Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

UNLABELLED: Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. IMPORTANCE: Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage.

Engineering Saccharomyces pastorianus for the co-utilisation of xylose and cellulose from biomass.

BACKGROUND: Lignocellulosic biomass is a viable source of renewable energy for bioethanol production. For the efficient conversion of biomass into bioethanol, it is essential that sugars from both the cellulose and hemicellulose fractions of lignocellulose be utilised. RESULTS: We describe the development of a recombinant yeast system for the fermentation of cellulose and xylose, the most abundant pentose sugar in the hemicellulose fraction of biomass. The brewer's yeast Saccharomyces pastorianus was chosen as a host as significantly higher recombinant enzyme activities are achieved, when compared to the more commonly used S. cerevisiae. When expressed in S. pastorianus, the Trichoderma reesei xylose oxidoreductase pathway was more efficient at alcohol production from xylose than the xylose isomerase pathway. The alcohol yield was influenced by the concentration of xylose in the medium and was significantly improved by the additional expression of a gene encoding for xylulose kinase. The xylose reductase, xylitol dehydrogenase and xylulose kinase genes were co-expressed with genes encoding for the three classes of T. reesei cellulases, namely endoglucanase (EG2), cellobiohydrolysase (CBH2) and ß-glucosidase (BGL1). The initial metabolism of xylose by the engineered strains facilitated production of cellulases at fermentation temperatures. The sequential metabolism of xylose and cellulose generated an alcohol yield of 82% from the available sugars. Several different types of biomass, such as the energy crop Miscanthus sinensis and the industrial waste, brewer's spent grains, were examined as biomass sources for fermentation using the developed yeast strains. Xylose metabolism and cell growth were inhibited in fermentations carried out with acid-treated spent grain liquor, resulting in a 30% reduction in alcohol yield compared to fermentations carried out with mixed sugar substrates. CONCLUSIONS: Reconstitution of complete enzymatic pathways for cellulose hydrolysis and xylose utilisation in S. pastorianus facilitates the co-fermentation of cellulose and xylose without the need for added exogenous cellulases and provides a basis for the development of a consolidated process for co-utilisation of hemicellulose and cellulose sugars.

Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts.

Molecular mimics of the tumour antigen MUC1.

A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1) from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

Phage display biopanning identifies the translation initiation and elongation factors (IF1α-3 and eIF-3) as components of Hsp70-peptide complexes in breast tumour cells.

The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70-peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a 'pull-down' assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.

The PolyA tail length of yeast histone mRNAs varies during the cell cycle and is influenced by Sen1p and Rrp6p.

Yeast histone mRNAs are polyadenylated, yet factors such as Rrp6p and Trf4p, required for the 3'-end processing of non-polyadenylated RNAs, contribute to the cell cycle regulation of these transcripts. Here, we investigated the role of other known 3'-end processing/transcription termination factors of non-polyadenylated RNA in the biogenesis of histone mRNAs, specifically the Nab3p/Nrd1p/Sen1p complex. We also re-evaluated the polyadenylation status of these mRNAs during the cell cycle. Our analysis reveals that yeast histone mRNAs have shorter than average PolyA tails and the length of the PolyA tail varies during the cell cycle; S-phase histone mRNAs possess very short PolyA tails while in G1, the tail length is relatively longer. Inactivation of either Sen1p or Rrp6p leads to a decrease in the PolyA tail length of histone mRNAs. Our data also show that Sen1p contributes to 3'-end processing of histone primary transcripts. Thus, histone mRNAs are distinct from the general pool of yeast mRNAs and 3'-end processing and polyadenylation contribute to the cell cycle regulation of these transcripts.

Lager yeasts possess dynamic genomes that undergo rearrangements and gene amplification in response to stress.

A long-term goal of the brewing industry is to identify yeast strains with increased tolerance to the stresses experienced during the brewing process. We have characterised the genomes of a number of stress-tolerant mutants, derived from the lager yeast strain CMBS-33, that were selected for tolerance to high temperatures and to growth in high specific gravity wort. Our results indicate that the heat-tolerant strains have undergone a number of gross chromosomal rearrangements when compared to the parental strain. To determine if such rearrangements can spontaneously arise in response to exposure to stress conditions experienced during the brewing process, we examined the chromosome integrity of both the stress-tolerant strains and their parent during a single round of fermentation under a variety of environmental stresses. Our results show that the lager yeast genome shows tremendous plasticity during fermentation, especially when fermentations are carried out in high specific gravity wort and at higher than normal temperatures. Many localised regions of gene amplification were observed especially at the telomeres and at the rRNA gene locus on chromosome XII, and general chromosomal instability was evident. However, gross chromosomal rearrangements were not detected, indicating that continued selection in the stress conditions are required to obtain clonal isolates with stable rearrangements. Taken together, the data suggest that lager yeasts display a high degree of genomic plasticity and undergo genomic changes in response to environmental stress.

A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-gamma (IFN-gamma) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.

Aneuploidy and copy number breakpoints in the genome of lager yeasts mapped by microarray hybridisation.

Competitive comparative genome hybridisation (CCGH) to Saccharomyces cerevisiae DNA microarrays and quantitative real-time polymerase chain reaction (qRT-PCR) assays are used to examine the copy number of S. cerevisiae-like genes, at single gene resolution, of two bottom-fermenting lager yeast strains, CMBS-33 and 6701. Using the S. cerevisiae gene order for each chromosome, we observe that the copy number for contiguous groups of S. cerevisiae-like genes is similar in both strains. However, discrete changes in copy number occur at distinct loci, indicating the aneuploid nature of the lager yeast genomes. The majority of loci where copy number changes occur are conserved in both strains. We also identify a large segment of S. cerevisiae DNA on chromosome XVI that fails to hybridise to genomic DNA from both lager strains, suggesting that this region may have diverged significantly or is absent in the lager yeast strains. Furthermore, very low levels of mRNA transcripts are detected from this region of the genome. Interestingly, the increased gene copy number observed elsewhere (e.g. chromosome III) does not correlate specifically with increased gene expression under fermentation conditions, suggesting that dosage compensation may play a role in controlling gene expression in these strains.