Extracellular Heme Uptake and the Challenge of Bacterial Cell Membranes.

Iron is essential for the survival of most bacteria but presents a significant challenge given its limited bioavailability. Furthermore, the toxicity of iron combined with the need to maintain physiological iron levels within a narrow concentration range requires sophisticated systems to sense, regulate, and transport iron. Most bacteria have evolved mechanisms to chelate and transport ferric iron (Fe3+) via siderophore receptor systems, and pathogenic bacteria have further lowered this barrier by employing mechanisms to utilize the host's hemoproteins. Once internalized, heme is cleaved by both oxidative and nonoxidative mechanisms to release iron. Heme, itself a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such, pathogenic bacteria have evolved sophisticated cell surface signaling and transport systems to obtain heme from the host. In this review, we summarize the structure and function of the heme-sensing and transport systems of pathogenic bacteria and the potential of these systems as antimicrobial targets.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

A new antibiotic traps lipopolysaccharide in its intermembrane transporter.

Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

Structures and mechanisms of the Arabidopsis auxin transporter PIN3.

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development1,2. Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static.

Structural insights into the inhibition of glycine reuptake.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action2,4. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder2,5. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6-12. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors15,16. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

Targeting cholesterol homeostasis in hematopoietic malignancies.

Cholesterol is a vital lipid for cellular functions. It is necessary for membrane biogenesis, cell proliferation, and differentiation. In addition to maintaining cell integrity and permeability, increasing evidence indicates a strict link between cholesterol homeostasis, inflammation, and hematological tumors. This makes cholesterol homeostasis an optimal therapeutic target for hematopoietic malignancies. Manipulating cholesterol homeostasis by either interfering with its synthesis or activating the reverse cholesterol transport via the engagement of liver X receptors affects the integrity of tumor cells both in vitro and in vivo. Cholesterol homeostasis has also been manipulated to restore antitumor immune responses in preclinical models. These observations have prompted clinical trials involving acute myeloid leukemia to test the combination of chemotherapy with drugs interfering with cholesterol synthesis (ie, statins). We review the role of cholesterol homeostasis in hematopoietic malignancies as well as in cells of the tumor microenvironment and discuss the potential use of lipid modulators for therapeutic purposes.

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner.

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and ß-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

Effects of Three Kinds of Carbohydrate Pharmaceutical Excipients-Fructose, Lactose and Arabic Gum on Intestinal Absorption of Gastrodin through Glucose Transport Pathway in Rats.

BACKGROUND: Some glucoside drugs can be transported via intestinal glucose transporters (IGTs), and the presence of carbohydrate excipients in pharmaceutical formulations may influence the absorption of them. This study, using gastrodin as probe drug, aimed to explore the effects of fructose, lactose, and arabic gum on intestinal drug absorption mediated by the glucose transport pathway. METHODS: The influence of fructose, lactose, and arabic gum on gastrodin absorption was assessed via pharmacokinetic experiments and single-pass intestinal perfusion. The expression of sodium-dependent glucose transporter 1 (SGLT1) and sodium-independent glucose transporter 2 (GLUT2) was quantified via RT‒qPCR and western blotting. Alterations in rat intestinal permeability were evaluated through H&E staining, RT‒qPCR, and immunohistochemistry. RESULTS: Fructose reduced the area under the curve (AUC) and peak concentration (Cmax) of gastrodin by 42.7% and 63.71%, respectively (P < 0.05), and decreased the effective permeability coefficient (Peff) in the duodenum and jejunum by 58.1% and 49.2%, respectively (P < 0.05). SGLT1 and GLUT2 expression and intestinal permeability remained unchanged. Lactose enhanced the AUC and Cmax of gastrodin by 31.5% and 65.8%, respectively (P < 0.05), and increased the Peff in the duodenum and jejunum by 33.7% and 26.1%, respectively (P < 0.05). SGLT1 and GLUT2 levels did not significantly differ, intestinal permeability increased. Arabic gum had no notable effect on pharmacokinetic parameters, SGLT1 or GLUT2 expression, or intestinal permeability. CONCLUSION: Fructose, lactose, and arabic gum differentially affect intestinal drug absorption through the glucose transport pathway. Fructose competitively inhibited drug absorption, while lactose may enhance absorption by increasing intestinal permeability. Arabic gum had no significant influence.

Selective Aster inhibitors distinguish vesicular and nonvesicular sterol transport mechanisms.

The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.

Cathelicidin and PMB neutralize endotoxins by multifactorial mechanisms including LPS interaction and targeting of host cell membranes.

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer.

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS-PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS-wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)-localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration-approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

Kinetic Properties and Pharmacological Modulation of High- and Low-Affinity Dopamine Transport in Striatal Astrocytes of Adult Rats.

Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [3H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [3H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders.

A Quantity-Dependent Nonlinear Model of Sodium Cromoglycate Suppression on Beta-Conglycinin Transport.

Understanding the transport mechanism is crucial for developing inhibitors that block allergen absorption and transport and prevent allergic reactions. However, the process of how beta-conglycinin, the primary allergen in soybeans, crosses the intestinal mucosal barrier remains unclear. The present study indicated that the transport of beta-conglycinin hydrolysates by IPEC-J2 monolayers occurred in a time- and quantity-dependent manner. The beta-conglycinin hydrolysates were absorbed into the cytoplasm of IPEC-J2 monolayers, while none were detected in the intercellular spaces. Furthermore, inhibitors such as methyl-beta-cyclodextrin (MßCD) and chlorpromazine (CPZ) significantly suppressed the absorption and transport of beta-conglycinin hydrolysates. Of particular interest, sodium cromoglycate (SCG) exhibited a quantity-dependent nonlinear suppression model on the absorption and transport of beta-conglycinin hydrolysates. In conclusion, beta-conglycinin crossed the IPEC-J2 monolayers through a transcellular pathway, involving both clathrin-mediated and caveolae-dependent endocytosis mechanisms. SCG suppressed the absorption and transport of beta-conglycinin hydrolysates by the IPEC-J2 monolayers by a quantity-dependent nonlinear model via clathrin-mediated and caveolae-dependent endocytosis. These findings provide promising targets for both the prevention and treatment of soybean allergies.

Effects of Commonly used Surfactants, Poloxamer 188 and Tween 80, on the Drug Transport Capacity of Intestinal Glucose Transporters.

Some glycoside drugs can be transported through intestinal glucose transporters (IGTs). The surfactants used in oral drug preparations can affect the function of transporter proteins. This study aimed to investigate the effect of commonly used surfactants, Poloxamer 188 and Tween 80, on the drug transport capacity of IGTs. Previous studies have shown that gastrodin is the optimal drug substrate for IGTs. Gastrodin was used as a probe drug to evaluate the effect of these two surfactants on intestinal absorption in SD rats through pharmacokinetic and in situ single-pass intestinal perfusion. Then, the effects of the two surfactants on the expression of glucose transporters and tight-junction proteins were examined using RT-PCR and western blotting. Additionally, the effect of surfactants on intestinal permeability was evaluated through hematoxylin-eosin staining. The results found that all experimental for Poloxamer 188 (0.5%, 2.0% and 8.0%) and Tween 80 (0.1% and 2.0%) were not significantly different from those of the blank group. However, the AUC(0-∞) of gastrodin increased by approximately 32% when 0.5% Tween 80 was used. The changes in IGT expression correlated with the intestinal absorption of gastrodin. A significant increase in the expression of IGTs was observed at 0.5% Tween 80. In conclusion, Poloxamer 188 had minimal effect on the drug transport capacity of IGTs within the recommended limits of use. However, the expression of IGTs increased in response to 0.5% Tween 80, which significantly enhanced the drug transport capacity of IGTs. However, 0.1% and 2.0% Tween 80 had no significant effect.

Flavonols regulate root hair development by modulating accumulation of reactive oxygen species in the root epidermis.

Reactive oxygen species (ROS) are signaling molecules produced by tissue-specific respiratory burst oxidase homolog (RBOH) enzymes to drive development. In Arabidopsis thaliana, ROS produced by RBOHC was previously reported to drive root hair elongation. We identified a specific role for one ROS, H2O2, in driving root hair initiation and demonstrated that localized synthesis of flavonol antioxidants control the level of H2O2 and root hair formation. Root hairs form from trichoblast cells that express RBOHC and have elevated H2O2 compared with adjacent atrichoblast cells that do not form root hairs. The flavonol-deficient tt4 mutant has elevated ROS in trichoblasts and elevated frequency of root hair formation compared with the wild type. The increases in ROS and root hairs in tt4 are reversed by genetic or chemical complementation. Auxin-induced root hair initiation and ROS accumulation were reduced in an rbohc mutant and increased in tt4, consistent with flavonols modulating ROS and auxin transport. These results support a model in which localized synthesis of RBOHC and flavonol antioxidants establish patterns of ROS accumulation that drive root hair formation.

Neuraminidase B controls neuraminidase A-dependent mucus production and evasion.

Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase A (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions.