Antimicrobial activity of endophytic bacterial populations isolated from medical plants of Iran

Endophytic actinobacteria colonize inside the plant tissues without causing damages to the host plant. Since these microorganisms colonize in the different parts of plants and can stop plant disease, they have been applied as biological agents for controlling human diseases. The aim of this study was molecular identification and measuring the antimicrobial activity of endophytic Actinomycetes isolated from medicinal plants of Iran. Materials and Methods: The total of 23 medicinal plant samples were collected, sterilized, and crushed. Small pieces of the crushed samples were then cultured directly on three selective media. Grown colonies were identified by 16S rRNA gene sequencing method. Each isolate was cultured in TSB medium and then antimicrobial compound was extracted using ethyl acetate and tested against the target bacteria. Results: Sixteen out of 23 bacterial isolates (69%) exhibited antimicrobial activity against the selected pathogenic bacteria, such as Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Citrobacter freundii, Proteus mi-rabilis, Shigella flexneri and Escherichia coli. Conclusion: Our Study showed a high phylogenetic diversity and the potent antibiotic activity of endophytic bacteria in medicinal plants of Iran. © 2017, Tehran University of Medical Science. All rights reserved

Autophagy induction regulates influenza virus replication in a time-dependent manner

Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research.Methodology. In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26 %. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50 % infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. Conclusion. This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production

Mutagenesis and functional studies of the HIV-1 vpr gene and Vpr protein obtained from South African virus strains

Thesis (PhD)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations.AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies

Defining the Interactions and Role of DCAF1/VPRBP in the DDB1-Cullin4A E3 Ubiquitin Ligase Complex Engaged by HIV-1 Vpr to Induce a G₂ Cell Cycle Arrest

<div><p>HIV viral protein R (Vpr) induces a cell cycle arrest at the G₂/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G₂ arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G₂ arrest.</p></div

Effect of mutations in the N-terminal F/YxxF/Y motifs of DCAF1 on Vpr and DDB1 binding.

<p><b>A.</b> HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD F1060A/Y1063A (lanes 5 and 6), Myc-DCAF1 WD F1077A/F1080A (lanes 7 and 8) or with Myc-DCAF1 WD Y1120A/F1123A (lanes 9 and 10)-encoding plasmids in the presence of empty vector (lanes 1, 3, 5, 7 and 9) or HA-Vpr-expressing plasmid (lanes 2, 4, 6, 8 and 10). Immunoprecipitations and Western Blot detection were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089195#pone-0089195-g002" target="_blank">figure 2B</a>. * denotes the light chain of the IgG used for immunoprecipitation. <b>B.</b> Quantitation of the DDB1 and HA-Vpr binding. Quantitation was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089195#pone-0089195-g002" target="_blank">figure 2C</a>.</p

The FDKF motif at position 1255-1258 is not required for efficient recruitment of Vpr.

<p><b>A.</b> HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD F1255A/F1258A at two different concentrations (lanes 5 to 8), or with Myc-DCAF1 WD F1077A/F1080A (lanes 9 and 10)-encoding plasmid in the presence of empty vector (lanes 1, 3, 5, 7 and 8) or HA-Vpr-expressing plasmids (lanes 2, 4, 6, 8, and 10). Immunoprecipitations and Western Blot detection were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089195#pone-0089195-g002" target="_blank">figure 2B</a>. * denotes the light chain of the IgG used for immunoprecipitation. # represents non-specific immunoprecipitated proteins. <b>B</b>. Quantitation of the DDB1 and HA-Vpr binding. Quantitation was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089195#pone-0089195-g002" target="_blank">figure 2C</a>.</p

Delineation of the minimal domain of DCAF1 that interacts with HIV-1 Vpr and DDB1.

<p><b>A.</b> Schematic representation of Myc-DCAF1 WT (1-1507), Myc-DCAF1 WD (1041-1393) and Myc-DCAF1 1377 (1041-1377). The different domains of DCAF1 with their amino-acid positions are highlighted. Additionally, the region targeted by the full length DCAF1-specific bp3 siRNA is highlighted in red (see below). <b>B-C</b>. HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 (1-1507) (lanes 3 and 4), Myc-DCAF1 WD (1041-1393) (lanes 5 and 6) or with Myc-DCAF1 1377 (1041-1377) (lanes 7 and 8) -encoding plasmids in the presence of empty vector (lanes 1, 3, 5 and 7) or HA-tagged Vpr-expressing plasmid (lanes 2, 4, 6, and 8). Total amounts of DNA were adjusted with empty vector so that similar quantities of plasmids were transfected in each sample. <b>B.</b> Immunoprecipitations using anti-Myc antibody were performed on cell extracts using protein-A sepharose beads. The levels of HA-Vpr, endogenous DDB1, Myc-DCAF1 proteins and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. <b>C.</b> Quantitation of DDB1 binding efficiency. Band signals corresponding to DDB1 in immunocomplexes were scanned by laser densitometry. The ratio of DDB1 signal over that of precipitated Myc-DCAF1 1507 or Myc-DCAF1WD was calculated and expressed as the percentage of that obtained in the absence of Vpr, which was assigned a value of 100%. Error bars indicate the standard error of the mean (SEM) from the quantitative analysis of three independent experiments. Statistical analysis was performed as described in the Experimental Procedures (p<0.05; ns; non significant). <b>D</b>. Immunoprecipitation using anti-HA antibody was performed on cell extracts using anti-HA antibody-coupled agarose beads. The levels of HA-Vpr, endogenous DDB1, endogenous DCAF1, Myc-DCAF1 proteins and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. The data shown here are representative of results obtained in three independent experiments. * denotes the light chain of the IgG used for immunoprecipitation. # represents non-specific immunoprecipitated proteins. <b>E.</b> Structural and molecular features of the DCAF1 WD minimal domain. Consensus secondary structure prediction of DCAF1 WD 1041-1393 was generated using the PSI-PRED server and structural data obtained from the 3D modelization. Orange lines highlight the predicted β-sheet structures while the green line and the green amino-acid residues highlight α-helices and the putative H-box motif (see below), respectively. The F/YxxF/Y repeats are highlighted in purple whereas the WDxR motifs are highlighted in red.</p