Analysis of Polycaprolactone Microfibers as Biofilm Carriers for Biotechnologically Relevant Bacteria.

Polymeric electrospun fibers are becoming popular in microbial biotechnology because of their exceptional physicochemical characteristics, biodegradability, surface-to-volume ratio, and compatibility with biological systems, which give them a great potential as microbial supports to be used in production processes or environmental applications. In this work, we analyzed and compared the ability of Escherichia coli, Pseudomonas putida, Brevundimonas diminuta, and Sphingobium fuliginis to develop biofilms on different types of polycaprolactone (PCL) microfibers. These bacterial species are relevant in the production of biobased chemicals, enzymes, and proteins for therapeutic use and bioremediation. The obtained results demonstrated that all selected species were able to attach efficiently to the PCL microfibers. Also, the ability of pure cultures of S. fuliginis (former Flavobacterium sp. ATCC 27551, a very relevant strain in the bioremediation of organophosphorus compounds) to form dense biofilms was observed for the first time, opening the possibility of new applications for this microorganism. This material showed to have a high microbial loading capacity, regardless of the mesh density and fiber diameter. A comparative analysis between PCL and polylactic acid (PLA) electrospun microfibers indicated that both surfaces have a similar bacterial loading capacity, but the former material showed higher resistance to microbial degradation than PLA.

Colonization of Electrospun Polycaprolactone Fibers by Relevant Pathogenic Bacterial Strains.

Electrospun biodegradable polymers have emerged as promising materials for their applications in several fields, including biomedicine and food industry. For this reason, the susceptibility of these materials to be colonized by different pathogens is a critical issue for public health, and their study can provide future knowledge to develop new strategies against bacterial infections. In this work, the ability of three pathogenic bacterial species ( Pseudomonas aeruginosa, Acinetobacter baumannii, and Listeria monocytogenes) to adhere and form biofilm in electrospun polycaprolactone (PCL) microfibrous meshes was investigated. Bacterial attachment was analyzed in meshes with different microstructure, and comparisons with other materials (borosilicate glass and electrospun polylactic acid (PLA)) fibers were assessed. Analysis included colony forming unit (CFU) counts, scanning electron microscopy (SEM), and crystal violet (CV) staining. All the obtained data suggest that PCL meshes, regardless of their microstructure, are highly susceptible to be colonized by the pathogenic relevant bacteria used in this study, so a pretreatment or a functionalization with compounds that present some antimicrobial activity or antibiofilm properties is highly recommended before their application. Moreover, an experiment designed to simulate a chronic wound environment was used to demonstrate the ability of these meshes to detach biofilms from the substratum where they have developed, thus making them promising candidates to be used in wound cleaning and disinfection.

Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Nonsense-mediated mRNA decay controls the changes in yeast ribosomal protein pre-mRNAs levels upon osmotic stress.

The expression of ribosomal protein (RP) genes requires a substantial part of cellular transcription, processing and translation resources. Thus, the RP expression must be tightly regulated in response to conditions that compromise cell survival. In Saccharomyces cerevisiae cells, regulation of the RP gene expression at the transcriptional, mature mRNA stability and translational levels during the response to osmotic stress has been reported. Reprogramming global protein synthesis upon osmotic shock includes the movement of ribosomes from RP transcripts to stress-induced mRNAs. Using tiling arrays, we show that osmotic stress yields a drop in the levels of RP pre-mRNAs in S. cerevisiae cells. An analysis of the tiling array data, together with transcription rates data, shows a poor correlation, indicating that the drop in the RP pre-mRNA levels is not merely a result of the lowered RP transcription rates. A kinetic study using quantitative RT-PCR confirmed the decrease in the levels of several RP-unspliced transcripts during the first 15 minutes of osmotic stress, which seems independent of MAP kinase Hog1. Moreover, we found that the mutations in the components of the nonsense-mediated mRNA decay (NMD), Upf1, Upf2, Upf3 or in exonuclease Xrn1, eliminate the osmotic stress-induced drop in RP pre-mRNAs. Altogether, our results indicate that the degradation of yeast RP unspliced transcripts by NMD increases during osmotic stress, and suggest that this might be another mechanism to control RP synthesis during the stress response.

Yeast mRNA cap-binding protein Cbc1/Sto1 is necessary for the rapid reprogramming of translation after hyperosmotic shock.

In response to osmotic stress, global translation is inhibited, but the mRNAs encoding stress-protective proteins are selectively translated to allow cell survival. To date, the mechanisms and factors involved in the specific translation of osmostress-responsive genes in Saccharomyces cerevisiae are unknown. We find that the mRNA cap-binding protein Cbc1 is important for yeast survival under osmotic stress. Our results provide new evidence supporting a role of Cbc1 in translation initiation. Cbc1 associates with polysomes, while the deletion of the CBC1 gene causes hypersensitivity to the translation inhibitor cycloheximide and yields synthetic "sickness" in cells with limiting amounts of translation initiator factor eIF4E. In cbc1Δ mutants, translation drops sharply under osmotic stress, the subsequent reinitiation of translation is retarded, and "processing bodies" containing untranslating mRNAs remain for long periods. Furthermore, osmostress-responsive mRNAs are transcriptionally induced after osmotic stress in cbc1Δ cells, but their rapid association with polysomes is delayed. However, in cells containing a thermosensitive eIF4E allele, their inability to grow at 37ºC is suppressed by hyperosmosis, and Cbc1 relocalizes from nucleus to cytoplasm. These data support a model in which eIF4E-translation could be stress-sensitive, while Cbc1-mediated translation is necessary for the rapid translation of osmostress-protective proteins under osmotic stress.

The bidirectional cytomegalovirus immediate/early promoter is regulated by Hog1 and the stress transcription factors Sko1 and Hot1 in yeast.

The work presented here intends to address the question of whether the immediate/early promoter of cytomegalovirus (CMV), which is widely used for expressing transgenes in eukaryotic cells, yields a constitutive expression of the transgenes under stress conditions in Saccharomyces cerevisiae cells. This information would also be relevant because in the tetracycline-regulated expression (tetO) system, which is one of the first choices for studying gene function from yeast to human cells, the CMV promoter controls the expression of the tetO transactivator. We found that the CMV promoter in yeast cells is bidirectionally induced by osmotic stress and in glycerol media. The mitogen-activated protein (MAP) kinase Hog1 controls CMV activation by osmotic stress through the ATF/CRE-related transcription factor Sko1 and the yeast osmostress factor Hot1. Our results indicate that the CMV and tetO expression systems respond to external signals and this should be considered before using these systems in yeast. Moreover, our results also suggest that CMV could be regulated by the intracellular glucose concentration in human cells.

Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae.

Hyperosmotic stress yields reprogramming of gene expression in Saccharomyces cerevisiae cells. Most of this response is orchestrated by Hog1, a stress-activated, mitogen-activated protein kinase (MAPK) homologous to human p38. We investigated, on a genomic scale, the contribution of changes in transcription rates and mRNA stabilities to the modulation of mRNA amounts during the response to osmotic stress in wild-type and hog1 mutant cells. Mild osmotic shock induces a broad mRNA destabilization; however, osmo-mRNAs are up-regulated by increasing both transcription rates and mRNA half-lives. In contrast, mild or severe osmotic stress in hog1 mutants, or severe osmotic stress in wild-type cells, yields global mRNA stabilization and sequestration of mRNAs into P-bodies. After adaptation, the absence of Hog1 affects the kinetics of P-bodies disassembly and the return of mRNAs to translation. Our results indicate that regulation of mRNA turnover contributes to coordinate gene expression upon osmotic stress, and that there are both specific and global controls of mRNA stability depending on the strength of the osmotic stress.