Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide–tetrazolium coupling reactions for quantitative l-lactate analysis

In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2⁻⁶ U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD⁺ (2 μl, 1.5 × 10⁻⁵ M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.     

Protein Kinase C-Dependent Growth-Associated Protein 43 Phosphorylation Regulates Gephyrin Aggregation at Developing GABAergic Synapses

Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABA_A receptors (GABA_ARs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43^S41A, but not PKC phosphorylation-mimicking GAP43^S41D, interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43^S41D, but not GAP43^S41A, inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABA_ARs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses.     

Lipoteichoic acid induces HO-1 expression via the TLR2/MyD88/c-Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth muscle cells

Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. Increasing reports have indicated that lipoteichoic acid (LTA) exerts as LPS as an immune system-stimulating agent and plays a role in the pathogenesis of severe inflammatory responses induced by Gram-positive bacterial infection. We report that LTA is an inducer of HO-1 expression mediated through the signaling pathways in human tracheal smooth muscle cells (HTSMCs). LTA-induced HO-1 protein levels, mRNA expression, and promoter activity were attenuated by transfection with dominant negative mutants of TLR2 and MyD88, by pretreatment with the inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride (DPI) and apocynin (APO)), and reactive oxygen species (ROS) scavenger (N-acetyl-l-cysteine) or by transfection with small interfering RNAs of Src and NF-E2-related factor 2 (Nrf2). LTA-stimulated translocation of p47(phox) and Nrf2 or ROS production was attenuated by transfection with dominant negative mutants of TLR2, MyD88, and c-Src and by pretreatment with DPI or APO. Furthermore, LTA-induced TLR2, MyD88, TNFR-associated factor (TRAF)6, c-Src, and p47(phox) complex formation was revealed by immunoprecipitation using an anti-TLR2 or anti-c-Src Ab followed by Western blot analysis against an anti-TLR2, anti-MyD88, anti-TRAF6, anti-c-Src, or anti-p47(phox) Ab. These results demonstrated that LTA-induced ROS generation was mediated through the TLR2/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiates the activation of Nrf2, and ultimately induces HO-1 expression in HTSMCs.     

T-cell tropism and the role of ORF66 protein in pathogenesis of varicella-zoster virus infection

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-gamma. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.     

Intracellular GSH and ascorbate inhibit radical-induced protein chain peroxidation in HL-60 cells

The results of this study suggest that the well-documented loss of GSH and ascorbate in organisms under oxidative stress may be mainly due to their reactions with protein radicals and/or peroxides. Protein hydroperoxides were generated in HL-60 cells exposed to radiation-generated hydroxyl radicals. We found for the first time evidence of chain peroxidation of the proteins in cells, with each hydroxyl radical leading to the formation of about 10 hydroperoxides. Protein peroxidation showed a lag, probably due to the endogenous antioxidant enzymes, with simultaneous loss of the intracellular GSH. Enhancement of the GSH levels by N-acetylcysteine decreased the formation of hydroperoxides, while treatment with l-buthionine sulfoximine had the opposite effect. The effect of variation of GSH levels on the formation of the peroxidized proteins is explained primarily by reduction of the protein hydroperoxides by GSH. Loading of the cells with ascorbate resulted in reduction of the amounts of protein hydroperoxides generated by the radiation, which was proportional to the intracellular ascorbate concentration. In contrast to the GSH, inhibition of protein hydroperoxide formation in the presence of the high (mM) intracellular ascorbate levels achieved was mainly due to the direct scavenging of hydroxyl radicals by the vitamin.     

Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-κB activation

The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.     

Vascular endothelial growth factor-C modulates proliferation and chemoresistance in acute myeloid leukemic cells through an endothelin-1-dependent induction of cyclooxygenase-2

High-level expression of vascular endothelial growth factor (VEGF)-C is associated with chemoresistance and adverse prognosis in acute myeloid leukemia (AML). Our previous study has found that VEGF-C induces cyclooxygenase-2 (COX-2) expression in AML cell lines and significant correlation of VEGF-C and COX-2 in bone marrow specimens. COX-2 has been reported to mediate the proliferation and drug resistance in several solid tumors. Herein, we demonstrated that the VEGF-C-induced proliferation of AML cells is effectively abolished by the depletion or inhibition of COX-2. The expression of endothelin-1 (ET-1) rapidly increased following treatment with VEGF-C. We found that ET-1 was also involved in the VEGF-C-mediated proliferation of AML cells, and that recombinant ET-1 induced COX-2 mRNA and protein expressions in AML cells. Treatment with the endothelin receptor A (ETRA) antagonist, BQ 123, or ET-1 shRNAs inhibited VEGF-C-induced COX-2 expression. Flow cytometry and immunoblotting revealed that VEGF-C induces S phase accumulation through the inhibition of p27 and the upregulation of cyclin E and cyclin-dependent kinase-2 expressions. The cell-cycle-related effects of VEGF-C were reversed by the depletion of COX-2 or ET-1. The depletion of COX-2 or ET-1 also suppressed VEGF-C-induced increases in the bcl-2/bax ratio and chemoresistance against etoposide and cytosine arabinoside in AML cells. We also demonstrated VEGF-C/ET-1/COX-2 axis-mediated chemoresistance in an AML xenograft mouse model. Our findings suggest that VEGF-C induces COX-2-mediated resistance to chemotherapy through the induction of ET-1 expression. Acting as a key regulator in the VEGF-C/COX-2 axis, ET-1 represents a potential target for ameliorating resistance to chemotherapy in AML patients.     

Specific inactivation of an antifungal bacterial siderophore by a fungal plant pathogen

Bacteria and fungi secrete many natural products that inhibit each other’s growth and development. The dynamic changes in secreted metabolites that occur during interactions between bacteria and fungi are complicated. Pyochelin is a siderophore produced by many Pseudomonas and Burkholderia species that induces systemic resistance in plants and has been identified as an antifungal agent. Through imaging mass spectrometry and metabolomics analysis, we found that Phellinus noxius, a plant pathogen, can modify pyochelin and ent-pyochelin to an esterification product, resulting in reduced iron-chelation and loss of antifungal activity. We also observed that dehydroergosterol peroxide, the fungal metabolite, is only accumulated in the presence of pyochelin produced through bacteria–fungi interactions. For the first time, we show the fungal transformation of pyochelin in the microbial interaction. Our findings highlight the importance of understanding the dynamic changes of metabolites in microbial interactions and their influences on microbial communities.Subject terms: Microbial ecology, Metabolomics

Microorganisms use various strategies to establish themselves within an ecological niche while facing keen competition in the environment. Natural products such as antibiotics, quorum sensing molecules, and siderophores are crucial in microbial interactions [1–3]. Certain microorganisms are equipped with uptake systems that enable them to acquire siderophores, even by those that may not produce them [4]. For example, pyochelin is a siderophore produced by many Pseudomonas and Burkholderia strains. Such bacterial strains are commonly found in soils, as endophytes, and from the rhizosphere where they may inhibit plant pathogens [5, 6].Burkholderia cenocepacia 869T2 was isolated as an endophyte and showed beneficial abilities to control banana Fusarium wilt [7]. It harbors many biosynthetic gene clusters of secondary metabolites, such as pyochelin, pyrrolnitrin, and pyrroloquinoline quinone [8]. Recently, we found that this strain could temporarily inhibit the growth of P. noxius, a fungal pathogen of brown root rot disease, which is prevalent in tropical and subtropical regions and has a wide host range covering over 200 plant species [9]. However, in the competition between fungi and bacteria, P. noxius can resist this inhibition and overwhelm bacterial colonies after 1–2 weeks under dual-culture conditions (Fig. S1). These results imply that fungi might have resistance responses and undergo metabolic changes in bacteria–fungi interactions [10]. Here we unveiled metabolic changes in the competitive interaction between B. cenocepacia 869T2 and P. noxius 2252 using the matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS) [11, 12].We specifically monitored the metabolites in the inhibition region of B. cenocepacia 869T2 and P. noxius 2252 dual-culture using MALDI-TOF IMS. Several induced or enzymatically modified metabolites were detected, including m/z 275, 362, 383, and 427 (Fig. 1A). In particular, pyochelin (m/z 325), surrounding the B. cenocepacia 869T2 colony, showed asymmetric distribution in dual-culture samples. Near the P. noxius 2252 mycelia, a new metabolite with m/z 383 was detected with a complementary distribution to pyochelin (Fig. 1A). In LC-MS/MS-based molecular networking analysis [13], we found that this new metabolite structure is an esterification product of pyochelin and glycolic acid, which we named pyochelin-GA (Fig. 1B). We then constructed a pchF-null mutant strain, ΔpchF, which cannot produce pyochelin, and then dual cultured it with P. noxius. Pyochelin and pyochelin-GA were not observed in the MALDI-TOF IMS and LC-MS analysis of dual-culture samples (Fig. 1A and Fig. S2). We further inoculated P. noxius 2252 with pyochelin-GA-free extract harvested from B. cenocepacia 869T2 single culture, and the complementary distribution of pyochelin and pyochelin-GA was observed by MALDI-TOF IMS again (Fig. S3). These results demonstrated that pyochelin-GA was transformed from pyochelin by P. noxius 2252, rather than produced by B. cenocepacia 869T2 under dual-culture conditions.Open in a separate windowFig. 1Metabolic changes in the bacteria–fungi interaction.A Spatial distribution of selected mass signals (m/z) in MALDI-TOF IMS analysis of Phellinus noxius 2252 (Pn2252) dual-cultured with Burkholderia cenocepacia 869T2 (869T2) and a pchF-null mutant strain (Δ pchF). B Molecular networking analysis of pyochelin and analogs from the dual-culture sample. The red node is pyochelin, and the green node is pyochelin-GA. The structures of pyochelin, pyochelin-GA, and dehydroergosterol peroxide (DHEP), together with their mass signals in MALDI-TOF IMS, are shown. C Iron-chelating abilities of pyochelin and pyochelin-GA were evaluated by Chrome Azurol S liquid assay using different concentrations (2.5, 1.25, 0.63, 0.31, and 0.16 mM, n = 3). Proportions of siderophore units are shown in Fig. S14. D Fungal transformation of pyochelin and ent-pyochelin by treating P. noxius 2252 with ethyl acetate crude extracts of B. cenocepacia 869T2, Pseudomonas aeruginosa PAO1, and P. protegens Pf-5 for 8 h. LC-MS was used to monitor the signals of pyochelin (red), ent-pyochelin (blue), and transformation product 383 (black).The chemical structure of pyochelin-GA was further confirmed via total synthesis, NMR, and LC-MS/MS analysis (Supplementary Material and Methods, and Figs. S4–7). The purified pyochelin and pyochelin-GA were also evaluated for their iron-chelating ability. Chrome Azurol S assay indicated that pyochelin had the dose-dependent iron-chelating ability, but pyochelin-GA had lower iron-binding efficiency (Fig. 1C, Fig. S8). Pyochelin chelates iron in the extracellular medium and transports it into cells via the specific outer membrane transporter FptA. The X-ray structure of FptA-pyochelin-Fe indicated that the terminal carboxylic acid of pyochelin plays an essential role in the iron uptake ability [14, 15]. Our docking analysis suggested that the glycolic ester moiety of pyochelin-GA would affect the binding pocket shape of FptA and result in different binding properties compared to FptA-pyochelin (Fig. S9).Pyochelin and ent-pyochelin are produced independently by different biosynthetic gene clusters in Pseudomonas species [16]. To determine whether P. noxius 2252 can transform both enantiomers via this esterification process, we treated P. noxius 2252 with the extracts of pyochelin producers (P. aeruginosa PAO1 and B. cenocepacia 869T2) and an ent-pyochelin producer (P. protegens Pf-5). After 8 h of treatment, both pyochelin and ent-pyochelin were converted to pyochelin-GA (or ent-pyochelin-GA) (Fig. 1D), demonstrating this is a non-stereospecific transformation.To better understand the iron-chelating ability of pyochelin, we used pyochelin and pyochelin-GA to treat P. noxius 2252 under iron-deficiency conditions, by adding the iron chelator deferoxamine, and iron-rich conditions by adding FeCl₃ (Fig. 2). Pyochelin-GA did not affect the growth of P. noxius 2252 under all conditions. However, P. noxius 2252 was more sensitive to pyochelin in iron-deficient conditions and more resistant to pyochelin in iron-rich conditions, demonstrating that iron availability directly affected the tolerance of P. noxius 2252 to pyochelin. A similar phenomenon was reported previously for Aspergillus fumigatus [17].Open in a separate windowFig. 2Pyochelin inhibition of mycelial growth of Phellinus noxius 2252 is inversely associated with iron concentration.Pyochelin-GA did not have an inhibition effect on P. noxius 2252. Potato dextrose agar (PDA) with deferoxamine (DFO; 200 and 400 µM) was used to mimic iron-deficiency conditions. Iron-rich conditions was prepared by adding FeCl₃ (200 and 400 µM) in PDA. P. noxius 2252 was treated with 0.03, 0.06, 0.12, and 0.24 µmol of pyochelin or pyochelin-GA at 30 °C for 24 h. The antifungal assay was performed in two biological replicates.Using MALDI-TOF IMS analysis of the dual-culture of B. cenocepacia 869T2 and P. noxius 2252, we observed that several metabolites (e.g., m/z 275, 362, and 427) were only observed in the boundary of fungal mycelia (Fig. 1A). Although those metabolites were not detected in the dual-culture of ΔpchF and P. noxius 2252 (Fig. 1A), they were present when we treated P. noxius 2252 with pyochelin (Fig. S10). We identified the metabolite associated with m/z 427 as dehydroergosterol peroxide (DHEP) (Fig. S11), which was initially oxidized from ergosterol and dehydroergosterol [18]. Pyochelin can enhance intercellular reactive oxygen species (ROS) and ultimately disrupts membrane integrity, leading to cell death [17, 19, 20]. To clarify whether ROS induced the accumulation of DHEP, we treated P. noxius 2252 with pyochelin, pyochelin-GA, and 2,2′-bipyridyl (an iron chelator). Pyochelin and 2,2′-bipyridyl showed antifungal effects on P. noxius 2252 and induced ROS production (Fig. S12). However, the accumulation of DHEP in P. noxius 2252 was only associated with pyochelin treatment (Fig. S13). The induction of ROS in P. noxius 2252 by pyochelin and pyochelin-GA was not significantly different (Fig. S14). Therefore, we predict that pyochelin-induced accumulation of DHEP in P. noxius 2252 is independent of ROS production and iron-deficiency.Overall, we demonstrate that pyochelin transformation by fungi, in the interaction between pyochelin-producing bacteria and the plant pathogen P. noxius transforms pyochelin and ent-pyochelin into pyochelin-GA (and ent-pyochelin-GA). This product no longer functions as an iron chelator and no longer shows antifungal activity. The production of a fungal metabolite, dehydroergosterol peroxide, was induced explicitly by pyochelin through an unknown mechanism. These results highlight the importance of monitoring dynamic changes of metabolites in situ to better understand the functions and influences of metabolites on microbial community interactions.