看護学部教官業績目録 ; 2003年1月～12月

Acinetobacter baumannii ATCC 19606 tolerates loss of lipopolysaccharide (LPS) caused by inactivation of early LPS pathway genes. However, mutations in pathway genes encoding steps downstream of LpxD have not been reported, implying that later biosynthetic steps may be essential for viability. Here, we determined if LpxH, the UDP-2,3-diacylglucosamine hydrolase that generates UMP-2,3-diacylglucosamine 1-phosphate (lipid X), was essential in A. baumannii ATCC 19606. Multiple attempts to disrupt lpxH on the genome were unsuccessful. When expression of LpxH was placed under control of an isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter, the cells failed to grow under standard laboratory conditions without IPTG induction. Growth under LpxH depletion conditions (-IPTG) was rescued by chemical inhibition of LpxC, upstream of LpxH, indicating that toxic accumulation of LPS pathway intermediates underlies LpxH essentiality. Consistent with this, the levels of LpxH substrate (product of LpxD) and a C14:0(3-OH) acyl variant of the LpxD substrate had accumulated in cells that were depleted of LpxH causing a growth defect. Intriguingly, under these partial depletion conditions, there was also a smaller but reproducible accumulation of the downstream pathway intermediates disaccharide 1-monophosphate and lipid IVA suggesting a complex downstream response to LpxH depletion

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region

Cell: Career Under A Microscope

Daryl Richie has prepared a first-person account of his career journey. This has already been approved through OAK by Jenn Leeds

Perils of an industrial job interview, don't forget to bring a water

Daryl completed his Ph.D. in Pathobiology and Molecular Medicine at the University of Cincinnati with a primary emphasis on fungal pathogenesis under the guidance of David Askew, Ph.D. He currently works at Novartis Institutes for BioMedical Research as an investigator in Bacterial Genetics & Physiology where his work is focused around developing microbiological assays to support target identification and validation of antibacterials

The Non-Specific effect of Efungumab on Amphotericin B Minimum Inhibitory Concentration

Background: Mycograb C28Y is a recombinant human antibody fragment thought to target HSP-90. Absence of in vivo efficacy led us to reevaluate its in vitro activity. Methods: Interaction between amphotericin B (AMB) and Mycograb C28Y were characterized following CLSI guidelines. Results: Addition of Mycograb C28Y and non-specific proteins resulted in a four fold decrease in MIC as compared to AMB alone. Conclusion: Potentiation of AMB by Mycograb C28Y appears to be a non-specific protein effect

Characterization of an Acinetobacter baumannii lptD Deletion Strain; Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate complete loss of lipopolysaccharide (LPS) resulting from inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC mutant under the growth conditions tested, but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from loss of LPS synthesis. Consistent with this, ATP leakage and NPN fluorescence assays indicated a more severe impact of lptD deletion than lpxC deletion on inner and outer membrane permeability, respectively. Targeted LCMS analysis of LPS intermediates from UDP-3-O-[(3R)-3-hydroxylauroyl]-N-acetyl-α-D-glucosamine through lipid IVA, showed that loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulations or mislocalizations caused by blockage of later LPS pathway steps impact envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes including LpxC and Fab B/F in the ΔlptD strain partially rescued growth and permeability defects

Characterization of an A. baumannii lptD Deletion Strain; Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate complete loss of lipopolysaccharide (LPS) resulting from inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC::KmR mutant under the growth conditions tested, but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from loss of LPS synthesis. Consistent with this, adenosine triphosphate (ATP) leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than lpxC deletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography–mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-D-glucosamine through lipid IVA, showed that loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by blockage of later LPS pathway steps impact envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes including LpxC and Fab B/F in the lptD::KmR strain partially rescued growth and permeability defects

Characterization of an Acinetobacter baumannii lptD deletion strain: Permeability defects and response to inhibition of lipopolysaccharide and fatty acid biosynthesis

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes anOMprotein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-Dglucosamine through lipid IVA showed that the loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in the lptD deletion strain partially rescued growth and permeability defects