capable of wild-type levels of growth in iron depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, ΔfpuA bacteria exhibited a significant decrease in growth under low iron conditions when compared to wild-type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of ΔfpuA to import this siderophore. ΔfpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake.

In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. Th... more In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATPbinding cassette (ABC)-import systems. Either of two distinct permeases, FpuB or FatCD, are required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron-depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse ar... more 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.

PLoS Pathogens, 2014

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular de... more 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 Gramnegative 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 Nterminal 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 Derl2dependent pathway exploited by retrograde trafficking toxins.

Proceedings of the National Academy of Sciences, 2013

Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results:... more Background: asbABCDEF mediates petrobactin production and facilitates anthrax virulence. Results: Purified AsbA-E proteins reconstituted petrobactin assembly in vitro. The crystal structure and enzymatic studies of AsbB highlight its function and role in the siderophore pathway. Conclusion: AsbB characterization demonstrated reaction flexibility and substrate positions in the binding pocket. Significance: Siderophore synthetases represent promising antimicrobial targets, and characterization of these versatile enzymes enables creation of novel compounds.

PLoS Pathogens, 2014

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular de... more 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 Gramnegative 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 Nterminal 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 Derl2dependent pathway exploited by retrograde trafficking toxins.

Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in suscep... more Infection with mouse adenovirus type 1 (MAV-1) results in fatal acute encephalomyelitis in susceptible mouse strains via infection of brain endothelial cells. Wild-type (wt) MAV-1 causes less brain inflammation than an early region 3 (E3) null virus in C57BL/6 mice. A mouse brain microvascular endothelial cell line infected with wt MAV-1 had higher expression of mRNAs for the proinflammatory chemokines CCL2 and CCL5 than mock-and E3 null virus-infected cells. Primary mouse brain endothelial cells infected with wt virus had elevated levels of CCL2 compared to mock-or E3 null virus-infected cells. Infection of C57BL/6 mice with wt MAV-1 or the E3 null virus caused a dose-dependent breakdown of the blood-brain barrier, primarily due to direct effects of virus infection rather than inflammation. The tight junction proteins claudin-5 and occludin showed reduced surface expression on primary mouse brain endothelial cells following infection with either wt MAV-1 or the E3 null virus. mRNAs and protein for claudin-5, occludin, and zona occludens 2 were also reduced in infected cells. MAV-1 infection caused a loss of transendothelial electrical resistance in primary mouse brain endothelial cells that was not dependent on E3 or on MAV-1-induced CCL2 expression. Taken together, these results demonstrate that MAV-1 infection caused breakdown of the blood-brain barrier accompanied by decreased surface expression of tight junction proteins. Furthermore, while the MAV-1-induced pathogenesis and inflammation were dependent on E3, MAV-1-induced breakdown of the blood-brain barrier and alteration of endothelial cell function were not dependent on E3 or CCL2.

Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbe... more Iron acquisition mechanisms play an important role in the pathogenesis of many infectious microbes. In Bacillus anthracis, the siderophore petrobactin is required for both growth in iron depleted conditions and for full virulence of the bacterium. Here we demonstrate the roles of two putative petrobactin binding proteins FatB and FpuA (encoded by GBAA5330 and GBAA4766, respectively) in Bacillus anthracis iron acquisition and pathogenesis. Markerless deletion mutants were created using allelic exchange. The ΔfatB strain was capable of wild-type levels of growth in iron depleted conditions, indicating that FatB does not play an essential role in petrobactin uptake. In contrast, ΔfpuA bacteria exhibited a significant decrease in growth under low iron conditions when compared to wild-type bacteria. This mutant could not be rescued by the addition of exogenous purified petrobactin. Further examination of this strain demonstrated increased levels of petrobactin accumulation in the culture supernatants, suggesting no defect in siderophore synthesis or export but, instead, an inability of ΔfpuA to import this siderophore. ΔfpuA spores were also significantly attenuated in a murine model of inhalational anthrax. These results provide the first genetic evidence demonstrating the role of FpuA in petrobactin uptake.

In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. Th... more In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATPbinding cassette (ABC)-import systems. Either of two distinct permeases, FpuB or FatCD, are required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron-depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis.