Subclinical peripheral inflammation has systemic effects impacting central nervous system proteome in budgerigars.

Regulation of neuroimmune interactions varies across avian species. Little is presently known about the interplay between periphery and central nervous system (CNS) in parrots, birds sensitive to neuroinflammation. Here we investigated the systemic and CNS responses to dextran sulphate sodium (DSS)- and lipopolysaccharide (LPS)-induced subclinical acute peripheral inflammation in budgerigar (Melopsittacus undulatus). Three experimental treatment groups differing in DSS and LPS stimulation were compared to controls. Individuals treated with DSS showed significant histological intestinal damage. Through quantitative proteomics we described changes in plasma (PL) and cerebrospinal fluid (CSF) composition. In total, we identified 180 proteins in PL and 978 proteins in CSF, with moderate co-structure between the proteomes. Between treatments we detected differences in immune, coagulation and metabolic pathways. Proteomic variation was associated with the levels of pro-inflammatory cytokine mRNA expression in intestine and brain. Our findings shed light on systemic impacts of peripheral low-grade inflammation in birds.

Varroa destructor parasitism and Deformed wing virus infection in honey bees are linked to peroxisome-induced pathways.

The ectoparasitic mite Varroa destructor transmits and triggers viral infections that have deleterious effects on honey bee colonies worldwide. We performed a manipulative experiment in which worker bees collected at emergence were exposed to Varroa for 72 h, and their proteomes were compared with those of untreated control bees. Label-free quantitative proteomics identified 77 differentially expressed A. mellifera proteins (DEPs). In addition, viral proteins were identified by orthogonal analysis, and most importantly, Deformed wing virus (DWV) was found at high levels/intensity in Varroa-exposed bees. Pathway enrichment analysis suggested that the main pathways affected included peroxisomal metabolism, cyto-/exoskeleton reorganization, and cuticular proteins. Detailed examination of individual DEPs revealed that additional changes in DEPs were associated with peroxisomal function. In addition, the proteome data support the importance of TGF-ß signaling in Varroa-DWV interaction and the involvement of the mTORC1 and Hippo pathways. These results suggest that the effect of DWV on bees associated with Varroa feeding results in aberrant autophagy. In particular, autophagy is selectively modulated by peroxisomes, to which the observed proteome changes strongly corresponded. This study complements previous research with different study designs and suggests the importance of the peroxisome, which plays a key role in viral infections.

Predicting Blomia tropicalis allergens using a multiomics approach.

BACKGROUND: The domestic mite Blomia tropicalis is a major source of allergens in tropical and subtropical regions. Despite its great medical importance, the allergome of this mite has not been sufficiently studied. Only 14 allergen groups have been identified in B. tropicalis thus far, even though early radioimmunoelectrophoresis techniques (27 uncharacterized allergen complexes) and comparative data based on 40 allergen groups officially recognized by the World Health Organization (WHO)/IUIS in domestic astigmatid mites suggest the presence of a large set of additional allergens. METHODS: Here, we employ a multiomics approach to assess the allergome of B. tropicalis using genomic and transcriptomic sequence data and perform highly sensitive protein abundance quantification. FINDINGS: Among the 14 known allergen groups, we confirmed 13 (one WHO/IUIS allergen, Blo t 19, was not found) and identified 16 potentially novel allergens based on sequence similarity. These data indicate that B. tropicalis shares 27 known/deduced allergen groups with pyroglyphid house dust mites (genus Dermatophagoides). Among these groups, five allergen-encoding genes are highly expressed at the transcript level: Blo t 1, Blo t 5, Blo t 21 (known), Blo t 15, and Blo t 18 (predicted). However, at the protein level, a different set of most abundant allergens was found: Blo t 2, 10, 11, 20 and 21 (mite bodies) or Blo t 3, 4, 6 and predicted Blo t 13, 14 and 36 (mite feces). INTERPRETATION: We report the use of an integrated omics method to identify and predict an array of mite allergens and advanced, label-free proteomics to determine allergen protein abundance. Our research identifies a large set of novel putative allergens and shows that the expression levels of allergen-encoding genes may not be strictly correlated with the actual allergenic protein abundance in mite bodies.

Imidacloprid increases the prevalence of the intestinal parasite Lotmaria passim in honey bee workers.

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 µg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

Identification of potential molecular targets for the treatment of cluster 1 human pheochromocytoma and paraganglioma via comprehensive proteomic characterization.

BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. METHODS: We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. RESULTS: The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. CONCLUSIONS: Application of a combined proteomic strategy recently presented as "Pitchfork" enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.

Phosphorylation of tyrosine 90 in SH3 domain is a new regulatory switch controlling Src kinase.

The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

Variation in mouse chemical signals is genetically controlled and environmentally modulated.

In most mammals and particularly in mice, chemical communication relies on the detection of ethologically relevant fitness-related cues from other individuals. In mice, urine is the primary source of these signals, so we employed proteomics and metabolomics to identify key components of chemical signalling. We show that there is a correspondence between urinary volatiles and proteins in the representation of genetic background, sex and environment in two house mouse subspecies Mus musculus musculus and M. m. domesticus. We found that environment has a strong influence upon proteomic and metabolomic variation and that volatile mixtures better represent males while females have surprisingly more sex-biased proteins. Using machine learning and combined-omics techniques, we identified mixtures of metabolites and proteins that are associated with biological features.

Proteomic insight into the interaction of Paenibacillus larvae with honey bee larvae before capping collected from an American foulbrood outbreak: Pathogen proteins within the host, lysis signatures and interaction markers.

American foulbrood (AFB) is a devastating disease of honey bees. There remains a gap in the understanding of the interactions between the causative agent and host, so we used shotgun proteomics to gain new insights. Nano-LC-MS/MS analysis preceded visual description and Paenibacillus larvae identification in the same individual sample. A further critical part of our methodology was that larvae before capping were used as the model stage. The identification of the virulence factors SplA, PlCBP49, enolase, and DnaK in all P. larvae-positive samples was consistent with previous studies. Furthermore, the results were consistent with the array of virulence factors identified in an in vitro study of P. larvae exoprotein fractions. Although an S-layer protein and a putative bacteriocin were highlighted as important, the microbial collagenase ColA and InhA were not found in our samples. The most important virulence factor identified was isoform of neutral metalloproteinase (UniProt: V9WB82), a major protein marker responsible for the shift in the PCA biplot. This protein is associated with larval decay and together with other virulence factors (bacteriocin) can play a key role in protection against secondary invaders. Overall, this study provides new knowledge on host-pathogen interactions and a new methodical approach to study the disease.

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion.

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells - postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 106 µm3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.

Label-free proteomic analysis reveals differentially expressed Wolbachia proteins in Tyrophagus putrescentiae: Mite allergens and markers reflecting population-related proteome differences.

Tyrophagus putrescentiae is an astigmatid mite of great economic, medical and veterinary importance. The microbiome, especially intracellular bacteria, may affect allergy/allergen expression. We targeted Wolbachia proteins, allergen comparisons and markers in Wolbachia-mite interactions in three mite populations. A decoy database was constructed by proteogenomics using the T. putrescentiae draft genome, Wolbachia transcriptome assembly and current T. putrescentiae-related sequences in GenBank. Among thousands of mite-derived proteins, 18 Wolbachia proteins were reliably identified. We suggest that peroxiredoxin, bacterioferritin, ankyrin repeat domain-containing protein and DegQ family serine endoprotease indicate a higher-level bacterium-bacterium-host interaction. We produced evidence that the host-Wolbachia interaction is modulated through pattern recognition receptors (PRRs), mannose-binding lectins/mannose receptors, the cholinergic anti-inflammatory pathway with TNF-α, and others. We observed Tyr p 3 suppression in mites with Wolbachia, linking trypsin to PRR modulation. Nine out of the 12 current WHO/IUIS official allergens were reliably identified, but the remaining three allergens, Tyr p 1, 8 and 35, were detected as only trace hits. This study provides numerous markers for further Wolbachia-host interaction research. For accuracy, mite allergens should be considered according to abundance in species, but mite populations/strains, as well as their microbiome structure, may be key factors. SIGNIFICANCE: The astigmatid mites occurring in homes are significant producers of allergens that are highly dangerous to humans and domesticated animals. Mites are tightly associated with microorganisms that affect their biology and consequently allergy signatures. Mite populations were found to be infected with certain intracellular bacteria, but some populations lacked an intracellular bacterium. Our previous research showed that some populations of Tyrophagus putrescentiae are infected with Wolbachia, but some populations host additional bacteria of interest. Thus, there are not only interactions between the mites and Wolbachia but also likely an additional level of interaction that can be found in the interaction between different bacteria in the mites. These "higher-level" signatures and consequences that bacteria affect, including allergen production, are not understood in mites. In this study, we identified Wolbachia-specific proteins in mites for the first time. This study provides Wolbachia- and mite-derived markers that can be clues for describing "higher-level" mite-bacterium-bacterium interactions. Indeed, the microbiome contribution to allergies can potentially be derived directly from bacterial proteins, especially if they are abundant.