Search

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses.

Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

We designed an efficient transformation system for Candida guilliermondii based on a ura5 ATCC 6260 derived recipient strain and a URA5 recyclable selection marker. This "URA5 blaster" disruption system represents a powerful tool to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3'UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast "TRP blaster" disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created "TRP5 blaster" disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.

Candida guilliermondii is an emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains a promising biotechnological model for the industrial production of value-added metabolites. In the present study, we developed a recipient strain as well as a set of plasmids for construction of fluorescent protein (FP) fusions in this species. We demonstrated that C. guilliermondii phosphoglycerate kinase transcription-regulating sequences allow a constitutive expression of codon-optimized green, cyan, yellow and mCherry FP genes in C. guilliermondii cells and the fluorescence signal could be directly observed at the colony and blastospore level by epifluorescence microcopy. To illustrate differential targeting of the FPs into specified cellular compartments, we studied and validated the expected subcellular localization of various C. guilliermondii predicted proteins fused to FPs. Furthermore, co-expression experiments of various couples of FP-tagged C. guilliermondii predicted proteins in the same cell showed that the fluorescence of each FP could be detected independently, providing firm evidences that YFP/CFP and GFP/mCherry pairs can be used for dual labeling in C. guilliermondii cells. This technical advance will facilitate future studies of protein co-expression and co-localization in C. guilliermondii and will give precious help for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering.

International audience; Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

Adult bone marrow (BM)-derived stem cells, including hematopoietic stem cells (HSCs) and MSCs, represent an important source of cells for the repair of a number of damaged tissues. In contrast to HSCs, the soluble factors able to induce MSC migration have not been extensively studied. In the present work, we compared the in vitro migration capacity of human BM-derived MSCs, preincubated or not with the inflammatory cytokines interleukin 1beta (IL1beta) and tumor necrosis factor alpha (TNFalpha), in response to 16 growth factors (GFs) and chemokines. We show that BM MSCs migrate in response to many chemotactic factors. The GFs platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1) are the most potent, whereas the chemokines RANTES, macrophage-derived chemokine (MDC), and stromal-derived factor-1 (SDF-1) have limited effect. Remarkably, preincubation with TNFalpha leads to increased MSC migration toward chemokines, whereas migration toward most GFs is unchanged. Consistent with these results, BM MSCs express the tyrosine kinase receptors PDGF-receptor (R) alpha, PDGF-Rbeta, and IGF-R, as well as the RANTES and MDC receptors CCR2, CCR3, and CCR4 and the SDF-1 receptor CXCR4. TNFalpha increases CCR2, CCR3, and CCR4 expression (as opposed to that of CXCR4), together with RANTES membrane binding. These data indicate that the migration capacity of BM MSCs is under the control of a large range of receptor tyrosine kinase GFs and CC and CXC chemokines. Most chemokines are more effective on TNFalpha-primed cells. Our results suggest that the mobilization of MSCs and their subsequent homing to injured tissues may depend on the systemic and local inflammatory state. Disclosure of potential conflicts of interest is found at the end of this article.

Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.