Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics

Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC₅₀ value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.     

Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

Isolation of low-pathogenic H7N3 avian influenza from wild birds in Peru

Wild birds serve as natural reservoirs and sometimes harbor low-pathogenic avian influenza viruses. However, mutation of the virus can result in highly pathogenic strains, often more common among H5 and H7 genotypes. We report the isolation of a low-pathogenic H7N3 avian influenza in a Peruvian wetland.     

Sentinel surveillance of influenza-like-illness in two cities of the tropical country of Ecuador: 2006-2010

Background

Tropical countries are thought to play an important role in the global behavior of respiratory infections such as influenza. The tropical country of Ecuador has almost no documentation of the causes of acute respiratory infections. The objectives of this study were to identify the viral agents associated with influenza like illness (ILI) in Ecuador, describe what strains of influenza were circulating in the region along with their epidemiologic characteristics, and perform molecular characterization of those strains.

Methodology/Findings

This is a prospective surveillance study of the causes of ILI based on viral culture of oropharyngeal specimens and case report forms obtained in hospitals from two cities of Ecuador over 4 years. Out of 1,702 cases of ILI, nine viral agents were detected in 597 patients. During the time of the study, seven genetic variants of influenza circulated in Ecuador, causing six periods of increased activity. There appeared to be more heterogeneity in the cause of ILI in the tropical city of Guayaquil when compared with the Andean city of Quito.

Conclusions/Significance

This was the most extensive documentation of the viral causes of ILI in Ecuador to date. Influenza was a common cause of ILI in Ecuador, causing more than one outbreak per year. There was no well defined influenza season although there were periods of time when no influenza was detected alternating with epidemics of different variant strains.     

Sentinel Surveillance of Influenza-Like Illness in Two Hospitals in Maracay,Venezuela: 2006–2010

Background

Limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical South American countries such as Venezuela. The objective of the present study was to examine the epidemiology of influenza-like illness (ILI) in two hospitals in Maracay, Venezuela.

Methodology/Principal Findings

We performed a prospective surveillance study of persons with ILI who presented for care at two hospitals in Maracay, Venezuela, from October 2006 to December 2010. A respiratory specimen and clinical information were obtained from each participant. Viral isolation and identification with immunofluorescent antibodies and molecular methods were employed to detect respiratory viruses such as adenovirus, influenza A and B, parainfluenza, and respiratory sincytial virus, among others. There were 916 participants in the study (median age: 17 years; range: 1 month – 86 years). Viruses were identified in 143 (15.6%) subjects, and one participant was found to have a co-infection with more than one virus. Influenza viruses, including pandemic H1N1 2009, were the most frequently detected pathogens, accounting for 67.4% (97/144) of the viruses detected. Adenovirus (15/144), parainfluenza virus (13/144), and respiratory syncytial virus (11/144) were also important causes of ILI in this study. Pandemic H1N1 2009 virus became the most commonly isolated influenza virus during its initial appearance in 2009. Two waves of the pandemic were observed: the first which peaked in August 2009 and the second - higher than the preceding - that peaked in October 2009. In 2010, influenza A/H3N2 re-emerged as the most predominant respiratory virus detected.

Conclusions/Significance

Influenza viruses were the most commonly detected viral organisms among patients with acute febrile respiratory illnesses presenting at two hospitals in Maracay, Venezuela. Pandemic H1N1 2009 influenza virus did not completely replace other circulating influenza viruses during its initial appearance in 2009. Seasonal influenza A/H3N2 was the most common influenza virus in the post-pandemic phase.     

Circulating strains of human respiratory syncytial virus in central and south America

Human respiratory syncytial virus (HRSV) is a major cause of viral lower respiratory tract infections among infants and young children. HRSV strains vary genetically and antigenically and have been classified into two broad subgroups, A and B (HRSV-A and HRSV-B, respectively). To date, little is known about the circulating strains of HRSV in Latin America. We have evaluated the genetic diversity of 96 HRSV strains by sequencing a variable region of the G protein gene of isolates collected from 2007 to 2009 in Central and South America. Our results show the presence of the two antigenic subgroups of HRSV during this period with the majority belonging to the genotype HRSV-A2.