Biology subjects

Lu, M.

Publications and source records attributed to Lu, M..

At least 127 records · Page 7Linked to original sources

bioRxiv2021

Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

immunology↗

bioRxiv2021

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and represents a therapeutic target in myelofibrosis

Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-{kappa}B pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

cancer biology↗

bioRxiv2021

Total virome characterizations of game animals in China reveals a spectrum of emerging viral pathogens

Game animals are wildlife species often traded and consumed as exotic food, and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1725 game animals, representing 16 species and five mammalian orders, sampled across China. From this we identified 71 mammalian viruses, with 45 described for the first time. Eighteen viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high risk viruses. We identified the transmission of Bat coronavirus HKU8 from a bat to a civet, as well as cross-species jumps of coronaviruses from bats to hedgehogs and from birds to porcupines. We similarly identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence. HighlightsO_LI1725 game animals from five mammalian orders were surveyed for viruses C_LIO_LI71 mammalian viruses were discovered, 18 with a potential risk to humans C_LIO_LICivets harbored the highest number of potential high risk viruses C_LIO_LIA species jump of an alphacoronavirus from bats to a civet was identified C_LIO_LIH9N2 influenza virus was detected in a civet and an Asian badger C_LIO_LIHumans viruses were also identified in game animals C_LI

zoology↗

bioRxiv2021

A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis

Background and RationaleHepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development. MethodsHB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. ResultsWe found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers. ConclusionWe demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.

cancer biology↗

bioRxiv2021

SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses. Structural studies of soluble S variants have revealed an increased propensity towards conformations accessible to receptor human Angiotensin-Converting Enzyme 2 (hACE2). However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Forster Resonance Energy Transfer (smFRET) studies of S variants containing critical mutations, including D614G and E484K, in the context of virus particles. Investigated variants predominantly occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited decelerated transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

microbiology↗

bioRxiv2021

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and thrombosis

SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1{beta}, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

immunology↗

bioRxiv2021

High-throughput super-resolution single particle trajectory analysis reconstructs organelle dynamics and membrane re-organization

Super-resolution imaging can generate thousands of single-particle trajectories. These data can potentially reconstruct subcellular organization and dynamics, as well as measure disease-linked changes. However, computational methods that can derive quantitative information from such massive datasets are currently lacking. Here we present data analysis and algorithms that are broadly applicable to reveal local binding and trafficking interactions and organization of dynamic sub-cellular sites. We applied this analysis to the endoplasmic reticulum and neuronal membrane. The method is based on spatio-temporal time window segmentation that explores data at multiple levels and detects the architecture and boundaries of high density regions in areas that are hundreds of nanometers. By statistical analysis of a large number of datapoints, the present method allows measurements of nano-region stability. By connecting highly dense regions, we reconstructed the network topology of the ER, as well as molecular flow redistribution, and the local space explored by trajectories. Segmenting trajectories at appropriate scales extracts confined trajectories, allowing quantification of dynamic interactions between lysosomes and the ER. A final step of the method reveals the motion of trajectories relative to the ensemble, allowing reconstruction of dynamics in normal ER and the atlastin-null mutant. Our approach allows users to track previously inaccessible large scale dynamics at high resolution from massive datasets. The SPtsAnalysis algorithm is available as an ImageJ plugin that can be applied by users to large datasets of overlapping trajectories and offer a standard of SPTs metrics.

cell biology↗

bioRxiv2021

Regional Cell Atlas of Human Intestine Shapes Distinct Immune Surveillance

Regional intestinal immune surveillance remains obscure. In this study, we integrated single-cell RNA sequencing and spatial transcriptomics to create a regional atlas of fetal and adult intestines, consisting of 59 cell subsets, of which eight new subsets and ILCs transition states were identified. Results revealed that microenvironment determines in-situ cell differentiation and shapes the regional molecular characteristics, allowing different intestinal segments with diverse functions. We characterized the regional expression of mucins, immunoglobulins, and antimicrobial peptides (AMPs) and their shift during development and in inflammatory bowel disease. Notably, -defensins expressed most abundantly in small intestinal LGR5+ stem cells, rather than in Paneth cells, and down-regulated as cell maturing. Common upstream transcription factors controlled the AMPs expression, illuminating the concurrent change of AMPs during epithelial differentiation, and the spatial co-expression patterns. We demonstrated the correspondence of cell focus of risk genes to diseases location susceptibility and identified distinct cell-cell crosstalk and spatial heterogeneity of immune cell homing in different gut segments. Overall, a cross-spatiotemporal approach to transcriptomes at single-cell resolution revealed that the regional milieu of the human intestine determined cellular and molecular cues of immune surveillance, dictating gut homeostasis and disease.

cell biology↗

bioRxiv2021

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

microbiology↗

bioRxiv2021

A highly conserved lipase deacylates oxidized phospholipids and ameliorates acute lung injury

Oxidized phospholipids have diverse biological activities, many of which can be pathological, yet how they are inactivated in vivo is not fully understood. Here we present evidence that a previously unsuspected host lipase, acyloxyacyl hydrolase (AOAH), can play a significant role in reducing the pro-inflammatory activities of two prominent products of phospholipid oxidation, 1-palmitoyl-2-glutaryl-sn-glycero-3- phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). AOAH removed the sn-2 and sn-1 acyl chains from both lipids and reduced their ability to induce macrophage inflammasome activation and cell death in vitro and acute lung injury in vivo. In addition to transforming Gram-negative bacterial lipopolysaccharide from stimulus to inhibitor, its most studied activity, AOAH can inactivate these important danger-associated molecular pattern (DAMP) molecules and reduce tissue inflammation and cell death.

immunology↗

bioRxiv2021

Dynamics of glioma-associated microglia and macrophages reveals their divergent roles in the immune response of brain

Glioma microenvironment contains numerous myeloid cells, including brain-resident microglia and recruited monocytes and macrophages (Mo/M{varphi}). When studied collectively, these cells presented pro-tumor effects. Yet, little is known about the differences among these myeloid populations. Using single-cell sequencing analysis, we studied the phenotypic characteristics, spatial variances, and dynamic changes of these relatively heterogeneous cell populations. Microglia populations with distinct spatial distribution presented different functional states, including tumor-associated subsets with phagocytic and lipid metabolism signature. Notably, this subset of glioma-associated microglia shared similar trait in a diverse spectrum of neuropathogenesis. In contrast, Mo/M{varphi} highly expressed genes related to angiogenesis, tumor invasion, and immune evasion. Moreover, identifying the Mo/M{varphi} subsets had prognostic and classificatory value in clinical application. These results thus eliminate the long-existing ambiguity about the role of microglia and Mo/M{varphi} in glioma pathogenesis, and reveal their prognostic and therapeutic value for glioma patients.

immunology↗

bioRxiv2021

Novel Interpretable Tissue-Specific and Multi-Tissue Transcriptomic Clocks to Infer Aging Mechanisms

1.Aging is characterized as a progressive decline in fitness that ultimately results in death. We set out to build both tissue-specific and multi-tissue transcriptomic clocks to make global tissue age predictions in individuals from GTEx. Existing work in the field primarily uses epigenetic clocks as predictors of age, but these models have known issues and are significantly less interpretable than their transcriptomic counterparts. Due to their transcriptomic nature, we can use these models to directly infer mechanisms of aging from their features. Linear regression remains the current standard analysis technique, but we improved upon its baseline performance with modern techniques, exploring both XGBoost and MLPs. We also experimented with using deconvolved cell data for predictions, which account for cellular composition and reduce signal distortion from rare cell types. Since it is known that the heterogeneity of cell types in particular tissues can lead to noise in these models, we proposed using deconvolution as a potential remedy for this problem. Our results found that MLPs are not well suited for the task due to a lack of training data, but the use of XGBoost is effective at improving the baseline performance of predictions of existing tissue-specific clocks. These models allowed us to directly compute genes most important to age prediction in our models, and we showed that multiple genes found have been independently identified elsewhere to show evidence of correlation with age. Given the small size of our datasets, we were unable to make conclusive determinations about multi-tissue predictors, but preliminary results suggest that the technique shows promise and is worthy of future investigation. Likewise, given our limited deconvolved cell data, we did not currently observe strong results, but we again note that this is an area in need of further investigation. By improving upon the performance of existing models, we demonstrated that a novel machine learning technique, XGBoost, can be an effective technique to further our understanding of aging mechanisms by extraction of the most relevant genes found in those models. This is significant because the genetic causes of aging are still not fully understood, and research in the field of aging is lacking in comparison to other domains. As the problem of identifying tissues that age at different rates is of specific interest, our tissue-specific models potentially have other applications in this domain, including informing pathologies in tissues that are found to be aging faster, or analyzing how people with similar ages can have vastly different tissue ages. An extended technical presentation of this work can be found here, and a highly simplified non-technical overview presentation can be found here.

genomics↗

bioRxiv2021

Asymmetric structures and conformational plasticity of the uncleaved full-length human immunodeficiency virus (HIV-1) envelope glycoprotein trimer

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, "closed" (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCEThe envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.

molecular biology↗

bioRxiv2021

Occurrence of COVID-19 symptoms during SARS-CoV-2 infection defines waning of humoral immunity

Approximately half of the SARS-CoV-2 infections occur without apparent symptoms, raising questions regarding long-term humoral immunity in asymptomatic individuals. Plasma levels of immunoglobulin G (IgG) and M (IgM) against the viral spike or nucleoprotein were determined for 25,091 individuals enrolled in a surveillance program in Wuhan, China. We compared 405 asymptomatic individuals with 459 symptomatic COVID-19 patients. The well-defined duration of the SARS-CoV-2 endemic in Wuhan allowed a side-by-side comparison of antibody responses following symptomatic and asymptomatic infections without subsequent antigen re-exposure. IgM responses rapidly declined in both groups. However, both the prevalence and durability of IgG responses and neutralizing capacities correlated positively with symptoms. Regardless of sex, age, and body weight, asymptomatic individuals lost their SARS-CoV-2-specific IgG antibodies more often and rapidly than symptomatic patients. These findings have important implications for immunity and favour immunization programs including individuals after asymptomatic infections. One-Sentence SummaryPrevalence and durability of SARS-CoV-2-specific IgG responses and neutralizing capacities correlate with COVID-19 symptoms.

immunology↗

bioRxiv2021

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

immunology↗

bioRxiv2021

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.

immunology↗

bioRxiv2021

Complex response of beta diversity to dispersal in meta-community models

Dispersal is one of the most important drivers of community assembly. The conventional belief that dispersal leads to biotic homogenization (lower beta diversity) has been recently challenged by an experiment conducted in nectar microbes (Vannette & Fukami, 2017), showing that dispersal could lead to community divergence. In this paper, I re-examined the relationship between beta diversity and local dispersal in a range of theoretical models: from the classic island biogeography model and meta-population model to a meta-community model that incorporates biotic interactions. I find that the emergence of hump-shaped beta diversity-dispersal relationship is closely related to local dispersal (rather than global dispersal), non-neutrality and biotic interactions. The results reveal rich metacommunity dynamics in relation to dispersal types and biotic interactions which might be overlooked in previous theoretical and empirical studies. The findings call for more realistic experimental manipulations on dispersals in future community assembly studies.

ecology↗

bioRxiv2021

Metagenomic screening for lipolytic genes reveals an ecology-clustered distribution pattern

Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique genes encoding lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integral classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.

microbiology↗