M. Marta Guarna

An anti-infective peptide that selectively modulates the innate immune response

We show that an innate defense–regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris.

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera)

BackgroundDisease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies.ResultsWe sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae.ConclusionsOur results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

Alternative reading frame selection mediated by a tRNA-like domain of an internal ribosome entry site

The dicistrovirus intergenic region internal ribosome entry site (IRES) utilizes a unique mechanism, involving P-site tRNA mimicry, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG codon in the ribosomal A site. A subgroup of dicistrovirus genomes contains an additional stem-loop 5′-adjacent to the IRES and a short open reading frame (ORFx) that overlaps the viral structural polyprotein ORF (ORF2) in the +1 reading frame. Using mass spectrometry and extensive mutagenesis, we show that, besides directing ORF2 translation, the Israeli acute paralysis dicistrovirus IRES also directs ORFx translation. The latter is mediated by a U∶G base pair adjacent to the P-site tRNA-mimicking domain. An ORFx peptide was detected in virus-infected honey bees by multiple reaction monitoring mass spectrometry. Finally, the 5′ stem-loop increases IRES activity and may couple translation of the two major ORFs of the virus. This study reveals a novel viral strategy in which a tRNA-like IRES directs precise, initiator Met-tRNA-independent translation of two overlapping ORFs.

Ecological Adaptation of Diverse Honey Bee (Apis mellifera) Populations

Background Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology. Proteomics provides tools unique among the major ‘omics disciplines for identifying the mechanisms employed by an organism in adapting to environmental challenges. Results Through proteome profiling of adult honey bee midgut from geographically dispersed, domesticated populations combined with multiple parallel statistical treatments, the data presented here suggest some of the major cellular processes involved in adapting to different climates. These findings provide insight into the molecular underpinnings that may confer an advantage to honey bee populations. Significantly, the major energy-producing pathways of the mitochondria, the organelle most closely involved in heat production, were consistently higher in bees that had adapted to colder climates. In opposition, up-regulation of protein metabolism capacity, from biosynthesis to degradation, had been selected for in bees from warmer climates. Conclusions Overall, our results present a proteomic interpretation of expression polymorphisms between honey bee ecotypes and provide insight into molecular aspects of local adaptation or selection with consequences for honey bee management and breeding. The implications of our findings extend beyond apiculture as they underscore the need to consider the interdependence of animal populations and their agro-ecological context.

Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells.

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype–based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

A Bio-Economic Case Study of Canadian Honey Bee (Hymenoptera: Apidae) Colonies: Marker-Assisted Selection (MAS) in Queen Breeding Affects Beekeeper Profits

Abstract Over the past decade in North America and Europe, winter losses of honey bee (Hymenoptera: Apidae) colonies have increased dramatically. Scientific consensus attributes these losses to multifactorial causes including altered parasite and pathogen profiles, lack of proper nutrition due to agricultural monocultures, exposure to pesticides, management, and weather. One method to reduce colony loss and increase productivity is through selective breeding of queens to produce disease-, pathogen-, and mite-resistant stock. Historically, the only method for identifying desirable traits in honey bees to improve breeding was through observation of bee behavior. A team of Canadian scientists have recently identified markers in bee antennae that correspond to behavioral traits in bees and can be tested for in a laboratory. These scientists have demonstrated that this marker-assisted selection (MAS) can be used to produce hygienic, pathogen-resistant honey bee colonies. Based on this research, we present a beekeeping case study where a beekeeper’s profit function is used to evaluate the economic impact of adopting colonies selected for hygienic behavior using MAS into an apiary. Our results show a net profit gain from an MAS colony of between 2% and 5% when Varroa mites are effectively treated. In the case of ineffective treatment, MAS generates a net profit benefit of between 9% and 96% depending on the Varroa load. When a Varroa mite population has developed some treatment resistance, we show that MAS colonies generate a net profit gain of between 8% and 112% depending on the Varroa load and degree of treatment resistance.

Expression biomarkers used for the selective breeding of complex polygenic traits

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and quantitative trait loci (QTLs), is also now applied broadly in livestock breeding programs; however, selection on protein or mRNA expression markers have not been proved useful yet. Here we demonstrate the utility of protein markers to select for disease-resistant behaviour in the European honey bees (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis and trans effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting stock performed as well or better than bees selected using phenotype–based assessment of this trait, when challenged with disease. This is the first demonstration of the efficacy of protein markers for selective breeding in any agricultural species, plant or animal. Significance statement The honey bee has been in the news a lot recently, largely because of world-wide die-offs due to the parasitic Varroa mite, which is becoming resistant to the chemical controls the bee industry uses. In this study, we show that robust expression biomarkers of a disease-resistance trait can be used, in an out-bred population, to select for that trait. After three generations of selection, the resulting stock performed as well or better than bees selected using the phenotypic best method for assessing this trait when challenged with disease. This is the first demonstration of an expression marker for selective breeding in any agricultural species, plant or animal. This also represents a completely novel way to select for highly polygenic traits.