HemU and TonB1 contribute to hemin acquisition in Stenotrophomonas maltophilia.

Introduction: The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Methods: Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Results: Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. Conclusion: HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.

Characterization of the broad-spectrum antibacterial activity of bacteriocin-like inhibitory substance-producing probiotics isolated from fermented foods.

Antimicrobial peptides, such as bacteriocin, produced by probiotics have become a promising novel class of therapeutic agents for treating infectious diseases. Selected lactic acid bacteria (LAB) isolated from fermented foods with probiotic potential were evaluated for various tests, including exopolysaccharide production, antibiotic susceptibility, acid and bile tolerance, antibacterial activity, and cell adhesion and cytotoxicity to gastric cell lines. Six selected LAB strains maintained their high viability under gastrointestinal conditions, produced high exopolysaccharides, showed no or less cytotoxicity, and adhered successfully to gastric cells. Furthermore, three strains, Weissella confusa CYLB30, Lactiplantibacillus plantarum CYLB47, and Limosilactobacillus fermentum CYLB55, demonstrated a strong antibacterial effect against drug-resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica serovar Choleraesuis, Enterococcus faecium, and Staphylococcus aureus. Whole genome sequencing was performed on these three strains using the Nanopore platform; then, the results showed that all three strains did not harbor genes related to toxins, superantigens, and acquired antimicrobial resistance, in their genome. The bacteriocin gene cluster was found in CYLB47 genome, but not in CYLB30 and CYLB55 genomes. In SDS-PAGE, the extract of CYLB30 and CYLB47 bacteriocin-like inhibitory substance (BLIS) yielded a single band with a size of less than 10 kDa. These BLIS inhibited the growth and biofilm formation of drug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA), causing membrane disruption and inhibiting adhesion ability to human skin HaCaT cells. Moreover, CYLB30 and CYLB47 BLIS rescued the larvae after being infected with P. aeruginosa and MRSA infections. In conclusion, CYLB30 and CYLB47 BLIS may be potential alternative treatment for multidrug-resistant bacteria infections.

Modulatory role of SmeQ in SmeYZ efflux pump-involved functions in Stenotrophomonas maltophilia.

BACKGROUND: SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ-mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB'FA and sbiAB operons, and decreased intracellular iron level. OBJECTIVES: To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ-mediated pleiotropic defects. METHODS: The presence of operons was verified using RT-PCR. The role of SmeQ in ΔsmeYZ-mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein-protein interactions. Gene expression was quantified using quantitative RT-PCR (RT-qPCR). RESULTS: SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB'FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. CONCLUSIONS: SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ, entSCEBB'FA and sbiAB operons in smeYZ mutants.

Implication of the σE Regulon Members OmpO and σN in the ΔompA299-356-Mediated Decrease of Oxidative Stress Tolerance in Stenotrophomonas maltophilia.

Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299-356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299-356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299-356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299-356. KJΔOmpA299-356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299-356. Next, the involvement of the three σ factors in the ΔompA299-356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299-356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299-356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal ß-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to ß-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.

Identification of a novel interaction site between the large hepatitis delta antigen and clathrin that regulates the assembly of genotype III hepatitis delta virus.

BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.

Whole-genome-sequence-based characterization of an NDM-5-producing uropathogenic Escherichia coli EC1390.

BACKGROUND: Urinary tract infection (UTI) is one of the most common outpatient bacterial infections. In this study, we isolated and characterized an extensively-drug resistant (XDR) NDM-5-producing Escherichia coli EC1390 from a UTI patient by using whole-genome sequencing (WGS) in combination with phenotypic assays. METHODS: Antimicrobial susceptibility to 23 drugs was determined by disk diffusion method. The genome sequence of EC1390 was determined by Nanopore MinION MK1C platform. Conjugation assays were performed to test the transferability of EC1390 plasmids to E. coli recipient C600. Phenotypic assays, including growth curve, biofilm formation, iron acquisition ability, and cell adhesion, were performed to characterize the function of EC1390 plasmids. RESULTS: Our results showed that EC1390 was only susceptible to tigecycline and colistin, and thus was classified as XDR E. coli. A de novo genome assembly was generated using Nanopore 73,050 reads with an N50 value of 20,936 bp and an N90 value of 7,624 bp. WGS analysis showed that EC1390 belonged to the O101-H10 serotype and phylogenetic group A E. coli. Moreover, EC1390 contained 2 conjugative plasmids with a replicon IncFIA (pEC1390-1 with 156,286 bp) and IncFII (pEC1390-2 with 71,840 bp), respectively. No significant difference was observed in the bacterial growth rate in LB broth and iron acquisition ability between C600, C600 containing pEC1390-1, C600 containing pEC1390-2, and C600 containing pEC1390-1 and pEC1390-2. However, the bacterial growth rate in nutrition-limited M9 broth was increased in C600 containing pEC1390-2, and the cell adhesion ability was increased in C600 containing both pEC1390-1 and pEC1390-2. Moreover, these plasmids modulated the biofilm formation under different conditions. CONCLUSIONS: In summary, we characterized the genome of XDR-E. coli EC1390 and identified two plasmids contributing to the antimicrobial resistance, growth of bacteria in a nutrition-limited medium, biofilm formation, and cell adhesion.

Involvement of the hemP-hemA-smlt0796-smlt0797 Operon in Hemin Acquisition by Stenotrophomonas maltophilia.

The hemin acquisition system of Stenotrophomonas maltophilia was elucidated in this study. To identify the TonB-dependent outer membrane receptor for hemin in S. maltophilia, the hemin acquisition systems of Pseudomonas aeruginosa were referenced. PhuR, HasA, and HxuA are three known TonB-dependent outer membrane receptors involved in hemin acquisition by P. aeruginosa. Thus, HemA (Smlt0795) and Smlt2937, the orthologs of PhuR and HasA/HxuA in S. maltophilia, were first considered. KJΔEnt, a stenobactin-null strain, was used as the parental strain for the hemin utilization assay. Deletion of hemA, but not Smlt2937, of KJΔEnt impaired hemin acquisition under iron-depleted conditions, indicating that HemA is the TonB-dependent receptor for hemin uptake. The hemA gene is a member of the hemP-hemA-smlt0796-smlt0797 operon, whose expression was upregulated in a fur mutant and under iron-depleted conditions. The contribution of the hemP-hemA-smlt0796-smlt0797 operon to hemin acquisition was investigated by in-frame deletion mutant construction and hemin utilization assays. Inactivation of hemP, smlt0796, and smlt0797 of KJΔEnt insignificantly affected hemin acquisition under iron-depleted conditions. However, hemP deletion in a fur mutant increased hemin acquisition under iron-depleted conditions. Collectively, we revealed that (i) HemA likely functions as the outer membrane receptor for hemin uptake; (ii) HemP, a predicted transcriptional factor, apparently functions as a repressor of the expression of the hemA transcript; and (iii) in a fur mutant, HemP has a negative impact on hemin acquisition under iron-depleted conditions. IMPORTANCE Stenotrophomonas maltophilia is an emerging multidrug-resistant opportunistic pathogen, increasing the difficulty of treatment of this infection. Iron is a critical element for bacterial viability. Heme is the most abundant iron source in the human host; thus, heme is the major iron source for a pathogen in the infection niche. Blocking iron acquisition from heme can be an alternative strategy to control S. maltophilia infection. Although several hemin acquisition systems have been reported in various pathogens, very little is known about the hemin acquisition systems of S. maltophilia. By in-frame deletion mutant construction and hemin utilization assays, we demonstrated that HemA (Smlt0795) is the TonB-dependent outer membrane receptor for hemin uptake and that HemP (Smlt0794), a predicted transcriptional factor, had a negative impact on hemin acquisition in a fur mutant. The negative regulatory role of HemP in hemin acquisition is first reported.

Bisdemethoxycurcumin suppresses human brain glioblastoma multiforme GBM 8401 cell migration and invasion via affecting NF-κB and MMP-2 and MMP-9 signaling pathway in vitro.

Human glioblastoma (GBM) is one of the common cancer death in adults worldwide, and its metastasis will lead to difficult treatment. Finding compounds for future to develop treatment is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was isolated from the rhizome of turmeric (Curcuma longa), which has been shown to against many human cancer cells. In the present study, we evaluated the antimetastasis activity of BDMC in human GBM cells. Cell proliferation, cell viability, cellular uptake, wound healing, migration and invasion, and western blotting were analyzed. Results indicated that BDMC at 1.5-3 µM significantly decreased the cell proliferation by MTT assay. BDMC showed the highest uptake by cells at 3 h. After treatment of BDMC at 12-48 h significantly inhibited cell motility in GBM 8401 cells by wound healing assay. BDMC suppressed cell migration and invasion at 24 and 48 h treatment by transwell chamber assay. BDMC significantly decreased the levels of proteins associated with PI3K/Akt, Ras/MEK/ERK pathways and resulted in the decrease in the expressions of NF-κB, MMP-2, MMP-9, and N-cadherin, leading to the inhibition of cell migration and invasion. These findings suggest that BDMC may be a potential candidate for the antimetastasis of human GBM cells in the future.

The fciTABC and feoABI systems contribute to ferric citrate acquisition in Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia. METHODS: Candidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2'-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein-protein interaction. RESULTS: Smlt2858 and Smlt2356, the homologues of FecA and FecC/D of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein-protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate. CONCLUSIONS: The FciTABC and FeoABI systems contribute to ferric citrate acquisition in S. maltophilia.

FadACB and smeU1VWU2X Contribute to Oxidative Stress-Mediated Fluoroquinolone Resistance in Stenotrophomonas maltophilia.

Pathogenic bacteria experience diverse stresses induced by host cells during infection and have developed intricate systems to trigger appropriate responses. Bacterial stress responses have been reported to defend against these stresses and cross-protect bacteria from antibiotic attack. In this study, we aimed to assess whether oxidative stress affects bacterial susceptibility to fluoroquinolone (FQ) and the underlying mechanism. Stenotrophomonas maltophilia, a species with high genetic diversity, is distributed ubiquitously and is an emerging multidrug-resistant opportunistic pathogen. FQs are among the limited antibiotic treatment options for S. maltophilia infection. The minimum inhibitory concentrations (MICs) of 103 S. maltophilia clinical isolates against ciprofloxacin (CIP) and levofloxacin (LVX) were determined using the agar dilution method in Mueller-Hinton plates with or without menadione (MD), a superoxide generator. The resistance rates for ciprofloxacin and levofloxacin were 40% and 18% in the MD-null group and increased to 91% and 23%, respectively, in the MD-treated group. Of the 103 isolates tested, 54% and 27% had elevated MICs against ciprofloxacin and levofloxacin, respectively, in the presence of MD. The involvement of oxidative stress responses in the MD-mediated FQ resistance was further assessed by mutants construction and viability assay. Among the 16 oxidative stress alleviation systems evaluated, fadACB and smeU1VWU2X contributed to MD-mediated FQ resistance. The antibiotic susceptibility test is an accredited clinical method to evaluate bacterial susceptibility to antibiotics in clinical practice. However, oxidative stress-mediated antibiotic resistance was not detected using this test, which may lead to treatment failure.

Demethoxycurcumin Promotes Macrophage Cell Population and Phagocytosis in WEHI-3 Cell-generated Leukemia BALB/c Mice In Vivo.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the components of curcuminoids, has antitumor activities in many human cancer cells and is known to induce apoptosis in human leukemia cells. However, there are no reports showing the effects of DMC on the immune response in leukemia mice in vivo. Herein, we evaluated the impact of DMC on immune responses in WEHI-3-generated leukemia mice in vivo. MATERIALS AND METHODS: Fifty male BALB/c mice were separated randomly into five groups. Group I is normal mice, and groups II-V mice of generated leukemia by WEHI-3 cells. Group II-V mice were intraperitoneally injected with dimethyl sulfoxide (DMSO, as the positive control), 15, 30, and 60 mg/kg of DMC, respectively, every two days for 14 days. The body weight, blood, peritoneal fluid, liver, and spleen were individually analyzed. RESULTS: DMC did not significantly affect animal appearance and body weight. It decreased liver and spleen weight at a high dose. DMC did not affect the cluster of differentiation 3 (CD3) and CD19 cell populations but induced decrease of CD11b at 30 mg/kg treatment. However, DMC at low dose significantly increased the cluster of macrophage (Mac-3) cell populations, but at high dose it decreased them. DMC increased macrophage phagocytosis from peripheral blood mononuclear cells at 15 mg/kg treatment and peritoneal cavity at 15, 30 and 60 mg/kg of DMC treatments. DMC did not significantly affect the cytotoxic activity of natural killer (NK) cells. Furthermore, DMC decreased B and T cell proliferation at high doses. CONCLUSION: DMC elevated macrophage phagocytosis in leukemia mice in vivo.

Ugonin J Acts as a SARS-CoV-2 3C-like Protease Inhibitor and Exhibits Anti-inflammatory Properties.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe "flu-like" symptoms that can progress to acute respiratory distress syndrome (ARDS), pneumonia, renal failure, and death. From the therapeutic perspective, 3-chymotrypsin-like protein (3CLpro) is a plausible target for direct-acting antiviral agents because of its indispensable role in viral replication. The flavonoid ugonin J (UJ) has been reported to have antioxidative and anti-inflammatory activities. However, the potential of UJ as an antiviral agent remains unexplored. In this study, we investigated the therapeutic activity of UJ against SARS-CoV-2 infection. Importantly, UJ has a distinct inhibitory activity against SARS-CoV-2 3CLpro, compared to luteolin, kaempferol, and isokaempferide. Specifically, UJ blocks the active site of SARS-CoV-2 3CLpro by forming hydrogen bonding and van der Waals interactions with H163, M165 and E166, G143 and C145, Q189, and P168 in subsites S1, S1', S2, and S4, respectively. In addition, UJ forms strong, stable interactions with core pharmacophore anchors of SARS-CoV-2 3CLpro in a computational model. UJ shows consistent anti-inflammatory activity in inflamed human alveolar basal epithelial A549 cells. Furthermore, UJ has a 50% cytotoxic concentration (CC50) and a 50% effective concentration (EC50) values of about 783 and 2.38 µM, respectively, with a selectivity index (SI) value of 329, in SARS-CoV-2-infected Vero E6 cells. Taken together, UJ is a direct-acting antiviral that obstructs the activity of a fundamental protease of SARS-CoV-2, offering the therapeutic potential for SARS-CoV-2 infection.

Role of AzoR, a LysR-type transcriptional regulator, in SmeVWX pump-mediated antibiotic resistance in Stenotrophomonas maltophilia.

BACKGROUND: The SmeVWX efflux pump of Stenotrophomonas maltophilia contributes to menadione (MD) tolerance and resistance to chloramphenicol, quinolones and tetracycline. The components of the SmeVWX efflux pump are encoded by a five-gene operon, smeU1VWU2X. We have previously demonstrated that the smeU1VWU2X operon is intrinsically unexpressed and inducibly expressed by MD via a SoxR- and SmeRv-involved regulatory circuit in S. maltophilia KJ. We also inferred that there should be other regulator(s) involved in MD-mediated smeU1VWU2X expression in addition to SoxR and SmeRv. OBJECTIVES: To identify novel regulator(s) involved in the regulation of MD-mediated smeU1VWU2X expression and elucidate the regulatory circuit. METHODS: A possible regulator candidate involved in the regulation of MD-mediated smeU1VWU2X expression was identified by a homologue search using the helix-turn-helix domain of SmeRv as a query. Gene expression was assessed using the promoter-xylE transcriptional fusion assay and quantitative RT-PCR. The impact of the regulator on SmeVWX pump-mediated functions was investigated via mutant construction and functional tests (antibiotic susceptibility and MD tolerance). RESULTS: AzoR (Smlt3089), a LysR-type transcriptional regulator, was investigated. In unstressed logarithmically grown cells, AzoR was abundantly expressed and functioned as a repressor, inhibiting the expression of the smeU1VWU2X operon. MD challenge attenuated azoR expression, thus derepressing the expression of the smeU1VWU2X operon in S. maltophilia KJ. AzoR down-regulation-mediated smeU1VWU2X expression was observed in quinolone-resistant and SmeVWX-overexpressing S. maltophilia clinical isolates. CONCLUSIONS: AzoR negatively regulates the expression of the smeU1VWU2X operon and SmeVWX pump-mediated antibiotic resistance in S. maltophilia.

Momordica cochinchinensis Aril Ameliorates Diet-Induced Metabolic Dysfunction and Non-Alcoholic Fatty Liver by Modulating Gut Microbiota.

Obesity and its associated conditions, such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are a particular worldwide health problem at present. Momordica cochinchinensis (MC) is consumed widely in Southeast Asia. However, whether it has functional effects on fat-induced metabolic syndrome remains unclear. This study was conducted to examine the prevention effect of Momordica cochinchinensis aril (MCA) on obesity, non-alcoholic fatty liver and insulin resistance in mice. MCA protected the mice against high-fat diet (HFD)-induced body weight gain, hyperlipidemia and hyperglycemia, compared with mice that were not treated. MCA inhibited the expansion of adipose tissue and adipocyte hypertrophy. In addition, the insulin sensitivity-associated index that evaluates insulin function was also significantly restored. MCA also regulated the secretion of adipokines in HFD-induced obese mice. Moreover, hepatic fat accumulation and liver damage were reduced, which suggested that fatty liver was prevented by MCA. Furthermore, MCA supplementation suppressed hepatic lipid accumulation by activation of the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) signaling pathway in the human fatty liver HuS-E/2 cell model. Our data indicate that MCA altered the microbial contents of the gut and modulated microbial dysbiosis in the host, and consequently is involved in the prevention of HFD-induced adiposity, insulin resistance and non-alcoholic fatty liver disease.

Demethoxycurcumin Suppresses Proliferation, Migration, and Invasion of Human Brain Glioblastoma Multiforme GBM 8401 Cells via PI3K/Akt Pathway.

BACKGROUND/AIM: Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro. MATERIALS AND METHODS: DMC at 1.0-3.0 µM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 µM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells. RESULTS: The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFR(Tyr1068), GRB2, Sos1, p-Raf, MEK, p-ERK1/2, PI3K, p-Akt/PKBα(Thr308), p-PDK1, NF-κB, TIMP-1, MMP-9, MMP-2, GSK3α/ß, ß-catenin, N-cadherin, and vimentin, but it elevated Ras and E-cadherin at 24 h treatment. CONCLUSION: DMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Branched I antigen regulated cell susceptibility against natural killer cytotoxicity through its N-linked glycosylation and overall expression.

Cell surface glycosylation has been known as an important modification process that can be targeted and manipulated by malignant cells to escape from host immunosurveillance. We previously showed that the blood group branched I antigen on the leukemia cell surface can regulate the cell susceptibility against natural killer (NK) cell-mediated cytotoxicity through interfering target-NK interaction. In this work, we first identified N-linkage as the major glycosylation linkage type for branched I glycan formation on leukemia cells, and this linkage was responsible for cell sensitivity against therapeutic NK-92MI targeting. Secondly, by examining different leukemia cell surface death receptors, we showed death receptor Fas had highest expressions in both Raji and TF-1a cells. Mutations on two Fas extracellular N-linkage sites (118 and 136) for glycosylation impaired activation of Fas-mediated apoptosis during NK-92MI cytotoxicity. Last, we found that the surface I antigen expression levels enable leukemia cells to respond differently against NK-92MI targeting. In low I antigen expressing K-562 cell, reduction of I antigen presence greatly reduced leukemia cell susceptibility against NK-92MI targeting. But in other high I antigen expressing leukemia cells, similar reduction in I antigen expression did not affect cell susceptibility.

Mangiferin induces immune responses and evaluates the survival rate in WEHI-3 cell generated mouse leukemia in vivo.

Mangiferin is a naturally occurring polyphenol, widely distributed in Thymeraceae families, and presents pharmacological activity, including anti-cancer activities in many human cancer cell lines. Mangiferin has also been reported to affect immune responses; however, no available information concerning the effects of mangiferin on immune reactions in leukemia mice in vivo. In the present study, we investigated the effects of mangiferin on leukemia WEHI-3 cell generated leukemia BLAB/c mice. Overall, the experiments were divided into two parts, one part was immune responses experiment and the other was the survival rate experiment. The immune responses and survival rate study, 40 mice for each part, were randomly separated into five groups (N = 8): Group I was normal animals and groups II-V WEHI-3 cell generated leukemia mice. Group II mice were fed normal diet as a positive control; group III, IV, and V mice received mangiferin at 40, 80, and 120 mg/kg, respectively, by intraperitoneal injection every 2 days for 20 days. Leukocytes cell population, macrophage phagocytosis, and NK cell activities were analyzed by flow cytometry. Isolated splenocytes stimulated with lipopolysaccharide (LPS) and concanavalin A (Con A) were used to determine the proliferation of B and T cells, respectively, and subsequently were analyzed by flow cytometry. Results indicated that mangiferin significantly increased body weight, decreased the liver and spleen weights of leukemia mice. Mangiferin also increased CD3 T-cell and CD19 B cell population but decreased Mac-3 macrophage and CD11b monocyte. Furthermore, mangiferin decreased phagocytosis of macrophages from PBMC and peritoneal cavity at 40, 80, and 120 mg/kg treatment. However, it also increased NK cell activity at 40 and 120 mg/kg treatment. There were no effects on T and B cell proliferation at three examined doses. In survival rate studies, mangiferin significantly elevated survival rate at 40 and 120 mg/kg treatment of leukemia mice in vivo.

Purple-leaf tea (Camellia sinensis L.) ameliorates high-fat diet induced obesity and metabolic disorder through the modulation of the gut microbiota in mice.

BACKGROUND: Obesity and its associated diseases have become a major world-wide health problem. Purple-leaf Tea (Camellia sinensis L.) (PLT), that is rich of anthocyanins, has been shown to have preventive effects on obesity and metabolic disorders. The intestinal microbiota has been shown to contribute to inflammation, obesity, and several metabolic disorders. However, whether PLT consumption could prevent obesity and diet-induced metabolic diseases by modulating the gut microbiota, is not clearly understood. METHODS: In this study, six-week-old male C57BL/6 J mice were fed a normal diet (ND) or a high fat diet (HFD) without or with PLT for 10 weeks. RESULTS: PLT modulated the gut microbiota in mice and alleviated the symptoms of HFD-induced metabolic disorders, such as insulin resistance, adipocyte hypertrophy, and hepatic steatosis. PLT increased the diversity of the microbiota and the ratio of Firmicutes to Bacteroidetes. f_Barnesiellaceae, g_Barnesiella, f_Ruminococcaceae, and f_Lachnospiraceae were discriminating faecal bacterial communities of the PLT mice that differed from the HFD mice. CONCLUSIONS: These data indicate that PLT altered the microbial contents of the gut and prevented microbial dysbiosis in the host, and consequently is involved in the modulation of susceptibility to insulin resistance, hepatic diseases, and obesity that are linked to an HFD.

PhoPQ two-component regulatory system plays a global regulatory role in antibiotic susceptibility, physiology, stress adaptation, and virulence in Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia, an opportunistic pathogen, is ubiquitously present in various environments, signifying its high capability of environmental adaptation. Two-component regulatory system (TCS) is a powerful implement to help organisms to survive in different environments. In clinic, treatment of S. maltophilia infection is difficult because it is naturally resistant to many antibiotics, highlighting the necessity to develop novel drugs or adjuvants. Given their critical and extensively regulatory role, TCS system has been proposed as a convincing target for novel drugs or adjuvants. PhoPQ TCS, a highly conserved TCS in several pathogens, plays crucial roles in low-magnesium adaption, polymyxin resistance, and virulence. In this study, we aimed to characterize the role of PhoPQ TCS of S. maltophilia in antibiotic susceptibility, physiology, stress adaptation, and virulence. RESULTS: To characterize PhoPQ system, phoP single mutant as well as phoP and phoQ double mutant were constructed. Distinct from most phoPQ systems of other microorganisms, two features were observed during the construction of phoP and phoQ single deletion mutant. Firstly, the phoQ mutant was not successfully obtained. Secondly, the compromised phenotypes of phoP mutant were not reverted by complementing an intact phoP gene, but were partially restored by complementing a phoPQ operon. Thus, wild-type KJ, phoP mutant (KJΔPhoP), phoPQ mutant (KJΔPhoPQ), and complemented strain (KJΔPhoPQ (pPhoPQ)) were used for functional assays, including antibiotic susceptibility, physiology (swimming motility and secreted protease activity), stress adaptation (oxidative, envelope, and iron-depletion stresses), and virulence to Caenorhabditis elegans. KJΔPhoPQ totally lost swimming motility, had enhanced secreted protease activity, increased susceptibility to antibiotics (ß-lactam, quinolone, aminoglycoside, macrolide, chloramphenicol, and sulfamethoxazole/ trimethoprim), menadione, H2O2, SDS, and 2,2'-dipyridyl, as well as attenuated virulence to C. elegans. Trans-complementation of KJΔPhoPQ with phoPQ reverted these altered phenotypes to the wild-type levels. CONCLUSIONS: Given the critical and global roles of PhoPQ TCS in antibiotic susceptibility, physiology, stress adaptation, and virulence, PhoPQ is a potential target for the design of drugs or adjuvants.

Casticin Inhibits In Vivo Growth of Xenograft Tumors of Human Oral Cancer SCC-4 Cells.

BACKGROUND/AIM: Casticin, one of the active components of Vitex rotundifolia L., presents biological and pharmacological activities including inhibition of migration, invasion and induction of apoptosis in numerous human cancer cells in vitro. This study aimed to assess the effects of casticin on tumor growth in a human oral cancer SCC-4 cell xenograft mouse model in vivo. MATERIALS AND METHODS: Twenty-four nude mice were injected subcutaneously with SCC-4 cells and when palpable tumors reached a volume of 100-120 mm3 the mice were randomly divided into three groups. The control (0.1% dimethyl sulfoxide), casticin (0.2 mg/kg), and casticin (0.4 mg/kg) groups were intraperitoneally injected every two days for 18 days. Tumor volume and body weights were measured every two days. RESULTS: Casticin significantly decreased tumor volume and weight in SCC-4 cell xenograft mice but there was no statistically significant difference between the body weights of control mice and mice treated with 0.2 mg/kg or 0.4 mg/kg casticin. Therefore, the growth of SCC-4 cells in athymic nude mice can be inhibited by casticin in vivo. CONCLUSION: These findings support further investigations in the potential use of casticin as an oral anti-cancer drug in the future.