Treitz C et al. (2015) J Proteomics "Quantitative proteome analysis of Caenorhabditis elegans upon exposure to nematicidal Bacillus thuringiensis."

Leippe M, Hockendorf A, Treitz C, Tholey A, Cassidy L

[J Proteomics, 2015]

UNLABELLED: Caenorhabditis elegans can be infected by a plethora of pathogens, most of them are also pathogenic for humans. Consequently, the nematode has emerged as a powerful surrogate host to model microbial human infectious diseases in a non-vertebrate, for the study of innate immunity and host-pathogen interactions. Signaling cascades are well investigated that face bacterial or fungal pathogens. We analyzed the downstream processes of these cascades, i.e. the differential expression of effector and regulatory molecules due to a microbial challenge with a pathogenic strain of the bacterium Bacillus thuringiensis (Bt) in comparison to a non-pathogenic Bt strain. The protein abundance profile of the nematode was studied by quantitative proteomics using iTRAQ labeling and 2D-LC-MS analysis. We developed (i) a novel method for the preparation of defined C. elegans samples; (ii) a pooling strategy for fractions in 2D-LC separation schemes; and (iii) an isobaric labeling scheme reducing the number of necessary LC-MS experiments. More than 3,600 proteins were quantified, 288 of which showed altered abundances, implicating protein classes such as lectins, lysozymes, and transthyretin-like proteins to be involved in the nematode innate immune defense. A number of gene products previously only identified by transcriptomic profiling could be verified at the protein level. Moreover, several other protein classes such as proteases, proteins related to autophagy and apoptosis, structural proteins, and proteins involved in chromatin organization were detected. The results provide an overview of the physiological response towards a pathogen at protein level in the important model organism C. elegans, giving insights into highly complex host-pathogen interactions. BIOLOGICAL SIGNIFICANCE: This study identified system-wide effects of Bt intoxication on C. elegans at protein level, expanding the catalogue of immune effectors potentially acting towards the pathogen, and provide verification for numerous gene products implicated in previous transcriptomic studies. The data present evidence in support of both a general defense response as well as a specific reaction against the Bt toxin within the nematode. The described findings will also contribute to a deeper understanding of host-microbe interaction in other organisms, including humans, and may provide key information that touches far reaching aspects of coevolutionary processes.

Steinbach MK et al. (2022) J Proteome Res "Digital Microfluidics Supported Microproteomics for Quantitative Proteome Analysis of Single Caenorhabditis elegans Nematodes."

Leipert J, Tholey A, Steinbach MK, Blurton C, Leippe M

[J Proteome Res, 2022]

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 <i>Caenorhabditis elegans</i> 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.

Cassidy L et al. (2018) Proteomics "The C. elegans Proteome Response to Naturally Associated Microbiome Members of the Genus Ochrobactrum."

Schulenburg H, Cassidy L, Petersen C, Leippe M, Dierking K, Tholey A, Treitz C

[Proteomics, 2018]

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 analysed 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 colonising its gut, via a comparative analysis of the nematode exposed to two naturally associated Ochrobactrum spp. (MYb71, MYb237) versus C. elegans grown on E. coli OP50. A total of 4,677 C. elegans proteins were identified, 3,941 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 signalling 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. This article is protected by copyright. All rights reserved.

Yang W et al. (2015) Dev Comp Immunol "Overlapping and unique signatures in the proteomic and transcriptomic responses of the nematode Caenorhabditis elegans toward pathogenic Bacillus thuringiensis."

Rosenstiel P, Esser D, Yang W, Tholey A, Dierking K, Leippe M, Schulenburg H

[Dev Comp Immunol, 2015]

Pathogen infection can activate multiple signaling cascades that ultimately alter the abundance of molecules in cells. This change can be measured both at the transcript and protein level. Studies analyzing the immune response at both levels are, however, rare. Here, we compare transcriptome and proteome data generated after infection of the nematode and model organism Caenorhabditis elegans with the Gram-positive pathogen Bacillus thuringiensis. Our analysis revealed a high overlap between abundance changes of corresponding transcripts and gene products, especially for genes encoding C-type lectin domain-containing proteins, indicating their particular role in worm immunity. We additionally identified a unique signature at the proteome level, suggesting that the C. elegans response to infection is shaped by changes beyond transcription. Such effects appear to be influenced by AMP-activated protein kinases (AMPKs), which may thus represent previously unknown regulators of C. elegans immune defense.

Leipert J et al. (2023) Angew Chem Int Ed Engl "Digital Microfluidics and Magnetic Bead-based Intact Proteoform Elution for Quantitative Top-down Nanoproteomics of Single C. elegans Nematodes."

Steinbach MK, Tholey A, Leipert J, Kaulich PT, Leippe M, Steer B, Blurton C, Winkels K

[Angew Chem Int Ed Engl, 2023]

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 abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.

Pees B et al. (2024) mBio "The Caenorhabditis elegans proteome response to two protective Pseudomonas symbionts."

Pees B, Peters L, Tholey A, Kissoyan KAB, Hamerich IK, Treitz C, Dierking K

[mBio, 2024]

The <i>Caenorhabditis elegans</i> natural microbiota isolates <i>Pseudomonas lurida</i> MYb11 and <i>Pseudomonas fluorescens</i> MYb115 protect the host against pathogens through distinct mechanisms. While <i>P. lurida</i> produces an antimicrobial compound and directly inhibits pathogen growth, <i>P. fluorescens</i> MYb115 protects the host without affecting pathogen growth. It is unknown how these two protective microbes affect host biological processes. We used a proteomics approach to elucidate the <i>C. elegans</i> response to MYb11 and MYb115. We found that both <i>Pseudomonas</i> isolates increase vitellogenin protein production in young adults, which confirms previous findings on the effect of microbiota on <i>C. elegans</i> reproductive timing. Moreover, the <i>C. elegans</i> responses to MYb11 and MYb115 exhibit common signatures with the response to other vitamin B₁₂-producing bacteria, emphasizing the importance of vitamin B₁₂ in <i>C. elegans</i>-microbe metabolic interactions. We further analyzed signatures in the <i>C. elegans</i> response specific to MYb11 or MYb115. We provide evidence for distinct modifications in lipid metabolism by both symbiotic microbes. We could identify the activation of host-pathogen defense responses as an MYb11-specific proteome signature and provide evidence that the intermediate filament protein IFB-2 is required for MYb115-mediated protection. These results indicate that MYb11 not only produces an antimicrobial compound but also activates host antimicrobial defenses, which together might increase resistance to infection. In contrast, MYb115 affects host processes such as lipid metabolism and cytoskeleton dynamics, which might increase host tolerance to infection. Overall, this study pinpoints proteins of interest that form the basis for additional exploration into the mechanisms underlying <i>C. elegans</i> microbiota-mediated protection from pathogen infection and other microbiota-mediated traits.IMPORTANCESymbiotic bacteria can defend their host against pathogen infection. While some protective symbionts directly interact with pathogenic bacteria, other protective symbionts elicit a response in the host that improves its own pathogen defenses. To better understand how a host responds to protective symbionts, we examined which host proteins are affected by two protective <i>Pseudomonas</i> bacteria in the model nematode <i>Caenorhabditis elegans</i>. We found that the <i>C. elegans</i> response to its protective symbionts is manifold, which was reflected in changes in proteins that are involved in metabolism, the immune system, and cell structure. This study provides a foundation for exploring the contribution of the host response to symbiont-mediated protection from pathogen infection.

Yang W et al. (2019) Front Microbiol "The Inducible Response of the Nematode Caenorhabditis elegans to Members of Its Natural Microbiota Across Development and Adult Life."

Dirksen P, Waschina S, Petersen C, Kaleta C, Dierking K, Zimmermann J, Tholey A, Yang W, Leippe M, Schulenburg H, Pees B, Rosenstiel P

[Front Microbiol, 2019]

The biology of all organisms is influenced by the associated community of microorganisms. In spite of its importance, it is usually not well understood how exactly this microbiota affects host functions and what are the underlying molecular processes. To rectify this knowledge gap, we took advantage of the nematode <i>Caenorhabditis elegans</i> as a tractable, experimental model system and assessed the inducible transcriptome response after colonization with members of its native microbiota. For this study, we focused on two isolates of the genus <i>Ochrobactrum</i>. These bacteria are known to be abundant in the nematode's microbiota and are capable of colonizing and persisting in the nematode gut, even under stressful conditions. The transcriptome response was assessed across development and three time points of adult life, using general and <i>C. elegans</i>-specific enrichment analyses to identify affected functions. Our assessment revealed an influence of the microbiota members on the nematode's dietary response, development, fertility, immunity, and energy metabolism. This response is mainly regulated by a GATA transcription factor, most likely ELT-2, as indicated by the enrichment of (i) the GATA motif in the promoter regions of inducible genes and (ii) of ELT-2 targets among the differentially expressed genes. We compared our transcriptome results with a corresponding previously characterized proteome data set, highlighting a significant overlap in the differentially expressed genes, the affected functions, and ELT-2 target genes. Our analysis further identified a core set of 86 genes that consistently responded to the microbiota members across development and adult life, including several C-type lectin-like genes and genes known to be involved in energy metabolism or fertility. We additionally assessed the consequences of induced gene expression with the help of metabolic network model analysis, using a previously established metabolic network for <i>C. elegans</i>. This analysis complemented the enrichment analyses by revealing an influence of the <i>Ochrobactrum</i> isolates on <i>C. elegans</i> energy metabolism and furthermore metabolism of specific amino acids, fatty acids, and also folate biosynthesis. Our findings highlight the multifaceted impact of naturally colonizing microbiota isolates on <i>C. elegans</i> life history and thereby provide a framework for further analysis of microbiota-mediated host functions.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Tangrodchanapong T et al. (2020) Front Pharmacol "Frondoside A Attenuates Amyloid- Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation."

Tangrodchanapong T, Sobhon P, Meemon K

[Front Pharmacol, 2020]

Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.

Viney ME et al. (2004) Naturwissenschaften "Is dauer pheromone of Caenorhabditis elegans really a pheromone?"

Viney ME, Franks NR

[Naturwissenschaften, 2004]

Animals respond to signals and cues in their environment. The difference between a signal (e.g. a pheromone) and a cue (e.g. a waste product) is that the information content of a signal is subject to natural selection, whereas that of a cue is not. The model free-living nematode Caenorhabditis elegans forms an alternative developmental morph (the dauer larva) in response to a so-called 'dauer pheromone', produced by all worms. We suggest that the production of 'dauer pheromone' has no fitness advantage for an individual worm and therefore we propose that 'dauer pheromone' is not a signal, but a cue. Thus, it should not be called a pheromone.