SLAMF7 (CD319) on activated CD8+ T cells transduces environmental cues to initiate cytotoxic effector cell responses.

CD8+ T-cell responses are meticulously orchestrated processes regulated by intercellular receptor:ligand interactions. These interactions critically control the dynamics of CD8+ T-cell populations that is crucial to overcome threats such as viral infections or cancer. Yet, the mechanisms governing these dynamics remain incompletely elucidated. Here, we identified a hitherto unknown T-cell referred function of the self-ligating surface receptor SLAMF7 (CD319) on CD8+ T cells during initiation of cytotoxic T-cell responses. According to its cytotoxicity related expression on T effector cells, we found that CD8+ T cells could utilize SLAMF7 to transduce environmental cues into cellular interactions and information exchange. Indeed, SLAMF7 facilitated a dose-dependent formation of stable homotypic contacts that ultimately resulted in stable cell-contacts, quorum populations and commitment to expansion and differentiation. Using pull-down assays and network analyses, we identified novel SLAMF7-binding intracellular signaling molecules including the CRK, CRKL, and Nck adaptors, which are involved in T-cell contact formation and may mediate SLAMF7 functions in sensing and adhesion. Hence, providing SLAMF7 signals during antigen recognition of CD8+ T cells enhanced their overall magnitude, particularly in responses towards low-affinity antigens, resulting in a significant boost in their proliferation and cytotoxic capacity. Overall, we have identified and characterized a potent initiator of the cytotoxic T lymphocyte response program and revealed advanced mechanisms to improve CD8+ T-cell response decisions against weak viral or tumor-associated antigens, thereby strengthening our defense against such adversaries.

The nuclear GYF protein CD2BP2/U5-52K is required for T cell homeostasis.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.

When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping.

In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.

Mutational Analysis of RIP Type I Dianthin-30 Suggests a Role for Arg24 in Endocytosis.

Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

Characterization of the plasma proteome from healthy adult dogs.

Introduction: Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. Methods: In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Results and discussion: Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS).

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS.

Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Protein-Templated Ugi Reactions versus In-Situ Ligation Screening: Two Roads to the Identification of SARS-CoV-2 Main Protease Inhibitors.

Protein-templated fragment ligation was established as a method for the rapid identification of high affinity ligands, and multicomponent reactions (MCR) such as the Ugi four-component reaction (Ugi 4CR) have been efficient in the synthesis of drug candidates. Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein-templated Ugi 4CR quantitatively using a fluorescence-based enzyme assay, HPLC-QTOF mass spectrometry (MS), and native protein MS with SARS-CoV-2 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three-component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non-templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in-situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS-CoV-2 protease inhibitors.

The population context is a driver of the heterogeneous response of epithelial cells to interferons.

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.

Staphylococcus aureus adapts to the immunometabolite itaconic acid by inducing acid and oxidative stress responses including S-bacillithiolations and S-itaconations.

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus. In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-ß-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-ß-lactamases (MBL) such as New Delhi metallo-ß-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential ß-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila.

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.

The MarR/DUF24-Family QsrR Repressor Senses Quinones and Oxidants by Thiol Switch Mechanisms in Staphylococcus aureus.

Aims: The MarR/DUF24-family QsrR and YodB repressors control quinone detoxification pathways in Staphylococcus aureus and Bacillus subtilis. In S. aureus, the QsrR regulon also confers resistance to antimicrobial compounds with quinone-like elements, such as rifampicin, ciprofloxacin, and pyocyanin. Although QsrR was shown to be inhibited by thiol-S-alkylation of its conserved Cys4 residue by 1,4-benzoquinone, YodB senses quinones and diamide by the formation of reversible intermolecular disulfides. In this study, we aimed at further investigating the redox-regulation of QsrR and the role of its Cys4, Cys29, and Cys32 residues under quinone and oxidative stress in S. aureus. Results: The QsrR regulon was strongly induced by quinones and oxidants, such as diamide, allicin, hypochlorous acid (HOCl), and AGXX® in S. aureus. Transcriptional induction of catE2 by quinones and oxidants required Cys4 and either Cys29' or Cys32' of QsrR for redox sensing in vivo. DNA-binding assays revealed that QsrR is reversibly inactivated by quinones and oxidants, depending on Cys4. Using mass spectrometry, QsrR was shown to sense diamide by an intermolecular thiol-disulfide switch, involving Cys4 and Cys29' of opposing subunits in vitro. In contrast, allicin caused S-thioallylation of all three Cys residues in QsrR, leading to its dissociation from the operator sequence. Further, the QsrR regulon confers resistance against quinones and oxidants, depending on Cys4 and either Cys29' or Cys32'. Conclusion and Innovation: QsrR was characterized as a two-Cys-type redox-sensing regulator, which senses the oxidative mode of quinones and strong oxidants, such as diamide, HOCl, and the antimicrobial compound allicin via different thiol switch mechanisms.

Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP.

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIß (CaMKIIß) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled CAMK2B alternative splicing with a fundamental function in learning and memory.

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Eating in a losing cause: limited benefit of modified macronutrient consumption following infection in the oriental cockroach Blatta orientalis.

BACKGROUND: Host-pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. RESULTS: We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. CONCLUSIONS: We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host's dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted.

Nephridiophagids (Chytridiomycota) reduce the fitness of their host insects.

Nephridiophagids are unicellular fungi (Chytridiomycota), which infect the Malpighian tubules of insects. While most life cycle features are known, the effects of these endobionts on their hosts remain poorly understood. Here, we present results on the influence of an infection of the cockroach Blattella germanica with Nephridiophaga blattellae (Ni = Nephridiophaga-infected) on physical, physiological, and reproductive fitness parameters. Since the gut nematode Blatticola blattae is a further common parasite of B. germanica, we included double infected cockroaches (N + Ni = nematode plus Ni) in selected experiments. Ni individuals had lower fat reserves and showed reduced mobility. The lifespan of adult hosts was only slightly affected in these individuals but significantly shortened when both Nephridiophaga and nematodes were present. Ni as well as N + Ni females produced considerably less offspring than parasite-free (P-free) females. Immune parameters such as the number of hemocytes and phenoloxidase activity were barely changed by Nephridiophaga and/or nematode infections, while the ability to detoxify pesticides decreased. Quantitative proteomics from hemolymph of P-free, Ni, and N + Ni populations revealed clear differences in the expression profiles. For Ni animals, for example, the down-regulation of fatty acid synthases corroborates our finding of reduced fat reserves. Our study clearly shows that an infection with Nephridiophaga (and nematodes) leads to an overall reduced host fitness.

Evolutionarily conserved properties of CLCA proteins 1, 3 and 4, as revealed by phylogenetic and biochemical studies in avian homologues.

Species-specific diversities are particular features of mammalian chloride channel regulator, calcium activated (CLCA) genes. In contrast to four complex gene clusters in mammals, only two CLCA genes appear to exist in chickens. CLCA2 is conserved in both, while only the galline CLCA1 (gCLCA1) displays close genetic distance to mammalian clusters 1, 3 and 4. In this study, sequence analyses and biochemical characterizations revealed that gCLCA1 as a putative avian prototype shares common protein domains and processing features with all mammalian CLCA homologues. It has a transmembrane (TM) domain in the carboxy terminal region and its mRNA and protein were detected in the alimentary canal, where the protein was localized in the apical membrane of enterocytes, similar to CLCA4. Both mammals and birds seem to have at least one TM domain containing CLCA protein with complex glycosylation in the apical membrane of enterocytes. However, some characteristic features of mammalian CLCA1 and 3 including entire protein secretion and expression in cell types other than enterocytes seem to be dispensable for chicken. Phylogenetic analyses including twelve bird species revealed that avian CLCA1 and mammalian CLCA3 form clades separate from a major branch containing mammalian CLCA1 and 4. Overall, our data suggest that gCLCA1 and mammalian CLCA clusters 1, 3 and 4 stem from a common ancestor which underwent complex gene diversification in mammals but not in birds.