Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter.

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Platelets Affect the Activity of Amino Acid Transporter SNAT4 in HuH-7 Human Hepatoma Cells.

Platelets have been reported to exert diverse actions besides hemostasis and thrombus formation in the body. However, whether platelets affect transporter activity remains to be determined. In this study, we examined the effects of platelets on the activity of amino acid transporter system A, which is known to be changed by various factors, and we clarified the mechanism by which platelets affect system A activity. Among system A subtypes, we found that sodium-coupled neutral amino acid transporter (SNAT) 4 played a central role in the transport activity of system A in HuH-7 human hepatoma cells. Interestingly, platelets showed a biphasic effect on system A activity: activated platelet supernatants (APS) including the granule contents released from platelets downregulated system A activity at lower concentrations and the downregulation was suppressed at higher concentrations. The downregulation was due to a decrease in the affinity of SNAT4 for its substrate and not a decrease in the SNAT4 abundance on the plasma membrane. In addition, APS did not decrease the expression level of SNAT4 mRNA. On the other hand, platelets did not affect system A activity when the platelet suspension was added to HuH-7 cells. These results indicate that platelets indirectly affect the transport activity of system A by releasing bioactive substances but do not directly affect it by binding to HuH-7 cells.

Transcriptome analysis reveals regulatory mechanisms of different drought-tolerant Gleditsia sinensis seedlings under drought stress.

BACKGROUND: Gleditsia sinensis is a significant tree species from both ecological and economic perspectives. However, its growth is hampered by temporary droughts during the seedling stage, thereby impeding the development of the G. sinensis industry. Drought stress and rehydration of semi-annual potted seedlings using an artificial simulated water control method. RNA sequencing (RNA-seq) analyses were conducted on leaves collected from highly resistant (HR) and highly susceptible (HS) seedling families at five different stages during the process of drought stress and rehydration to investigate their gene expression patterns. RESULTS: The differentially expressed genes (DEGs) were predominantly enriched in pathways related to "chloroplast" (GO:0009507), "photosynthesis" (GO:0015979), "plant hormone signal transduction" (map04075), "flavonoid biosynthesis" (map00941), "stress response", "response to reactive oxygen species (ROS)" (GO:0000302), "signal transduction" (GO:0007165) in G. sinensis HR and HS families exposed to mild and severe drought stress. Additionally, the pathways related to "plant hormone signal transduction" (map04075), and osmoregulation were also enriched. The difference in drought tolerance between the two families of G. sinensis may be associated with "transmembrane transporter activity" (GO:0022857), "stress response", "hormones and signal transduction" (GO:0007165), "cutin, suberine and wax biosynthesis" (map00073), "ribosome" (map03010), "photosynthesis" (map00195), "sugar metabolism", and others. An enrichment analysis of DEGs under severe drought stress suggests that the drought tolerance of both families may be related to "water-soluble vitamin metabolic process" (GO:0006767), "photosynthesis" (map00195), "plant hormone signal transduction" (map04075), "starch and sucrose metabolism" (map00500), and "galactose metabolism" (map00052). Osmoregulation-related genes such as delta-1-pyrroline-5-carboxylate synthase (P5CS), Amino acid permease (AAP), Amino acid permease 2 (AAP2) and Trehalose-phosphate synthase (TPS), as well as the antioxidant enzyme L-ascorbate peroxidase 6 (APX6), may be significant genes involved in drought tolerance in G. sinensis. Five genes were selected randomly to validate the RNA-seq results using quantitative real-time PCR (RT-qPCR) and they indicated that the transcriptome data were reliable. CONCLUSIONS: The study presents information on the molecular regulation of the drought tolerance mechanism in G. sinensis and provides a reference for further research on the molecular mechanisms involved in drought tolerance breeding of G. sinensis.

SLC6A14 promotes ulcerative colitis progression by facilitating NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory condition with frequent relapse and recurrence. Evidence suggests the involvement of SLC6A14 in UC pathogenesis, but the central regulator remains unknown. AIM: To explore the role of SLC6A14 in UC-associated pyroptosis. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR), immunoblotting, and immunohistochemical were used to assess SLC6A14 in human UC tissues. Lipopolysaccharide (LPS) was used to induce inflammation in FHC and NCM460 cells and model enteritis, and SLC6A14 levels were assessed. Pyroptosis markers were quantified using enzyme-linked immunosorbent assay, Western blotting, and qRT-PCR, and EdU incubation, CCK-8 assays and flow cytometry were used to examine proliferation and apoptosis. Mouse models of UC were used for verification. RESULTS: SLC6A14 was increased and correlated with NLRP3 in UC tissues. LPS-induced FHC and NCM460 cells showed increased SLC6A14 levels. Reducing SLC6A14 increased cell proliferation and suppressed apoptosis. Reducing SLC6A14 decreased pyroptosis-associated proteins (ASC, IL-1ß, IL-18, NLRP3). NLRP3 overexpression counteracted the effects of sh-SLC6A14 on LPS-induced FHC and NCM460 cell pyroptosis. SLC6A14 improved the mucosa in mice with dextran sulfate sodium-induced colitis. CONCLUSION: SLC6A14 promotes UC pyroptosis by regulating NLRP3, suggesting the therapeutic potential of modulating the SLC6A14/NLRP3 axis.

Genome-wide identification of AAAP gene family and expression analysis in response to saline-alkali stress in foxtail millet (Setaria italica L.).

Amino acid/auxin permease (AAAP) genes encode a large family of protein transporters that play important roles in various aspects of plant growth and development. Here, we performed genome-wide identification of members in the foxtail millet (Setaria italica L.) AAAP family (SiAAAP) and their saline-alkali stress-induced expression patterns, resulting in the identification of 65 SiAAAP genes, which could be divided into eight subfamilies. Except for SiAAAP65, the remaining 64 genes were located on nine chromosomes of foxtail millet. Gene structure and conserved motif analyses indicated that the members in the same subfamily are highly conserved. Gene duplication event analysis suggested that tandem duplication may be the main factor driving the expansion of this gene family, and Ka/Ks analysis indicated that all the duplicated genes have undergone purifying selection. Transcriptome analysis showed differential expression of SiAAAPs in roots, stems, leaves, and tassel inflorescence. Analysis of cis-acting elements in the promoter indicated that SiAAAPs contain stress-responsive cis-acting elements. Under saline-alkali stress, qRT-PCR analysis showed that SiAAP3, SiLHT2, and SiAAP16 were differentially expressed between salt-alkali tolerant millet variety JK3 and salt-alkali sensitive millet variety B175. These results suggest that these genes may be involved in or regulate the response to saline-alkali stress, providing a theoretical basis for further studying the function of SiAAAPs.

Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.

A Toxoplasma gondii putative amino acid transporter localizes to the plant-like vacuolar compartment and controls parasite extracellular survival and stage differentiation.

Toxoplasma gondii is a protozoan parasite that infects a broad spectrum of hosts and can colonize many organs and cell types. The ability to reside within a wide range of different niches requires substantial adaptability to diverse microenvironments. Very little is known about how this parasite senses various milieus and adapts its metabolism to survive, replicate during the acute stage, and then differentiate to the chronic stage. T. gondii possesses a lysosome-like organelle known as the plant-like vacuolar compartment (PLVAC), which serves various functions, including digestion, ion storage and homeostasis, endocytosis, and autophagy. Lysosomes are critical for maintaining cellular health and function by degrading waste materials and recycling components. To supply the cell with the essential building blocks and energy sources required for the maintenance of its functions and structures, the digested solutes generated within the lysosome are transported into the cytosol by proteins embedded in the lysosomal membrane. Currently, a limited number of PLVAC transporters have been characterized, with TgCRT being the sole potential transporter of amino acids and small peptides identified thus far. To bridge this knowledge gap, we used lysosomal amino acid transporters from other organisms as queries to search the T. gondii proteome. This led to the identification of four potential amino acid transporters, which we have designated as TgAAT1-4. Assessing their expression and sub-cellular localization, we found that one of them, TgAAT1, localized to the PLVAC and is necessary for normal parasite extracellular survival and bradyzoite differentiation. Moreover, we present preliminary data showing the possible involvement of TgAAT1 in the PLVAC transport of arginine.IMPORTANCEToxoplasma gondii is a highly successful parasite infecting a broad range of warm-blooded organisms, including about one-third of all humans. Although Toxoplasma infections rarely result in symptomatic disease in individuals with a healthy immune system, the incredibly high number of persons infected, along with the risk of severe infection in immunocompromised patients and the potential link of chronic infection to mental disorders, makes this infection a significant public health concern. As a result, there is a pressing need for new treatment approaches that are both effective and well tolerated. The limitations in understanding how Toxoplasma gondii manages its metabolism to adapt to changing environments and triggers its transformation into bradyzoites have hindered the discovery of vulnerabilities in its metabolic pathways or nutrient acquisition mechanisms to identify new therapeutic targets. In this work, we have shown that the lysosome-like organelle plant-like vacuolar compartment (PLVAC), acting through the putative arginine transporter TgAAT1, plays a pivotal role in regulating the parasite's extracellular survival and differentiation into bradyzoites.

The posttranslational regulation of amino acid transporters is critical for their function in the tumor microenvironment.

Amino acid transporters (AATs) facilitate nutrient uptake and nutrient exchange between cancer and stromal cells. The posttranslational modification (PTM) of transporters is an important mechanism that tumor-associated cells use to dynamically regulate their function and stability in response to microenvironmental cues. In this review, we summarize recent findings that demonstrate the significance of N-glycosylation, phosphorylation, and ubiquitylation for the function of AATs. We also highlight powerful approaches that hijack the PTM machinery that could be used as therapeutics or tools to modulate transporter activity.

Metabolic control from the endolysosome: lysosome-resident amino acid transporters open novel therapeutic possibilities.

Amino acid transporters are generally recognized as machinery that transport amino acids from the extracellular environment into the cytoplasm. Although their primary function is the uptake of amino acids to supply the cell with nutrients and energy, endolysosome-resident amino acid (EL-aa) transporters possess several unique functions in accordance with their localization in intracellular vesicular membranes. They play pivotal roles in the maintenance of metabolic homeostasis via direct involvement in the amino acid sensing pathway, which regulates the activity of mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cellular metabolism. Additionally, some EL-aa transporters contribute to the maintenance of dynamic homeostasis of endolysosomes, including the regulation of endolysosomal acidity, by carrying amino acids out of endolysosomes. In addition, EL-aa transporters act as a scaffold to gather signaling molecules and multiple enzymes to control cellular metabolism on the endolysosomal membrane. Among EL-aa transporters, solute carrier family 15 member 4 (SLC15A4) is preferentially expressed in immune cells, including macrophages, dendritic cells, and B cells, and plays a key role in the integration of metabolic and inflammatory signals. In this review, we summarize our recent findings on EL-aa transporter contributions to inflammatory and metabolic signaling in the endolysosomes of immune cells by focusing on the SLC15 family, including SLC15A4 and SLC15A3, and discuss their uniqueness and universality. We also discuss the potential of targeting these EL-aa transporters in immune cells for the development of novel therapeutic strategies for inflammatory diseases. Because these transporters are highly expressed in immune cells and significantly alter the functions of immune cells, targeting them would provide a great advantage in ensuring a wide safety margin.

ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis.

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane-localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone-substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3-AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3-AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

Disruption of the amino acid transporter CsAAP2 inhibits auxin-mediated root development in cucumber.

Amino acid transporters are the principal mediators of organic nitrogen distribution within plants and are essential for plant growth and development. Despite this importance, relatively few amino acid transporter genes have been explored and elucidated in cucumber (Cucumis sativus). Here, a total of 86 amino acid transporter genes were identified in the cucumber genome. We further identified Amino Acid Permease (AAP) subfamily members that exhibited distinct expression patterns in different tissues. We found that the CsAAP2 as a candidate gene encoding a functional amino acid transporter is highly expressed in cucumber root vascular cells. CsAAP2 knockout lines exhibited arrested development of root meristem, which then caused the delayed initiation of lateral root and the inhibition of root elongation. What is more, the shoot growth of aap2 mutants was strongly retarded due to defects in cucumber root development. Moreover, aap2 mutants exhibited higher concentrations of amino acids and lignin in roots. We found that the mutant roots had a stronger ability to acidize medium. Furthermore, in the aap2 mutants, polar auxin transport was disrupted in the root tip, leading to high auxin levels in roots. Interestingly, slightly alkaline media rescued their severely reduced root growth by stimulating auxin pathway.

Knock out of amino acid transporter gene OsLHT1 accelerates leaf senescence and enhances resistance to rice blast fungus.

Plant amino acid transporters regulate not only long-distance transport and reallocation of nitrogen (N) from source to sink organs, but also the amount of amino acids in leaves hijacked by invading pathogens. However, the function of amino acid transporters in plant defense responses to pathogen infection remains unknown. In this study, we found that the rice amino acid transporter gene OsLHT1 was expressed in leaves and up-regulated by maturation, N starvation, and inoculation of the blast fungus Magnaporthe oryzae. Knock out of OsLHT1 resulted in development stage- and N supply-dependent premature senescence of leaves at the vegetative growth stage. In comparison with the wild type, Oslht1 mutant lines showed sustained rusty red spots on fully mature leaf blades irrespective of N supply levels. Notably, no relationship between the severity of leaf rusty red spots and concentration of total N or amino acids was found in Oslht1 mutants at different developmental stages. Disruption of OsLHT1 altered transport and metabolism of amino acids and biosynthesis of flavones and flavonoids, enhanced expression of jasmonic acid- and salicylic acid-related defense genes, production of jasmonic acid and salicylic acid, and accumulation of reactive oxygen species. OsLHT1 inactivation dramatically prevented the leaf invasion by M. oryzae, a hemi-biotrophic ascomycete fungus. Overall, these results establish a link connecting the activity of an amino acid transporter with leaf metabolism and defense against rice blast fungus.

Contribution of the L-Type Amino Acid Transporter Family in the Diagnosis and Treatment of Prostate Cancer.

The L-type amino acid transporter (LAT) family contains four members, LAT1~4, which are important amino acid transporters. They mainly transport specific amino acids through cell membranes, provide nutrients to cells, and are involved in a variety of metabolic pathways. They regulate the mTOR signaling pathway which has been found to be strongly linked to cancer in recent years. However, in the field of prostate cancer (PCa), the LAT family is still in the nascent stage of research, and the importance of LATs in the diagnosis and treatment of prostate cancer is still unknown. Therefore, this article aims to report the role of LATs in prostate cancer and their clinical significance and application. LATs promote the progression of prostate cancer by increasing amino acid uptake, activating the mammalian target of rapamycin (mTOR) pathway and downstream signals, mediating castration-resistance, promoting tumor angiogenesis, and enhancing chemotherapy resistance. The importance of LATs as diagnostic and therapeutic targets for prostate cancer was emphasized and the latest research results were introduced. In addition, we introduced selective LAT1 inhibitors, including JPH203 and OKY034, which showed excellent inhibitory effects on the proliferation of various tumor cells. This is the future direction of amino acid transporter targeting therapy drugs.

Amino Acid Starvation-Induced Glutamine Accumulation Enhances Pneumococcal Survival.

Bacteria are known to cope with amino acid starvation by the stringent response signaling system, which is mediated by the accumulation of the (p)ppGpp alarmones when uncharged tRNAs stall at the ribosomal A site. While a number of metabolic processes have been shown to be regulatory targets of the stringent response in many bacteria, the global impact of amino acid starvation on bacterial metabolism remains obscure. This work reports the metabolomic profiling of the human pathogen Streptococcus pneumoniae under methionine starvation. Methionine limitation led to the massive overhaul of the pneumococcal metabolome. In particular, methionine-starved pneumococci showed a massive accumulation of many metabolites such as glutamine, glutamic acid, lactate, and cyclic AMP (cAMP). In the meantime, methionine-starved pneumococci showed a lower intracellular pH and prolonged survival. Isotope tracing revealed that pneumococci depend predominantly on amino acid uptake to replenish intracellular glutamine but cannot convert glutamine to methionine. Further genetic and biochemical analyses strongly suggested that glutamine is involved in the formation of a "prosurvival" metabolic state by maintaining an appropriate intracellular pH, which is accomplished by the enzymatic release of ammonia from glutamine. Methionine starvation-induced intracellular pH reduction and glutamine accumulation also occurred to various extents under the limitation of other amino acids. These findings have uncovered a new metabolic mechanism of bacterial adaptation to amino acid limitation and perhaps other stresses, which may be used as a potential therapeutic target for infection control. IMPORTANCE Bacteria are known to cope with amino acid starvation by halting growth and prolonging survival via the stringent response signaling system. Previous investigations have allowed us to understand how the stringent response regulates many aspects of macromolecule synthesis and catabolism, but how amino acid starvation promotes bacterial survival at the metabolic level remains largely unclear. This paper reports our systematic profiling of the methionine starvation-induced metabolome in S. pneumoniae. To the best of our knowledge, this represents the first reported bacterial metabolome under amino acid starvation. These data have revealed that the significant accumulation of glutamine and lactate enables S. pneumoniae to form a "prosurvival" metabolic state with a lower intracellular pH, which inhibits bacterial growth for prolonged survival. Our findings have provided insightful information on the metabolic mechanisms of pneumococcal adaptation to nutrient limitation during the colonization of the human upper airway.

Fetal membranes exhibit similar nutrient transporter expression profiles to the placenta.

INTRODUCTION: During pregnancy, the growth of the fetus is supported by the exchange of nutrients, waste, and other molecules between maternal and fetal circulations in the utero-placental unit. Nutrient transfer, in particular, is mediated by solute transporters such as solute carrier (SLC) and adenosine triphosphate-binding cassette (ABC) proteins. While nutrient transport has been extensively studied in the placenta, the role of human fetal membranes (FM), which was recently reported to have a role in drug transport, in nutrient uptake remains unknown. OBJECTIVES: This study determined nutrient transport expression in human FM and FM cells and compared expression with placental tissues and BeWo cells. METHODS: RNA sequencing (RNA-Seq) of placental and FM tissues and cells was done. Genes of major solute transporter groups, such as SLC and ABC, were identified. Proteomic analysis of cell lysates was performed via nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to confirm expression at a protein level. RESULTS: We determined that FM tissues and cells derived from the fetal membrane tissues express nutrient transporter genes, and their expression is similar to that seen in the placenta or BeWo cells. In particular, transporters involved in macronutrient and micronutrient transfer were identified in both placental and FM cells. Consistent with RNA-Seq findings, carbohydrate transporters (3), vitamin transport-related proteins (8), amino acid transporters (21), fatty acid transport-related proteins (9), cholesterol transport-related proteins (6) and nucleoside transporters (3) were identified in BeWo and FM cells, with both groups sharing similar nutrient transporter expression. CONCLUSION: This study determined the expression of nutrient transporters in human FMs. This knowledge is the first step in improving our understanding of nutrient uptake kinetics during pregnancy. Functional studies are required to determine the properties of nutrient transporters in human FMs.

A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion.

Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.

Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

The organic zinc with moderate chelation strength enhances the expression of related transporters in the jejunum and ileum of broilers.

Our previous study demonstrated that the zinc (Zn) proteinate with moderate chelation strength (Zn-Prot M) enhanced the Zn absorption in the small intestine partially via increasing the expression of some Zn and amino acid transporters in the duodenum of broilers. However, it remains unknown whether the Zn-Prot M could also regulate the expression of related transporters in the jejunum and ileum of broilers in the above enhancement of Zn absorption. The present study was conducted to investigate the effect of the Zn-Prot M on the expression of related transporters in the jejunum and ileum of broilers compared to the Zn sulfate (ZnS). Zinc-deficient broilers (13-d-old) were fed with the Zn-unsupplemented basal diets (control) or the basal diets supplemented with 60 mg Zn/kg as ZnS or Zn-Prot M for 26 d. The results showed that in the jejunum, compared to the control, supplementation of the organic or inorganic Zn increased (P < 0.05) mRNA and protein expression of b0,+-type amino acid transporter (rBAT), Zn transporter 10 (ZnT10), and peptide-transporter 1 (PepT1) mRNA expression and Zn transporter 7 (ZnT7) protein expression on d 28, while y+L-type amino transporter 2 (y+LAT2) mRNA and protein expression, and protein expression of ZnT7 and ZnT10 on 28 d and zrt-irt-like protein 3 (ZIP3) and zrt-irt-like protein 5 (ZIP5) on d 39 were higher (P < 0.05) for Zn-Prot M than for ZnS. In the ileum, Zn addition regardless of Zn source up-regulated (P < 0.05) mRNA expression of Zn transporter 9 (ZnT9) and ZIP3, ZIP5, and y+LAT2 protein expression on d 28, and PepT1 mRNA and protein expression, ZIP3 and y+LAT2 mRNA expression and ZnT10 protein expression on d 39. Furthermore, Zn transporter 4 (ZnT4) and ZnT9 mRNA expression and Zn transporter 1 (ZnT1) protein expression on d 28, and y+LAT2 mRNA expression and ZnT10 and PepT1 protein expression on d 39 were higher (P < 0.05) for Zn-Prot M than for ZnS. It was concluded that the Zn-Prot M enhanced the expression of the ZnT1, ZnT4, ZnT9, ZnT10, ZIP3, ZIP5, y+LAT2, and PepT1 in the jejunum or ileum of broilers compared to the ZnS.