Biology subjects

Shapira, M.

Publications and source records attributed to Shapira, M..

14 recordsLinked to original sources

bioRxiv2026

Environmental microbial extracts for longitudinal studies of gut microbiome assembly and maintenance

Animals harbor diverse gut microorganisms that influence host health and fitness. Synthetic microbial communities have been instrumental in enabling reductionist studies of host-microbiome interactions, but some questions require microbial communities with more natural-like complexity while preserving experimental tractability, in vivo monitoring, and quantitative analysis. Here, we describe a method optimized for longitudinal studies of host-microbiome-environment interactions in the nematode Caenorhabditis elegans. In this approach, complex microbial extracts (CMEs) are generated from environmental samples and applied to worm culture plates, providing a diverse yet experimentally convenient microbial environment. We show that CME composition remains stable during cold storage, enabling reproducible longitudinal experiments while minimizing confounding environmental drift over time. As a proof of principle, we apply this method to examine age-dependent changes in the worm gut microbiome, providing support for previous reports of age-dependent increase in the abundance of gut Enterobacteriaceae. CMEs provide a practical and reproducible framework that complements experiments using monocultures or synthetic communities, enabling longitudinal studies of host-microbiome interactions under conditions that better approximate natural microbial complexity.

ecology↗

bioRxiv2026

Attraction to secreted isoamyl alcohol as a signal for beneficial commensals

C. elegans, a bacterivore living in microbially-complex environments, harbors a characteristic community of gut bacteria that contribute to its health and fitness. What determines which environmental bacteria end up as commensals is largely unknown in C. elegans, as in other animals. Previous work found that gut Pantoea isolates supported rapid worm development and infection resistance, while environmental congenerics were inferior. Notably, worms were preferentially attracted to the more beneficial gut isolates. Using bioactivity-guided fractionation and gas chromatography-mass spectrometry analysis, we identified bacterially derived isoamyl alcohol (IAA) as a secreted volatile attractant underlying this preference. Screening of worm mutants implicated AWC sensory neuron-associated genes in preferential attraction to beneficial Pantoea and established a causal link between IAA sensing and colonization by beneficial strains. While IAA sensing was important for initial colonization, gut-associated Pantoea strains ultimately outcompeted environmental congenerics over time, indicating that microbiome assembly is shaped by two complementary processes: host behavioral preference for high-IAA producers and bacterial competitive fitness within the gut. While IAA is a product of leucine metabolism and may function as a nutritional cue, we found that it could also directly enhance host infection resistance, suggesting an additional role in modulating host physiology. Finally, knockout analysis identified a bacterial branched-chain amino acid aminotransferase homolog as important for IAA production. Together, these findings identify bacterial volatile sensing as an important and underexplored mechanism shaping microbiome composition and its contributions to host fitness.

microbiology↗

bioRxiv2025

Beyond translation initiation: dual regulatory interactions with phosphorylated and non-phosphorylated LeishIF4E1

Translation initiation in eukaryotes begins with the assembly of the cap-binding eIF4F complex at the 5 ends of mRNAs, through eIF4E. Among the six orthologs of eIF4E in Leishmania, LeishIF4E1 is intriguing, as it does not bind any Leishmania eIF4G. It is expressed in both life-forms, maintaining efficient cap-binding activity for translation initiation, unlike other LeishIF4Es. We identified two novel phosphorylation sites specifically S108, and S112, along with S203, which is conserved with the mammalian phosphorylation site. A non-phosphorylatable, tagged, mutant of LeishIF4E1 created by substituting the phosphorylated serine residues with alanine [S(108,112,203)A] was generated. The phosphorylation status did not affect the binding of LeishIF4E1 to translation initiation factors. However, the phosphorylated LeishIF4E1 interacted with proteins involved in DNA and chromatin structure, while non-phosphorylated LeishIF4E1 interacted with proteins that assist cells under stress and unfavorable conditions, particularly in gluconeogenesis. This suggests secondary regulatory roles for LeishIF4E1. RNA sequencing of the LeishIF4E1-associated transcripts revealed a tenfold reduction in transcripts binding to non-phosphorylated LeishIF4E1. GO enrichment analysis indicated distinct phosphorylation-dependent transcript specificities for each form. This study highlights the critical role of LeishIF4E1 phosphorylation in modulating the selectivity of protein interactome and transcript associations, extending the role of LeishIF4E1 beyond its well-established function in translation initiation. Author SummaryLeishmania are digenetic parasites that transition from sand fly vector to mammalian hosts, undergoing a developmental program of gene expression regulated at the level of translation. Protein synthesis is initiated by the assembly of multi-subunit complexes at the mRNA 5 cap structure and is recruited onto the ribosomes. There are six cap-binding proteins in Leishmania denoted LeishIF4Es1-6, and five LeishIF4G candidates. Among these, the function of LeishIF4E1 is intriguing, as it does not bind any LeishIF4G paralogs, and it is highly expressed in both parasite life forms. We examined how LeishIF4E1 phosphorylation modulates its interaction with proteins and transcripts, given that eIF4E phosphorylation in higher eukaryotes is known to regulate gene expression during malignancy. We identified three phosphorylation sites (S108, S112 and S203) and mutated them to A residues [S(108,112,203)A]. The mutated genes were transfected back into parasites for expression. Using pull-down assays, we identified the interacting proteins and associated transcripts for both the phosphorylated and non-phosphorylated LeishIF4E1 forms. The phosphorylated LeishIF4E1 pulled down proteins involved in DNA and chromatin structure, while the non-phosphorylated LeishIF4E1 interacted with proteins known to assist cells during stress and unfavorable conditions, especially gluconeogenesis. The transcript specificities differed markedly in the number of transcripts bound to both forms, shedding light on the importance of LeishIF4E1 phosphorylation. Furthermore, the phosphorylated form bound more transcripts encoding enzymes related to protein phosphorylation. We also showed that phosphorylation of LeishIF4E1 did not influence the cap-binding ability, as both the forms bind the m7GTP cap with parallel efficiencies. Our data suggest that the different cap-binding proteins in Leishmania are responsible for different processes and have a transcript-specific effect on gene expression.

microbiology↗

bioRxiv2024

Perception of Temperature Even in the Absence of Actual Change is Sufficient to Drive Transgenerational Epigenetic Inheritance

Can processes occurring in one individuals nervous system influence the physiology of the descendants? Here, we explored the hypothesis that parents sensation or perception of environmental cues can influence their offspring, extending across many subsequent generations. We show that in Caenorhabditis elegans, temperature perception by the AFD thermosensory neurons initiates a signaling cascade that, directly or indirectly, induces transgenerational changes in RNAi factors, small RNAs, and their target genes. Moreover, we identify secreted factors that enable this neuron-to-germline communication and trace the path of the epigenetic signal. We further model the process mathematically, and the model yields new predictions that we validate experimentally: blocking sensory input dampens RNAi inheritance initiated by exogenous double-stranded RNA (dsRNA). Together, our results demonstrate that sensory perception is sufficient to influence small RNA-mediated heritable gene expression memory.

genetics↗

bioRxiv2024

The West African lungfish secretes a living cocoon during aestivation with uncertain antimicrobial function

One of the most exceptional adaptations to extreme drought is found in the sister group to tetrapods, the lungfishes (Dipnoi), which can aestivate inside a mucus cocoon for multiple years at reduced metabolic rates with complete cessation of ingestion and excretion. However, the function of the cocoon tissue is not fully understood. Here we developed a new more natural laboratory protocol for inducing aestivation in the West African lungfish, Protopterus annectens, and investigated the structure and function of the cocoon. We used electron microscopy and imaging of live tissue-stains to confirm that the inner and outer layers of the paper-thin cocoon are composed primarily of living cells. However, we also repeatedly observed extensive bacterial and fungal growth covering the cocoon and found no evidence of anti-microbial activity in vitro against E. coli for the cocoon tissue in this species. This classroom discovery-based research, performed during a course-based undergraduate research experience course (CURE), provides a robust laboratory protocol for investigating aestivation and calls into the question the function of this bizarre vertebrate adaptation.

physiology↗

bioRxiv2023

Identification of intestinal mediators of Caenorhabditis elegans DBL-1/BMP immune signaling shaping gut microbiome composition

The composition of the gut microbiome is determined by a complex interplay of diet, host genetics, microbe-microbe competition, abiotic factors, and stochasticity. Previous studies have demonstrated the importance of host genetics in community assembly of the Caenorhabditis elegans gut microbiome and identified a pivotal role for DBL-1/BMP immune signaling in determining the abundance of gut Enterobacteriaceae, in particular of the genus Enterobacter. However, the effects of DBL-1 signaling on gut bacteria were found to depend on its activation in extra-intestinal tissues, suggesting that yet unidentified intestinal factors must mediate these effects. In the present study, we used RNA-seq gene expression analysis of wildtype, dbl-1 and sma-3 mutants, and dbl-1 over-expressors to identify genes regulated by DBL-1/BMP signaling that take part in interactions with gut commensals. Following confirmation of several putative targets by qRT-PCR, we carried out colonization experiments with respective mutants raised on monocultures as well as on defined bacterial communities. These experiments identified five intestinal DBL-1/BMP targets, predicted to be secreted, that showed increased Enterobacteriaceae abundance compared to wildtype. The extent of increases was for the most part lower than those seen in DBL-1 pathway mutants, suggesting that identified mediators are components of a DBL-1-regulated antibacterial cocktail, which may additively contribute to shaping of gut microbiome composition. IMPORTANCECompared to the roles of diet, environmental availability, or lifestyle in determining gut microbiome composition, that of genetic factors is the least understood and often underestimated. The identification of intestinal mediators acting downstream of DBL-1/BMP signaling to control enteric bacteria, describes a cocktail of effectors with distinct molecular functions, thus offering a glimpse into the genetic logic of microbiome control as well as a list of targets for future exploration of this logic.

microbiology↗

bioRxiv2023

Microbiome remodeling through bacterial competition and host behavior enables rapid adaptation to environmental toxins.

Human activity is altering the environment in a rapid pace, challenging the adaptive capacities of genetic variation within animal populations. Animals also harbor extensive gut microbiomes, which play diverse roles in host health and fitness and may help expanding host capabilities. The unprecedented scale of human usage of xenobiotics and contamination with environmental toxins describes one challenge against which bacteria with their immense biochemical diversity would be useful, by increasing detoxification capacities. To explore the potential of bacteria-assisted rapid adaptation, we used Caenorhabditis elegans worms harboring a defined microbiome, and neomycin as a model toxin, harmful for the worm host and neutralized to different extents by some microbiome members. Worms raised in the presence of neomycin showed delayed development and decreased survival but were protected when colonized by neomycin-resistant members of the microbiome. Two distinct mechanisms facilitated this protection: gut enrichment driven by altered bacterial competition for the strain best capable of modifying neomycin; and host avoidance behavior, which depended on the conserved JNK homolog KGB-1, enabling preference and acquisition of neomycin-protective bacteria. We further tested the consequences of adaptation, considering that enrichment for protective strains may represent dysbiosis. We found that neomycin-adapted gut microbiomes caused increased susceptibility to infection as well as an increase in gut lipid storage, suggesting metabolic remodeling. Our proof-of-concept experiments support the feasibility of bacteria-assisted host adaptation and suggest that it may be prevalent. The results also highlight trade-offs between toxin adaptation and other traits of fitness.

evolutionary biology↗

bioRxiv2023

An Enterobacteriaceae bloom in aging animals is restrained by the gut microbiome

The gut microbiome plays important roles in host function and health. Core microbiomes have been described for different species, and imbalances in their composition, known as dysbiosis, are associated with pathology. Changes in the gut microbiome and dysbiosis are common in aging, possibly due to multi-tissue deterioration, which includes metabolic shifts, dysregulated immunity, and disrupted epithelial barriers. However, the characteristics of these changes, as reported in different studies, are varied and sometimes conflicting. Using clonal populations of C. elegans to highlight trends shared among individuals, and employing NextGen sequencing, CFU counts and fluorescent imaging to characterize age-dependent changes in worms raised in different microbial environments, we identified an Enterobacteriaceae bloom as a common denominator in aging animals. Experiments using Enterobacter hormachei, a representative commensal, suggested that the Enterobacteriaceae bloom was facilitated by a decline in Sma/BMP immune signaling in aging animals and demonstrated its detrimental potential for increasing susceptibility to infection. However, such detrimental effects were context-dependent, mitigated by competition with commensal communities, highlighting the latter as determinants of healthy versus unhealthy aging, depending on their ability to restrain opportunistic pathobionts.

physiology↗

bioRxiv2022

Design and Immunogenicity of SARS-CoV-2 DNA vaccine encoding RBD-PVXCP fusion protein

The potential of immune evasive mutations accumulation of the SARS-CoV-2 virus has led to its rapid spread causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. Huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants renews interest in DNA vaccine technology. Here we report a rapid generation and immunological evaluation of novel DNA vaccine candidates against Wuhan-Hu-1 and Omicron variants, based on the RBD protein fused with the Potato virus X coat protein (PVXCP). Delivery of DNA vaccines using electroporation in a two-doses regimen induced high antibody titers and profound cellular response in mice. Antibody titers induced against Omicron variant of the vaccine were sufficient for the effective protection against both the Omicron and Wuhan-Hu-1 virus infections. PVXCP protein in the vaccine construct shifted immune response to the favorable Th1-like type and provided oligomerization of RBD-PVXCP protein. A naked DNA delivery by the needle-free injection device allowed us to achieve antibody titers comparable with the mRNA-LNP delivery in rabbits. This data identifies the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study.

immunology↗

bioRxiv2022

Development of an Escape-resistant SARS CoV-2 Neutralizing Synthetic Nanobody

An emerging COVID-19 pandemic resulted in a global crisis, but also accelerated vaccine development and antibody discovery. In this work, we identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD), by screening synthetic humanized antibody library with more than 1011 diversity. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, their affinities are weakly influenced by SARS-CoV-2 VoC mutations. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies respectively for emerging Omicron variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2020

ZnJ6 is a DnaJ-like Chaperone with Oxidizing Activity in the Thylakoid Membrane in Chlamydomonas reinhardtii

Assembly of photosynthetic complexes is sensitive to changes in light intensities, drought, and pathogens that induce a redox imbalance, and require a variety of substrate-specific chaperones to overcome the stress. Proteins with cysteine (C) residues and disulfide bridges are more responsive to the redox changes. This study reports on a thylakoid membrane-associated DnaJ-like protein, ZnJ6 (ZnJ6.g251716.t1.2) in Chlamydomonas reinhardtii. The protein has four CXXCX(G)X(G) motifs that form a functional zinc-binding domain. Site-directed mutagenesis (Cys to Ser) in all the CXXCX(G)X(G) motifs eliminates its zinc-binding ability. In vitro chaperone assays using recombinant ZnJ6 confirm that it is a chaperone that possesses both holding and oxidative refolding activities. Although mutations (Cys to Ser) do not affect the holding activity of ZnJ6, they impair its ability to promote redox-controlled reactivation of reduced and denatured RNaseA, a common substrate protein. The presence of an intact zinc-binding domain is also required for protein stability at elevated temperatures, as suggested by a single spectrum melting curve. Pull-down assays with recombinant ZnJ6 revealed that it interacts with oxidoreductases, photosynthetic proteins (mainly PSI), and proteases. Our in vivo experiments with Chlamydomonas reinhardtii insertional mutants ({Delta}ZnJ6) expressing a low level of ZnJ6, suggested that the mutant is more tolerant to oxidative stress. In contrast, the wild type has better protection at elevated temperature and DTT induced stress. We propose that DnaJ-like chaperone ZnJ6 assists in the prevention of protein aggregation, stress endurance, and maintenance of redox balance. One-sentence summaryZnJ6 is a redox-regulated DnaJ-like chaperone associated with the thylakoid membrane and involved in the prevention of protein aggregation and stress endurance.

plant biology↗

bioRxiv2020

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments by stage-differentiation, accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression, since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E candidates, each assumed to have specific functions, although overlaps are expected. It is noted that some of them can bind to a dedicated eIF4G candidate partners, and LeishIF4E2 does not bind any known eIF4G ortholog. LeishIF4E2 was previously shown to comigrate in the polysomal fractions of sucrose gradients, whereas initiation factors usually comigrate with pre-initiation and initiation complexes. Using the CRISPR-Cas9 methodology, we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe alterations in the morphology of mutant cells that turned round and equipped with a very short flagellum, but their growth rate and general translation remained unaffected. Proteomic analysis of the LeishIF4E2(+/-) mutant cells compared to wild type controls showed that the number of proteins that were upregulated exceeded the number of downregulated proteins, possibly indicating that a repressor function was eliminated. The upregulated proteins were related mainly to general metabolic processes, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included several groups, including cytoskeletal and ribosomal proteins. Despite the fact that only one of the two LeishIF4E2 alleles was deleted, the mutant cells were impaired in their ability to infect cultured macrophages. LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Although it could have a repressive function, we cannot exclude the possibility that it is responsible for translation of a specific set of transcripts. Overall, our results are in line with the possibility that the different LeishIF4Es are assigned unique functions. Author summaryLeishmania parasites cause a broad spectrum of diseases that lead to different pathological symptoms. During their life cycle, the parasites shuffle between sand-fly vectors and mammalian hosts, while adapting to changing environments via a stage specific program of gene expression that assists the survival of Leishmania under the changing conditions. Translation initiation plays a key role in control of gene expression, in Leishmania this is exemplified by the presence of multiple cap-binding complexes that interact with mRNAs. The parasites encode multiple paralogs of the cap-binding translation initiation factor eIF4E, and of its corresponding binding partner eIF4G, forming complexes with different potential functions. Using the CRISPR-Cas9 methodology, we generated a heterozygous mutant of the least studied cap-binding paralog, LeishIF4E2, eliminating one of its two alleles. The mutation caused morphological defects, resulting in short and rounded up cells with a significant reduction in their flagellar length. Although general translation rates and growth of the mutant parasite were not affected, total proteome analysis and translation assay suggested that LeishIF4E2 could possibly function as a translation repressor. Alternatively, LeishIF4E-2 could be responsible for promoting translation of a specific set of transcripts.

microbiology↗

bioRxiv2020

CeMbio - The C. elegans microbiome resource

The study of microbiomes by sequencing has revealed a plethora of correlations between microbial community composition and various life-history characteristics of the corresponding host species. However, inferring causation from correlation is often hampered by the sheer compositional complexity of microbiomes, even in simple organisms. Synthetic communities offer an effective approach to infer cause-effect relationships in host-microbiome systems. Yet the available communities suffer from several drawbacks, such as artificial (thus non-natural) choice of microbes, microbe-host mismatch (e.g. human microbes in gnotobiotic mice), or hosts lacking genetic tractability. Here we introduce CeMbio, a simplified natural Caenorhabditis elegans microbiota derived from our previous meta-analysis of the natural microbiome of this nematode. The CeMbio resource is amenable to all strengths of the C. elegans model system, strains included are readily culturable, they all colonize the worm gut individually, and comprise a robust community that distinctly affects nematode life-history. Several tools have additionally been developed for the CeMbio strains, including diagnostic PCR primers, completely sequenced genomes, and metabolic network models. With CeMbio, we provide a versatile resource and toolbox for the in-depth dissection of naturally relevant host-microbiome interactions in C. elegans. Dataset accession numbersWhole genome sequencing data (PRJNA624308); microbiome sequencing [PRJEB37101 and PRJEB37035]; data supplement on the GSA Figshare Portal.

microbiology↗

bioRxiv2019

mir-71 mediates age-dependent opposing contributions of the stress activated kinase KGB-1 in Caenorhabditis elegans

Studying the evolutionary processes that shaped aging offers a path for understanding the causes of aging. The Antagonistic Pleiotropy theory for the evolution of aging proposes that the inverse correlation between natural selection strength and aging allows positive selection of gene variants with early-life beneficial contributions to fitness in spite of detrimental late-life consequences. However, mechanistic understanding of how this principle manifests in aging is still lacking. We previously identified antagonistic pleiotropy in the function of the Caenorhabditis elegans JNK homolog KGB-1, which provided stress protection in developing larvae, but sensitized adults to stress and shortened their lifespan. To a large extent, KGB-1s contributions depended on age-dependent and opposing regulation of the stress transcription factor DAF-16, but the underlying mechanisms remained unknown. Here we describe a role for the microRNA mir-71 in mediating effects of KGB-1 on DAF-16 and on downstream phenotypes. Fluorescent imaging along with genetic and survival analyses revealed age-dependent regulation of mir-71 expression by KGB-1 - upregulation in larvae, but downregulation in adults, and showed that mir-71 was required both for late-life effects of KGB-1 (infection sensitivity and shortened lifespan), as well as for early life resistance to cadmium. While mir-71 disruption did not compromise development under protein folding stress (known to depend on KGB-1), disruption of the argonaute gene alg-1, a central component of the microRNA machinery, did. These results suggest that microRNAs play a role in mediating age-dependent antagonistic contributions of KGB-1 to survival, with mir-71 playing a central role and additional microRNAs contributing redundantly.

genetics↗