Digital Microfluidics and Magnetic Bead-Based Intact Proteoform Elution for Quantitative Top-down Nanoproteomics of Single C.†elegans Nematodes.

While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up proteomics workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40 % formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analyses of single Caenorhabditis elegans nematodes, thereby increasing the number of proteoform identifications compared to in-tube sample preparation by 46 %. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in the abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.

Digital Microfluidics Supported Microproteomics for Quantitative Proteome Analysis of Single Caenorhabditis elegans Nematodes.

Miniaturization of sample preparation, including omissible manual sample handling steps, is key for reproducible nanoproteomics, as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3-supported sample preparation, the numbers of identified peptides and proteins were markedly increased, while lower standard deviations between replicates were observed. We repeatedly identified up to 5000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, highlighting the potential of this technology for low-input proteomics studies. LC-MS data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD033143.

Validation of Top-Down Proteomics Data by Bottom-Up-Based N-Terminomics Reveals Pitfalls in Top-Down-Based Terminomics Workflows.

Bottom-up proteomics (BUP)-based N-terminomics techniques have become standard to identify protein N-termini. While these methods rely on the identification of N-terminal peptides only, top-down proteomics (TDP) comes with the promise to provide additional information about post-translational modifications and the respective C-termini. To evaluate the potential of TDP for terminomics, two established TDP workflows were employed for the proteome analysis of the nematode Caenorhabditis elegans. The N-termini of the identified proteoforms were validated using a BUP-based N-terminomics approach. The TDP workflows used here identified 1658 proteoforms, the N-termini of which were verified by BUP in 25% of entities only. Caveats in both the BUP- and TDP-based workflows were shown to contribute to this low overlap. In BUP, the use of trypsin prohibits the detection of arginine-rich or arginine-deficient N-termini, while in TDP, the formation of artificially generated termini was observed in particular in a workflow encompassing sample treatment with high acid concentrations. Furthermore, we demonstrate the applicability of reductive dimethylation in TDP to confirm biological N-termini. Overall, our study shows not only the potential but also current limitations of TDP for terminomics studies and also presents suggestions for future developments, for example, for data quality control, allowing improvement of the detection of protein termini by TDP.

Designed Trp-Cage Proteins with Antimicrobial Activity and Enhanced Stability.

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.

Microbes to-go: slugs as source for Caenorhabditis elegans microbiota acquisition.

Research on the Caenorhabditis elegans microbiota only recently started, with little known about how C. elegans acquires its microbiota. Slugs live in the same habitat as C. elegans and are known vectors for the worm. Hence, we wondered how the passage through a slug affects the C. elegans gut microbiota and whether worms can acquire bacteria from the slug. Using fluorescently labelled microbiota and 16S rRNA gene amplicon sequencing, we evaluated microbiota persistence and acquisition in C. elegans after slug passage. We compared C. elegans gut microbiomes isolated from wild-caught slugs to the microbiomes of worms after experimental slug passage to compare similarities and differences in microbiome composition. We found that microbiota persists in C. elegans while passing the slug gut and that worms simultaneously acquire additional bacteria species from the slug. Although the amplicon sequencing variant (ASV) richness of worms from the experiment did not exceed the richness of worms that naturally occur in slugs, we found a high number of shared ASVs indicating the importance of commonly associated microbiota. We demonstrate that C. elegans can take advantage of its passage through the slug by acquiring new potential microbiota without losing its native microbiota.

Alkaline guts contribute to immunity during exposure to acidified seawater in the sea urchin larva.

Larval stages of members of the Abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date, the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that, analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens. pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50-60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus, which elicits a coordinated immune response in the larvae of the sea urchin Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid-base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of larvae during Vibrio infection. Reductions in seawater pH resembling ocean acidification conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response or resilience of sea urchin larvae to a Vibrio infection under ocean acidification conditions. Our study addressed the evolutionary benefits of the alkaline midgut of Ambulacraria larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in seawater pH.

The Caenorhabditis elegans Proteome Response to Naturally Associated Microbiome Members of the Genus Ochrobactrum.

The nematode Caenorhabditis elegans interacts with a variety of bacteria as it feeds on microbes, and a number of these both associate and persist within the worm's intestine. Host-microbe interactions in C. elegans have been analyzed primarily at the transcriptome level with the host response often been monitored after challenge with pathogens. We assessed the proteome of C. elegans after growth on bacteria capable of colonizing its gut, via a comparative analysis of the nematode exposed to two naturally associated Ochrobactrum spp. (MYb71, MYb237) versus C. elegans grown on Escherichia coli OP50. A total of 4677 C. elegans proteins were identified, 3941 quantified. Significant alterations in protein abundances were observed for 122 proteins, 48 higher and 74 lower in abundance. We observed an increase in abundance of proteins potentially regulated via host signaling pathways, in addition to proteins involved in processing of foreign entities (e.g., lipase, proteases, glutathione metabolism). Decreased in abundance were proteins involved in both degradation and biosynthesis of amino acids, and enzymes associated with the degradation of peptidoglycan (lysozymes). The protein level differences between C. elegans grown on native microbiome members compared to the laboratory food bacterium may help to identify molecular processes involved in host-microbe interactions.

Miniaturized dispersive liquid-liquid microextraction and MALDI MS using ionic liquid matrices for the detection of bacterial communication molecules and virulence factors.

The identification and quantification of molecules involved in bacterial communication are major prerequisites for the understanding of interspecies interactions at the molecular level. We developed a procedure allowing the determination of 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and the virulence factor pyocyanin (PYO) formed by the Gram-negative bacterium Pseudomonas aeruginosa. The method is based on dispersive liquid-liquid microextraction from small supernatant volumes (below 10 µL) followed by quantitative matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The use of ionic liquid matrix led to a lowered limit of detection for pyocyanin and, due to suppression of matrix background signals, easy to interpret mass spectra compared to crystalline matrices. Using an isotope-labeled pyocyanin standard synthesized in small-scale synthesis, quantitative analysis spanning approximately one order of magnitude (0.5 to 250 fmol) was feasible. The method was successfully applied to the detection of the signaling molecules PQS and HHQ in cultures of P. aeruginosa strains isolated from sputum of cystic fibrosis patients and allowed a highly sensitive quantification of PYO from these cultures. Hence, the developed method bears the potential to be used for screening purposes in clinical settings and will help to decipher the molecular basis of bacterial communication. Graphical abstract Ionic liquid matrices for the detection and quantification of the toxin pyocyanin and other signaling molecules from P. aeruginosa by MALDI MS.

Mechanistic insights into the lipid interaction of an ancient saposin-like protein.

The members of the expanding family of saposin-like proteins (SAPLIPs) have various biological functions in plants, animals, and humans. In addition to a similar protein backbone, these proteins have in common the fact that they interact with lipid membranes. According to their phylogenetic position, it has long been thought that amoeboid protozoans produce archetypes of SAPLIPs and that these are lytic proteins that can perforate membranes of prokaryotic and eukaryotic target cells. Here, we show that an amoebic SAPLIP from Entamoeba invadens does not form lytic pores in membranes but displays several characteristics that are known from human saposins. The protein named invaposin changes the conformation from a closed to an open form in the presence of lipid membranes, acts in a pH-dependent manner, selectively binds anionic lipids, aggregates lipid vesicles of the preferred composition, and dimerizes upon acidification. Our data indicate that the principal features of the lipid-binding saposins evolved long before the appearance of the vertebrate lineage and push the origin of saposins even deeper down the phylogenetic tree to unicellular organisms.

Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba.

Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.

Potent antimicrobial peptides against Legionella pneumophila and its environmental host, Acanthamoeba castellanii.

Legionella pneumophila, the major causative agent of Legionnaires' disease, is most often found in the environment in close association with free-living amoebae, leading to persistence, spread, biocide resistance, and elevated virulence of the bacterium. In the present study, we evaluated the anti-Legionella and anti-Acanthamoeba activities of three alpha-helical antimicrobial peptides (AMPs), namely, NK-2, Ci-MAM-A24, and Ci-PAP-A22, already known for the extraordinary efficacy against other microbes. Our data represent the first demonstration of the activity of a particular AMP against both the human facultative intracellular pathogen L. pneumophila and its pathogenic host, Acanthamoeba castellanii. Interestingly, the most effective peptide, Ci-MAM-A24, was also found to reduce the Legionella cell number within amoebae. Accordingly, this peptide was immobilized on gold surfaces to assess its antimicrobial activity. Surfaces were characterized, and activity studies revealed that the potent bactericidal activity of the peptide was conserved after its immobilization. In the frame of elaborating anti-Legionella surfaces, Ci-MAM-A24 represents, by its direct and indirect activity against Legionella, a potent peptide template for biological control of the bacterium in plumbings.

Pore-forming toxins from pathogenic amoebae.

Some amoeboid protozoans are facultative or obligate parasites in humans and bear an enormous cytotoxic potential that can result in severe destruction of host tissues and fatal diseases. Pathogenic amoebae produce soluble pore-forming polypeptides that bind to prokaryotic and eukaryotic target cell membranes and generate pores upon insertion and oligomerization. This review summerizes the current knowledge of such small protein toxins from amoebae, compares them with related proteins from other species, focuses on their three-dimensional structures, and gives insights into divergent activation mechanisms. The potential use of pore-forming toxins in biotechnology will be briefly outlined.

EGFR signaling in the brain is necessary for olfactory learning in Drosophila larvae.

Signaling via the epidermal growth factor receptor (EGFR) pathway has emerged as one of the key mechanisms in the development of the central nervous system in Drosophila melanogaster. By contrast, little is known about the functions of EGFR signaling in the differentiated larval brain. Here, promoter-reporter lines of EGFR and its most prominent activating ligands, Spitz, Keren, and Vein, were used to identify the brain structures relevant for the EGFR pathway. Unexpectedly, promoter activity of all these pathway components was found in the mushroom bodies, which are known to be a higher brain center required for olfactory learning. We investigated the role of the EGFR pathway in this process by using different mutant larvae with reduced pan-neuronal EGFR signaling and those with reduced EGFR signaling in mushroom bodies only. Expression of a dominant-negative form of EGFR as well as silencing of the ligands via RNA interference was applied and resulted in significantly impaired olfactory learning performances. General defects in the ability to taste or smell as well as impaired EGFR signaling during embryonic development could be excluded as major reasons for this learning phenotype. In addition, targeted expression of a constitutively active form of the ligand Spitz also led to a significantly reduced learning ability. Thus, very low levels as well as very high levels of EGFR signaling are deleterious for olfactory learning and memory formation. We hypothesize that EGFR signaling in a certain range maintains a homeostatic situation in the mushroom bodies that is necessary for proper learning and memory.

Molecular and functional characterization of ILYS-5, a major invertebrate lysozyme of Caenorhabditis elegans.

To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is exposed to a variety of microbes due to its bacterivorous lifestyle, previous work on the components of its immune system mainly based on the description of transcriptional changes during bacterial challenges. Very few effector components of its immune system have been characterized so far. To investigate the role of lysozymes in terms of antibacterial defense and digestion, we studied a member of the widely neglected family of C. elegans invertebrate lysozymes (ILYS). We focused on the so far virtually undescribed ILYS-5, which we purified from protein extracts of C. elegans tracing its peptidoglycan-degrading activity and localized the tissue expression of the gene in vivo using a translational reporter construct. We recombinantly synthesized ILYS-5 and determined the physicochemical activity optimum and the antibacterial spectrum of a lysozyme from C. elegans for the first time. With an activity optimum at low ionic strength (≤100 mM) and at acidic pH (≤ pH 4.0), ILYS-5 is likely to be involved in killing and digestion of bacteria within acidified phagolysosomes and acidic regions of the gut, presumably secreted by lysosome-like vesicles. This notion is supported by potent activity against various live Gram-positive and Gram-negative bacteria. Notably, members of the natural associated microbiome of C. elegans are substantially less susceptible to ILYS-5. Ablation of the ilys-5 gene resulted in reduction of lifespan and fertility when cultured on the standard food bacterium Escherichia coli OP50, whereas exposure of the ilys-5 knock-out mutant to the host-associated bacterium Pseudomonas lurida MYb11 did not have a clear effect. These findings indicate a role of ILYS-5 in immunity and nutrition and a co-evolved adaptation of host and bacteria to the mutualistic nature of their interaction.

Structural analysis of the NK-lysin-derived peptide NK-2 upon interaction with bacterial membrane mimetics consisting of phosphatidylethanolamine and phosphatidylglycerol.

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil - helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.

Macin family of antimicrobial proteins combines antimicrobial and nerve repair activities.

The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions.

The equine antimicrobial peptide eCATH1 is effective against the facultative intracellular pathogen Rhodococcus equi in mice.

Rhodococcus equi, the causal agent of rhodococcosis, is a major pathogen of foals and is also responsible for severe infections in immunocompromised humans. Of great concern, strains resistant to currently used antibiotics have emerged. As the number of drugs that are efficient in vivo is limited because of the intracellular localization of the bacterium inside macrophages, new active but cell-permeant drugs will be needed in the near future. In the present study, we evaluated, by in vitro and ex vivo experiments, the ability of the alpha-helical equine antimicrobial peptide eCATH1 to kill intracellular bacterial cells. Moreover, the therapeutic potential of the peptide was assessed in experimental rhodococcosis induced in mice, while the in vivo toxicity was evaluated by behavioral and histopathological analysis. The study revealed that eCATH1 significantly reduced the number of bacteria inside macrophages. Furthermore, the bactericidal potential of the peptide was maintained in vivo at doses that appeared to have no visible deleterious effects for the mice even after 7 days of treatment. Indeed, daily subcutaneous injections of 1 mg/kg body weight of eCATH1 led to a significant reduction of the bacterial load in organs comparable to that obtained after treatment with 10 mg/kg body weight of rifampin. Interestingly, the combination of the peptide with rifampin showed a synergistic interaction in both ex vivo and in vivo experiments. These results emphasize the therapeutic potential that eCATH1 represents in the treatment of rhodococcosis.

The saposin-like protein SPP-12 is an antimicrobial polypeptide in the pharyngeal neurons of Caenorhabditis elegans and participates in defence against a natural bacterial pathogen.

Caenopores are antimicrobial and pore-forming polypeptides in Caenorhabditis elegans belonging to the saposin-like protein superfamily and are considered important elements of the nematode's intestinal immune system. In the present study, we demonstrate that, unlike the other members of the multifarious gene family (spps) coding for caenopores, spp-12 is expressed exclusively in two pharyngeal neurons. Recombinantly expressed SPP-12 binds to phospholipid membranes and forms pores in a pH-dependent manner characteristic of caenopores. Moreover, SPP-12 kills viable Gram-positive bacteria, yeast cells and amoebae by permeabilizing their membranes, suggesting a wide-target cell spectrum. A spp-12 knockout mutant is more susceptible to pathogenic Bacillus thuringiensis than wild-type worms and is tolerant to non-pathogenic bacteria. By contrast, SPP-1, a caenopore, whose gene is expressed only in the intestine and reported to be regulated by the same pathway as spp-12, is apparently non-protective against pathogenic B. thuringiensis, although it also does display antimicrobial activity. The transcription of spp-1 is down-regulated in wild-type worms in the presence of pathogenic B. thuringiensis and a spp-1 knockout mutant is hyposusceptible to this bacterium. This implies that SPP-12, but not SPP-1, contributes to resistance against B. thuringiensis, a natural pathogen of the nematode.

A guided tour through α-helical peptide antibiotics and their targets.

Nowadays, not only biologists, but also researchers from other disciplines such as chemistry, pharmacy, material sciences, or physics are working with antimicrobial peptides. This review is written for researchers and students working in or interested in the field of antimicrobial peptides - and especially those who do not have a profound biological background. To lay the ground for a thorough discussion on how AMPs act on cells, the architectures of mammalian and bacterial cell envelopes are described in detail because they are important targets of AMPs and provide the basis for their selectivity. The modes of action of α-helical AMPs (αAMPs) are not limited to different models of membrane permeabilization, but also include the disruption of intracellular processes, as well as the formation of fibrillary structures and their potential implications for antimicrobial activity. As biofilm-related infections are very difficult to treat with conventional antibiotics, they pose a major problem in the clinic. Therefore, this review also discusses the biological background of biofilm infections and the mode of actions of αAMPs against biofilms. The last chapter focusses on the design of αAMPs by providing an overview of historic milestones in αAMP design. It describes how modern αAMP design is aiming to produce peptides suitable to be applied in the clinic. Hence, the article concludes with a section on translational research discussing the prospects of αAMPs and remaining challenges on their way into the clinic.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.