Project description:The study aimed to investigate the effect of pH on Bacillus subtilis secretome into cell-free supernatant which is used as plant growth biostimulant
Project description:The plant pathogen Pseudomonas syringae pv.tomato (DC3000) causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS). In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp). Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant's capacity to recognize both pathogen and microbial determinants (PAMP/MAMP-triggered immunity). We have developed and employed MS-based shotgun and targeted proteomics to (i) elucidate the extracellular and secretome composition of DC3000 and (ii) evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopP1, HrpK1, HrpA1 and AvrPto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS) to quantify HrpA1 and AvrPto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid AvrPto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.
Project description:A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.
Project description:Bacterial vaginosis (BV) is the most common vaginal infection in reproductive women, which is characterized by depleted level of lactic acid bacteria and overgrowth of anaerobes such as Gardnerella vaginalis spp. Lactic acid bacteria have been known to be beneficial for amelioration of BV, since they produce antimicrobial substances against G. vaginalis spp. The objectives of this study were to characterize different fractions of cell-free supernatant of Lactobacillus paracasei CH88 (LCFS) and investigate antibacterial activity of the LCFS fractions against G. vaginalis in-vitro and in-vivo. Antibacterial activity of the LCFS was stable during thermal treatment up to 120 °C for 30 min and maintained at pH ranging from 3.0 to 13.0 except pH 5.0. Fraction below 3 kDa of the LCFS partially lost its antibacterial activity after treatment with proteolytic enzymes. Precipitated protein fraction below 3 kDa of the LCFS (< 3 kDa LCFSP) inhibited the growth and biofilm formation of G. vaginalis. Treatment of L. paracasei CH88 or the < 3 kDa LCFSP attenuated G. vaginalis-induced BV in mice by inhibiting the growth of G. vaginalis, reducing exfoliation of vaginal epithelial cells, and regulating immune response. These results suggest that L. paracasei CH88 may have potential in ameliorating G. vaginalis-induced BV.
Project description:Chemerin (CHEM) is a hormone mainly expressed in adipocytes involved in the regulation of energy homeostasis and inflammatory response. CHEM expression has been demonstrated in the structures of the porcine hypothalamic-pituitary-gonadal axis, as well as in the uterus, trophoblasts and conceptuses of pigs. In this study, we performed high-throughput proteomic analyses (liquid chromatography with tandem mass spectrometry, LC-MS/MS) to examine the influence of CHEM (400 ng/mL) on differentially regulated proteins (DRPs) in the porcine endometrial tissue explants during implantation (15 to 16 days of gestation). Among all 352 DRPs, 164 were up-regulated and 188 were down-regulated in CHEM-treated group. DRPs were assigned to 47 gene ontology (GO) terms (p-adjusted < 0.05). Validation of four DRPs (IFIT5, TGFβ1, ACO1 and PGRMC1) by Western blot analysis confirmed the veracity and accuracy of the LC-MS/MS method used in the present study. We suggest that CHEM, by modulating various protein expressions, takes part in the endometrial cell proliferation, migration and invasion at the time of implantation. It also regulates the endometrial immune response, sensitivity to P4 and the formation of new blood vessels. Additionally, CHEM appears to be an important factor involved in endothelial cell dysfunction during the pathogenesis of preeclampsia. The identification of a large number of DRPs under the influence of CHEM provides a valuable resource for understanding the molecular mechanisms of this hormone action during implantation, which is a prerequisite for better control of pig reproduction.
Project description:Background:Paenibacillus larvae is the etiological agent of American foulbrood (AFB) disease, the most lethal disease in honeybee (Apis mellifera) larvae. Aims:The aim of the present work was to study the antimicrobial effect of cell free supernatant (CFS) of probiotics on an Iranian isolate's biofilm formation. Methods:A local strain was identified by 16S rRNA sequencing. The antibacterial effect of some probiotics was evaluated through drop plate method, minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and time-kill assay. The biofilm formation ability of P. larvae and the inhibition of biofilm formation by CFS were studied by microplate and scanning electron microscopy (SEM). The nature of the secondary metabolites in CFS was examined by microscale optical density assay (MODA). Results:Alignment of the results of P. larvae KB10 (GenBank accession number MH000685.1) 16S rRNA with the database revealed more than 97% identity with P. larvae . The most antibacterial effect was observed in the CFS of Lactobacillus reuteri ATCC23272 with 12.75 ± 3.2 mm for zone of inhibition (ZOI) at 1000 µL/ml for MIC and MBC. Time-kill assay revealed that CFS eliminated 1.5 × 108 CFU/ml P. larvae KB10 at 2 h of exposure. Microtitre plate and SEM results revealed that CFS (at sub-MIC concentration) was able to inhibit biofilm formation by P. larvae. The results of MODA assay showed that antimicrobial activity were related to the production of organic acids. Conclusion:Cell free supernatant from L. reuteri ATCC 23272 had inhibitory effects on P. larvae KB10 growth and biofilm production due to its acidic nature. The obtained results can be used for antibiotic substitution in AFB control and treatment.
Project description:Frequently observed in tropical and sub-tropical regions, crops contamination by aflatoxin B1 (AFB1) produced by Aspergillus flavus, is emerging in Europe, due to climate change. Many alternative methods are currently developed to reduce the use of chemical inputs to prevent mycotoxin contamination, such as biocontrol agents (BCAs). Actinobacteria are known to produce many bioactive compounds and some of them are able to reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell free supernatant (CFS) from Streptomyces roseolus liquid culture on A. flavus development, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. To study the impact of Streptomyces roseolus cell free supernatant on global transcriptome of Aspergillus flavus we have employed whole genome microarray expression profiling.
Project description:Analysis of gene expression in RAW264.7 cells stimulated for osteoclastogenesis and then treated with cell culture supernatant from Lactobacillus reuteri. Results will offer insight into targeted mechanisms suppressing osteoclastogenesis
Project description:The analysis of mesenchymal stromal cells secretome is fundamental to identify key players of processes involving these cells. Truly secreted proteins may be difficult to detect in MS based analysis of conditioned media (CM) due to proteins supplemented with fetal bovine serum (FBS). We compared different growth conditions to determine the effect of varying FBS concentration on the number and quantity of truly secreted human proteins vs contaminating bovine proteins. The results suggest that to minimize interference cells should be grown in presence of FBS until confluence and transferred into a serum-free medium prior to secretome collection.