Zhang F et al. (2017) Front Microbiol "Caenorhabditis elegans as a Model for Microbiome Research."

Schulenburg H, Zhang F, Felix MA, Dierking K, Berg M, Shapira M, Samuel BS

[Front Microbiol, 2017]

The nematode Caenorhabditis elegans is used as a central model system across biological disciplines. Surprisingly, almost all research with this worm is performed in the absence of its native microbiome, possibly affecting generality of the obtained results. In fact, the C. elegans microbiome had been unknown until recently. This review brings together results from the first three studies on C. elegans microbiomes, all published in 2016. Meta-analysis of the data demonstrates a considerable conservation in the composition of the microbial communities, despite the distinct geographical sample origins, study approaches, labs involved and perturbations during worm processing. The C. elegans microbiome is enriched and in some cases selective for distinct phylotypes compared to corresponding substrate samples (e.g., rotting fruits, decomposing plant matter, and compost soil). The dominant bacterial groups include several Gammaproteobacteria (Enterobacteriaceae, Pseudomonaceae, and Xanthomonodaceae) and Bacteroidetes (Sphingobacteriaceae, Weeksellaceae, Flavobacteriaceae). They are consistently joined by several rare putative keystone taxa like Acetobacteriaceae. The bacteria are able to enhance growth of nematode populations, as well as resistance to biotic and abiotic stressors, including high/low temperatures, osmotic stress, and pathogenic bacteria and fungi. The associated microbes thus appear to display a variety of effects beneficial for the worm. The characteristics of these effects, their relevance for C. elegans fitness, the presence of specific co-adaptations between microbiome members and the worm, and the molecular underpinnings of microbiome-host interactions represent promising areas of future research, for which the advantages of C. elegans as an experimental system should prove of particular value.

Zhang F et al. (2007) Nature "Multimodal fast optical interrogation of neural circuitry."

Gottschalk A, Liewald JF, Deisseroth K, Zhang F, Wood PG, Bamberg E, Wang LP, Watzke N, Brauner M, Nagel G, Kay K

[Nature, 2007]

Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Zhang F et al. (2023) Front Microbiol "Insights into the genetic influences of the microbiota on the life span of a host."

Hu Y, Zhu Z, Shi H, Wang L, Fang Z, Du Y, Wang H, Zhang F, Jin J, Zhang Y, Pang Y, Ding X

[Front Microbiol, 2023]

<i>Escherichia coli</i> (<i>E. coli</i>) mutant strains have been reported to extend the life span of <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). However, the specific mechanisms through which the genes and pathways affect aging are not yet clear. In this study, we fed <i>Drosophila melanogaster</i> (fruit fly) various <i>E. coli</i> single-gene knockout strains to screen mutant strains with an extended lifespan. The results showed that <i>D. melanogaster</i> fed with <i>E. coli purE</i> had the longest mean lifespan, which was verified by <i>C. elegans</i>. We conducted RNA-sequencing and analysis of <i>C. elegans</i> fed with <i>E. coli purE</i> (a single-gene knockout mutant) to further explore the underlying molecular mechanism. We used differential gene expression (DGE) analysis, enrichment analysis, and gene set enrichment analysis (GSEA) to screen vital genes and modules with significant changes in overall expression. Our results suggest that <i>E. coli</i> mutant strains may affect the host lifespan by regulating the protein synthesis rate (<i>cfz-2</i>) and ATP level (<i>catp-4</i>). To conclude, our study could provide new insights into the genetic influences of the microbiota on the life span of a host and a basis for developing anti-aging probiotics and drugs.

Zhang, F. et al. (2019) International Worm Meeting "Insulin signaling drives Caenorhabditis elegans microbiome acquisition."

Felix, MA., Weckhorst, J., Ayoub, C., Samuel, B., Vidal, D., Assie, A., Zhang, F.

[International Worm Meeting, 2019]

Host genetic landscapes shape microbiome acquisition in the animal gut. To identify specific signaling pathways involved, we employed Caenorhabditis elegans and its native microbiome to model these host-microbiome relationships. We tested this by exposing a diverse panel of 38 C. elegans wild strains to the same model microbiome (BIGbiome) to examine natural variation in microbiome acquisition. By day 3 of adulthood the 38 strains could be clustered into three distinct gut microbiome types: Type 1 [8/38; 74%], Ochrobactrum-dominant and low colonization levels; Type 2 [N2 and 4 others], Bacteroidetes-dominant and high colonization levels; and type 3, low colonization levels with microbiomes similar to the lawn. Type 1 strains also exhibit faster growth rates than the others. C. elegans appears to regulate its microbiome by deterministically selecting the microbes that colonize the gut and/or their levels within the gut, which can impact its overall physiology. Using previously generated genomes of these 38 C. elegans strains, we sought to identify the genetic drivers of these gut types. By GWAS, we identified a strong QTL [-log(p-value)= 9.32] in Type 2 strains with genes that are often misregulated in daf-2/IGFR insulin receptor mutants (35%) and/or respond to xenobiotics (23%). Further, RNAseq analyses of Type 1 strains showed higher insulin ligand and xenobiotic response gene expression than the other types. Expression of many host genes [2344] also strongly correlated with specific gut bacterial abundance-e.g., insulin signaling negatively correlated with Ochrobactrum levels. Tests of genetic mutants in insulin signaling support their roles in modulating host resistance to microbiome colonization, as microbiome colonization is lower in daf-2 mutant than N2 controls. This is only partially suppressed in daf-2;daf-16 animals, suggesting other downstream transcriptional factors contribute to colonization resistance in N2. However, daf-2(RNAi) of Type 1 strains does not influence overall colonization levels, but does alter Ochrobactrum colonization specifically. Thus, insulin signaling may play a more specific role in shaping the microbiome in wild strains rather than the global colonization resistance observed in these and other N2 studies. Together, our study illustrates how host genetic variation contributes to distinct gut types in C. elegans, and establishes a platform for comprehensive identification of the determinants of host microbiome management.

Zhang F et al. (2020) Methods Mol Biol "High-Throughput Assessment of Changes in the Caenorhabditis elegans Gut Microbiome."

Vidal D, Loeza-Cabrera M, Weckhorst JL, Khodakova AS, Assie A, Samuel BS, Zhang F

[Methods Mol Biol, 2020]

The gut microbiome is animportant driver of host physiology and development. Altered abundance or membership of this microbe community can influence host health and disease progression, including the determination of host lifespan and healthspan. Here, we describe a robust pipeline to measure microbiome abundance and composition in the C. elegans gut that can be applied to examine the role of the microbiome on host aging or other physiologicprocesses.

Zhang, F. et al. (2017) International Worm Meeting "Dissecting the foundations of commensalism using C. elegans and its natural microbiome."

Weckhorst, J.L, Zhang, F., Ayoub, C.A, Felix, M.A., Samuel, B.S.

[International Worm Meeting, 2017]

Partnerships between animals and microbes are ancient and coevolved relationships are common and born out of mutual benefit. Proper establishment and maintenance of these relationships relies on strong lines of host-microbial communication. Our goal is to develop a comprehensive understanding of the host-microbial signaling pathways that regulate these processes. To do this, we employ the genetically tractable, high-throughput amenable and microbially 'tuned' nematode C. elegans as it harbors a simple community of microbes in the wild (its 'microbiome). Through collaborative meta-analyses, we defined its core microbiome that is largely distinct from that of corresponding substrates- Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Sphingomonadaceae, three Bacteroidetes families and several less numerous families. To comprehensively examine host selection of its microbiome, we defined a community of 68 bacterial isolates with >98% identify to meta-analyses from our collection [>550 microbes] and fed this model microbiome to wild C. elegans strains (42) for comparison. Using a high-throughput gut colonization method that we developed, we observed host strain specific colonization levels over a 30 fold range with N2 at an extreme. As in the wild, sequencing of animals over time demonstrated strong host role in selecting its microbiome distinct from the lawns. This process appears to be deterministic, as specific wild C. elegans strains are highly adapted to manage levels their native microbes- i.e., C. elegans JU1218 (an apple worm) is resistant to the deleterious effects of its native Bacteroidetes (Chryseobacterium sp. JUb44) and maintained lower levels of colonization. To dissect the genetics of these adaptations, we generated and screened an RNAi sub-library of 364 genes (based on functional SNPs between JU1218 and N2) for clones that improved the overall health and/or reduced colonization levels of the lab strain of C. elegans (N2) when challenged with the Chryseobacterium. The majority of the RNAi clones further enhanced JU1218 resistance, suggesting partial loss of functions in the wild strain, but also identified 26 promising candidates in signaling pathways (e.g., a GPCR and several transcription factors) influence Chryseobacterium impact on N2. Thus, through these and additional studies, we hope to leverage C. elegans as a robust natural genetic system to catalogue the pathways that are important managing microbiome form and function.

Zhang F et al. (2023) J Vis Exp "Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome."

Zhang F, Assie A, Samuel BS, Hosea CN, Blackburn D

[J Vis Exp, 2023]

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

Zhang F et al. (2018) Org Lett "Biemamides A-E, Inhibitors of the TGF- Pathway That Block the Epithelial to Mesenchymal Transition."

Ananiev GE, Hoffmann FM, Bugni TS, Rajski SR, Zhang F, Tsai IW, Braun DR

[Org Lett, 2018]

Screening of a marine natural products library for inhibitors of TGF- revealed five pyrimidinedione derivatives, biemamides A-E (1-5). The structures were determined by 2D NMR and HRMS experiments; absolute configurations were established by advanced Marfey's analysis and ECD calculations. Biemamides A-E specifically inhibited in vitro TGF- induced epithelial to mesenchymal transition in NMuMG cells. Additionally, using Caenorhabditis elegans, selected biemmamides were found to influence in vivo developmental processes related to body size regulation in a dose-dependent manner.

Zhang F et al. (2003) Genes Dev "A model of repression: CTD analogs and PIE-1 inhibit transcriptional elongation by P-TEFb."

Zhang F, Barboric M, Peterlin BM, Blackwell TK

[Genes Dev, 2003]

The positive transcription elongation factor b (P-TEFb) contains cyclin T1 (CycT1) and cyclin-dependent kinase 9 (Cdk9). For activating the expression of eukaryotic genes, the histidine-rich sequence in CycT1 binds the heptapeptide repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII), whereupon Cdk9 phosphorylates the CTD. We found that alanine-substituted heptapeptide repeats that cannot be phosphorylated also bind CycT1. When placed near transcription units, these CTD analogs block effects of P-TEFb. Remarkably, the transcriptional repressor PIE-1 from Caenorhabditis elegans behaves analogously. It binds CycT1 via an alanine-containing heptapeptide repeat and inhibits transcriptional elongation. Thus, our findings reveal a new mechanism by which repressors inhibit eukaryotic transcription.

Zhang F et al. (2021) Curr Biol "Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut."

Assie A, Weckhorst JL, Felix MA, Zhang F, Khodakova AS, Hosea C, Ayoub CA, Cabrera ML, Vidal Vilchis D, Samuel BS

[Curr Biol, 2021]

Host genetic landscapes can shape microbiome assembly in the animal gut by contributing to the establishment of distinct physiological environments. However, the genetic determinants contributing to the stability and variation of these microbiome types remain largely undefined. Here, we use the free-living nematode Caenorhabditis elegans to identify natural genetic variation among wild strains of C.elegans that drives assembly of distinct microbiomes. To achieve this, we first established a diverse model microbiome that represents the strain-level phylogenetic diversity naturally encountered by C.elegans in the wild. Using this community, we show that C.elegans utilizes immune, xenobiotic, and metabolic signaling pathways to favor the assembly of different microbiome types. Variations in these pathways were associated with enrichment for specific commensals, including the Alphaproteobacteria Ochrobactrum. Using RNAi and mutant strains, we showed that host selection for Ochrobactrum is mediated specifically by host insulin signaling pathways. Ochrobactrum recruitment is blunted in the absence of DAF-2/IGFR and modulated by the competitive action of insulin signaling transcription factors DAF-16/FOXO and PQM-1/SALL2. Further, the ability of C.elegans to enrich for Ochrobactrum as adults is correlated with faster animal growth rates and larger body size at the end of development. These results highlight a new role for the highly conserved insulin signaling pathways in the regulation of gut microbiome composition in C.elegans.