The ATP-Binding Cassette Transporter ABCB19 Regulates Postembryonic Organ Separation in Arabidopsis

The phytohormone auxin plays a critical role in plant development, including embryogenesis, organogenesis, tropism, apical dominance and in cell growth, division, and expansion. In these processes, the concentration gradient of auxin, which is established by polar auxin transport mediated by PIN-FORMED (PIN) proteins and several ATP-binding cassette/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) transporters, is a crucial signal. Here, we characterized the function of ABCB19 in the control of Arabidopsis organ boundary development. We identified a new abcb19 allele, abcb19-5, which showed stem-cauline leaf and stem-pedicel fusion defects. By virtue of the DII-VENUS marker, the auxin level was found to be increased at the organ boundary region in the inflorescence apex. The expression of CUP-SHAPED COTYLEDON2 (CUC2) was decreased, while no obvious change in the expression of CUC3 was observed, in abcb19. In addition, the fusion defects were greatly enhanced in cuc3 abcb19-5, which was reminiscent of cuc2 cuc3. We also found that some other organ boundary genes, such as LOF1/2 were down-regulated in abcb19. Together, these results reveal a new aspect of auxin transporter ABCB19 function, which is largely dependent on the positive regulation of organ boundary genes CUC2 and LOFs at the postembryonic organ boundary.     

Asymmetric Conformational Flexibility in the ATP-Binding Cassette Transporter HI1470/1

Putative metal-chelate-type ABC transporter HI1470/1 is homologous with vitamin B₁₂ importer BtuCD but exhibits a distinct inward-facing conformation in contrast to the outward-facing conformation of BtuCD. Normal-mode analysis of HI1470/1 reveals the intrinsic asymmetric conformational flexibility in this transporter and demonstrates that the transition from the inward-facing to the outward-facing conformation is realized through the asymmetric motion of individual subunits of the transporter. This analysis suggests that the asymmetric arrangement of the BtuC dimer in the crystal structure of the BtuCD-F complex represents an intermediate state relating HI1470/1 and BtuCD. Furthermore, a twisting motion between transmembrane domains and nucleotide-binding domains encoded in the lowest-frequency normal mode of this type of importer is found to contribute to the conformational transitions during the whole cycle of substrate transportation. A more complete translocation mechanism of the BtuCD type importer is proposed.     

转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展

：ABCC4、ABCC5是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和排泄等。ABCC4、ABCC5的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就转运体ABCC4和ABCC5介导的肿瘤多药耐药研究进展进行综述。     

转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展

ABCC4、ABCC5 是ABCC（ATP-binding cassette transporter family class C, ABCC）蛋白转运体家族的成员,介导众多内源性 代谢产物和外源性药物从细胞内向外转运。ABCC4和ABCC5 在体内分布广泛,参与机体对药物和内、外源物质的吸收、分布和 排泄等。ABCC4、ABCC5 的一些突变会引起转运体表达、功能的改变和机体对药物反应的改变。近年研究发现ABCC4、ABCC5 与某些肿瘤的多药耐药相关,转运体的过表达可以引起肿瘤细胞对多种肿瘤化疗药物的耐药性,导致临床化疗效果不佳。本文就 转运体ABCC4和ABCC5 介导的肿瘤多药耐药研究进展进行综述。     

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible “bulge” loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.     

Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus

Abstract The gene encoding the secreted alkaline protease, a suspected virulence factor of Aspergillus fumigatus , was inactivated by gene disruption. The disruption was performed by transformation of a pathogenic strain of the fungus with a linear DNA fragment carrying the gene from which the central part was replaced by the selectable Escherichia coli hygromycin B dominant resistance marker. Two transformants were shown to produce no alkaline protease. Restriction fragment analysis of the DNA of these two transformants was consistent for chromosomal integration of the disrupted gene by homologous recombination. Both isogenic alkaline protease-producing and non-producing A. fumigatus strains invaded lung tissues, causing comparable mortality in immunosuppressed mice. A significant residual proteolytic activity observed in alkaline protease non-producing strain cultures could play a role in the invasion of the tissues by the fungus.     

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.     

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum

In Dictyostelium, soluble cell adhesion molecule, DdCAD-1, regulates cell–cell interaction through an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes revealed that the potential DdCAD-1 interacting protein was between 64 and 98 kDa. To isolate and identify the anchoring protein, GST-DdCAD-1 and anchoring protein were cross-linked in vivo by chemical cross-linker and stable protein complex was isolated by co-immunoprecipitation assays. The protein cross-linked to DdCAD-1 was extracted from the gel slice and trypsinized. The peptides were subjected to analysis by mass spectrometry, which showed that the putative anchoring protein belongs to ATP-binding cassette transporter family.     

Development of Azole Resistance in Aspergillus fumigatus during Azole Therapy Associated with Change in Virulence

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.     

ABCA1基因多态性与冠心病相关性研究

为了探讨ATP-Binding Cassette Transporter 1（ABCA1）基因R219K多态在中国汉族人群中的分布及其与冠心病（coronary heart disease,CHD）的关系,采用PCR-RFLP方法,对396例CHD患者和417名正常人ABCA1基因R219K多态位点进行分析。结果表明,对照组R219K多态K等位基因及KK基因型的频率（0.465、0.228）较CHD组（0.381、0.162） 显著为高（P＜0.05）;根据发病年龄分组,早发CHD组K等位基因及KK基因型频率（0.34、0.111）明显低于晚发CHD组（0.419、0.205）和对照组（P＜0.05）,而在对照组和晚发CHD组间无此频率差异显著性（P＞0.05）;KK基因型患者血浆甘油三酯（TG）水平较RR基因型显著降低（P＜0.05）;不同基因型患者间血浆高密度脂蛋白胆固醇（HDL-C）水平差异无显著性（P＞0.05）。提示 ABCA1基因R219K多态与CHD存在相关性;KK基因型可能具有对抗动脉粥样硬化的作用,但这种作用不伴有血浆HDL-C水平的改变。     

Dsc Orthologs Are Required for Hypoxia Adaptation,Triazole Drug Responses,and Fungal Virulence in Aspergillus fumigatus

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.     

Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.     

Composite Survival Index to Compare Virulence Changes in Azole-Resistant Aspergillus fumigatus Clinical Isolates

Understanding resistance to antifungal agents in Aspergillus fumigatus is of increasing importance for the treatment of invasive infections in immunocompromised patients. Although a number of molecular resistance mechanisms are described in detail, the potential accompanying virulence changes and impact on clinical outcome have had little attention. We developed a new measure of survival, the composite survival index (CSI) to use as a measure of the virulence properties of A. fumigatus. Using a novel mathematical model we found a strong correlation between the in vitro growth characteristics and virulence in vivo expressed as CSI. Our model elucidates how three critical parameters (the lag phase (τ), decay constant (λ), and growth rate (ν)) interact with each other resulting in a CSI that correlated with virulence. Hence, strains with a long lag phase and high decay constant were less virulent in a murine model of invasive aspergillosis, whereas high virulence for isolates with a high CSI was associated in vitro with rapid growth and short lag phases. Resistant isolates with cyp51A mutations, which account for the majority of azole resistant aspergillosis cases, did not show a lower virulence compared to azole-susceptible isolates. In contrast, the CSI index revealed that a non-cyp51A-mediated resistance mechanism was associated with a dramatic decrease in CSI. Because of its predictive value, the mathematical model developed may serve to explore strain characteristics in vitro to predict virulence in vivo and significantly reduce the number of experimental animals required in such studies. The proposed measure of survival, the CSI can be used more in a general form in survival studies to explore optimal treatment options.     

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.Invasive aspergillosis is a fungal disease caused by Aspergillus species that primarily affects immunocompromised patients, such as those treated for hematological malignancy. Patients may become infected by inhalation of ambient air that contains fungal spores. The Aspergillus conidia can penetrate into the alveoli and if not effectively removed, may germinate, proliferate, and cause invasive aspergillosis. Mortality and morbidity due to invasive aspergillosis remain a significant problem.Triazoles, such as itraconazole (ITZ), voriconazole, and posaconazole, are used increasingly in the management of patients with this disease. Although the risk of resistance due to the increased use of triazoles is considered low (11), we recently observed ITZ resistance rapidly emerging in clinical Aspergillus fumigatus isolates (19, 22, 24, 25). Azole resistance was observed in up to 6% of patients in our hospital and in up to 14.5% of isolates sent to our laboratory from other hospitals in The Netherlands, which were obtained from patients with aspergillus disease (19). Furthermore, azole resistance has been reported in other European countries (3, 13, 19). The ITZ-resistant isolates also showed significantly reduced susceptibility to the other mold-active medical triazoles voriconazole and posaconazole (19). A substitution of leucine for histidine at codon 98 (L98H), combined with a 34-bp tandem repeat (designated TR) in the promoter region of the cyp51A gene (TR/L98H), which is the target for antifungal azoles, was found in 94% of isolates (14, 19, 24).Azole resistance can develop through the exposure of the fungus to azole compounds, which may occur in azole-treated patients or through the use of azole compounds in the environment. The dominance of a single resistance mechanism is difficult to explain by resistance development in individual azole-treated patients, as one would expect multiple resistance mechanisms to develop. Also, spread by person-to-person transmission of any Aspergillus isolate is highly unlikely. As inhalation of airborne aspergillus spores is the common route of infection for aspergillus diseases, we hypothesized that the dominance of a single resistance mechanism in clinical ITZ-resistant isolates was more consistent with acquisition from a common environmental source (19). If azole-resistant A. fumigatus is present in our environment, patients could inhale resistant spores and subsequently develop azole-resistant disease. Indeed, azole-resistant aspergillosis was reported in azole-naïve patients, indicating that resistance does not exclusively develop during azole therapy (24).Favorable conditions for resistance development are exposure to azole compounds and the presence of reproducing fungus (1). A. fumigatus is abundantly present in our environment as saprophytic, reproducing fungi, most notably in soil and compost. Furthermore, azoles are commonly used for plant protection as well as material preservation. Therefore, it appears that resistance development in A. fumigatus is feasible in the environment, and isolates that develop resistance to fungicides might be cross-resistant to medical triazoles.We investigated if A. fumigatus isolates that are present in our environment are resistant to medical triazoles and if they are cross-resistant to azole fungicides. Furthermore, we characterized the isolates by microsatellite typing in order to determine if they were genetically related to clinical A. fumigatus isolates previously obtained from patients cared for in our University Medical Center.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

A Single UDP-galactofuranose Transporter Is Required for Galactofuranosylation in Aspergillus fumigatus

Galactofuranose (Galf) containing molecules have been described at the cell surface of several eukaryotes and shown to contribute to the virulence of the parasite Leishmania major and the fungus Aspergillus fumigatus. It is anticipated that a number of the surface glycoconjugates such as N-glycans or glycolipids are galactofuranosylated in the Golgi apparatus. This raises the question of how the substrate for galactofuranosylation reactions, UDP-Galf, which is synthesized in the cytosol, translocates into the organelles of the secretory pathway. Here we report the first identification of a Golgi-localized nucleotide sugar transporter, named GlfB, with specificity for a UDP-Galf. In vitro transport assays established binding of UDP-Galf to GlfB and excluded transport of several other nucleotide sugars. Furthermore, the implication of glfB in the galactofuranosylation of A. fumigatus glycoconjugates and galactomannan was demonstrated by a targeted gene deletion approach. Our data reveal a direct connection between galactomannan and the organelles of the secretory pathway that strongly suggests that the cell wall-bound polysaccharide originates from its glycosylphosphatidylinositol-anchored form.     

Identification of Fibrinogen-Binding Proteins of Aspergillus fumigatus Using Proteomic Approach

Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.