Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2018

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass

BackgroundsPyricularia is a multispecies complex that could infect and cause severe blast disease on diverse hosts, including rice, wheat and many other grasses. Although the genome size of this fungal complex is small [~40 Mbp for Pyricularia oryzae (syn. Magnaporthe oryzae), and ~45 Mbp for P. grisea], the genome plasticity allows the fungus to jump and adapt to new hosts. Therefore, deciphering the genome basis of individual species could facilitate the evolutionary and genetic study of this fungus. However, except for the P. oryzae subgroup, many other species isolated from diverse hosts, such as the Pennisetum grasses, remain largely uncovered genetically.\n\nResultsHere, we report the genome sequence of a pyriform-shaped fungal strain P. penniseti P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. We performed a phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species and addressed P1609 into a Pyricularia subclade that is distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires were fairly different between P1609 and the P. oryzae strain 70-15, including the cloned avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements.\n\nConclusionTaken together, our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species.

genomics↗

bioRxiv2018

Population dynamics and transcriptomic responses of Pseudomonas aeruginosa in a complex laboratory microbial community

Pseudomonas aeruginosa is one of the dominant species when it co-exists with many other bacterial species in diverse environments. To understand its physiology and interactions with co-existing bacterial species in different conditions, we established physiologically reproducible eighteen-species communities, and found that P. aeruginosa became the dominant species in mixed-species biofilm community but not in the planktonic community. P. aeruginosa H1 type VI secretion system was highly induced in the mixed-species biofilm community compare to its mono-species biofilm, which was further demonstrated to play a key role for P. aeruginosa to gain fitness over other bacterial species. In addition, the type IV pili and Psl exopolysaccharide were shown to be required for P. aeruginosa to compete with other bacterial species in the biofilm community. Our study showed that the physiology of P. aeruginosa is strongly affected by interspecies interactions, and both biofilm determinants and H1 type VI secretion system contribute to P. aeruginosa fitness over other species in complex biofilm communities.\n\nImportancePseudomonas aeruginosa usually coexists with different bacterial species in natural environment. However, systematic comparative characterization of P. aeruginosa in complex microbial communities with its mono-species communities is lacking. We constructed mixed-species planktonic and biofilm communities consisting P. aeruginosa and seventeen other bacterial species to study the physiology and interaction of P. aeruginosa in complex multiple-species community. A single molecule detection platform, NanoString nCounter(R) 16S rRNA array, was used to shown that P. aeruginosa can become the dominant species in the biofilm communities while not in the planktonic communities. Comparative transcriptomic analysis and fluorescence-based quantification further revealed that P. aeruginosa H1 type VI secretion system and biofilm determinants are both required for its fitness in mixed-species biofilm communities.

microbiology↗

bioRxiv2018

Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome and controls the abundance of key regulators of cellular function by selective protein degradation, but how foldingdefective proteins in the secretory system are selected from the large and diverse constellation of membrane and secretory proteins and efficiently delivered to proteasomes in the cytosol is not well understood. To determine the basis of substrate selectivity in human cells, we developed a transcriptional shut off approach to conduct parallel, unbiased, genome-wide CRISPR analysis of structurally and topologically diverse ER-associated degradation (ERAD) clients. Highly quantitative screen metrics allowed precise dissection of entire pathways, enabling identification of unique substrate-specific combinations of recognition and ubiquitin conjugation modules. Our analysis identified cytosolic ubiquitin conjugating machinery that has not been previously linked to ERAD but collaborates with membrane-integrated ubiquitin ligases to conjugate branched or mixed ubiquitin chains to promote efficient and processive substrate degradation.

cell biology↗

bioRxiv2018

ALV-J and REV synergistically activate a new oncogene of KIAA1199 via NF-κB and EGFR signaling regulated by miR-147

The tumorigenesis is the result of the accumulation of multiple oncogenes and tumor suppressor genes changes. Co-infection of avian leucosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV), as two oncogenic retroviruses, showed synergistic pathogenic effects characterized by enhanced tumor initiation and progression. The molecular mechanism underlying synergistic effects of ALV-J and REV on the neoplasia remains unclear. Here, we found co-infection of ALV-J and REV enhanced the ability of virus infection, increased viral life cycle, maintained cell survival and enhanced tumor formation. We combined the high-throughput proteomic readout with a large-scale miRNA screening to identify which molecules are involved in the synergism. Our results revealed co-infection of ALV-J and REV activated a latent oncogene of KIAA1199 and inhibited the expression of tumor suppressor miR-147. Further, enhanced KIAA1199, down-regulated miR-147, activated NF-{kappa}B and EGFR were demonstrated in co-infected tissues and tumor. Mechanistically, we showed ALV-J and REV synergistically enhanced KIAA1199 by activation of NF-{kappa}B and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 was contributed to maintain the NF-{kappa}B/KIAA1199/EGFR pathway crosstalk by targeting the 3UTR region sequences of NF-{kappa}B p50 and KIAA1199. Our results contributed to the understanding of the molecular mechanisms of viral synergistic tumorgenesis, which provided the evidence that suggested the synergistic actions of two retroviruses could result in activation of latent pro-oncogenes.\n\nAuthor summaryThe tumorigenesis is the result of the accumulation of multiple oncogenes and tumor suppressor genes changes. Co-infection with ALV-J and REV showed synergistic pathogenic effects characterized by enhanced tumor progression, however, the molecular mechanism on the neoplasia remains unclear. Our results revealed co-infection of ALV-J and REV promotes tumorigenesis by both induction of a latent oncogene of KIAA1199 and suppression of the expression of tumor suppressor miR-147. Mechanistic studies revealed that ALV-J and REV synergistically enhance KIAA1199 by activation of NF-{kappa}B and EGFR signalling pathway, and the suppression of tumor suppressor miR-147 was contributed to maintain the NF-{kappa}B/KIAA1199/EGFR pathway crosstalk by targeting the 3UTR region sequences of NF-{kappa}B p50 and KIAA1199. These results provided the evidence that suggested the synergistic actions of two retroviruses could result in activation of latent pro-oncogenes, indicating the potential preventive target and predictive factor for ALV-J and REV induced tumorigenesis.

molecular biology↗

bioRxiv2018

Inductive reasoning differs between taxonomic and thematic contexts: Electrophysiological evidence

Inductive reasoning can be performed in different contexts, but it is unclear whether the neural mechanism of inductive reasoning performed in a thematic context (e.g., panda has x, so bamboo has x) is the same as that performed in a taxonomic context (e.g., panda has x, so bear has x). In the present study, participants were required to judge whether a conclusion was acceptable or not based on its premise, for which the taxonomic or thematic distances between premise and conclusion objects were either far or near. The ERP results indicated that the effect of reasoning context (taxonomic vs. thematic) was initially observed in the P2 component; while the distance effect (far vs. near) was observed in N400 and late components. Moreover, the distance effect on thematic-based inductive reasoning was found in the frontal and frontal-central brain regions, while the distance effect in taxonomic-based inductive reasoning conditions was found in the central-parietal and parietal regions. These results support the view that inductive reasoning is performed differently under different semantic contexts.

neuroscience↗

bioRxiv2018

NOVEL INSIGHTS INTO SELECTION FOR ANTIBIOTIC RESISTANCE IN COMPLEX MICROBIAL COMMUNITIES

Recent research has demonstrated selection for antibiotic resistance occurs at very low antibiotic concentrations in single species experiments, but the relevance of these findings when species are embedded in complex microbial communities is unclear. We show the strength of selection for naturally occurring resistance alleles in a complex community remains constant from low sub-inhibitory to above clinically relevant concentrations. Selection increases with antibiotic concentration before reaching a plateau where selection remains constant over a two order magnitude concentration range. This is likely to be due to cross-protection of the susceptible bacteria in the community following rapid extracellular antibiotic degradation by the resistant population, shown experimentally through a combination of chemical quantification and bacterial growth experiments. Metagenome and 16S rRNA analyses on sewage-derived bacterial communities evolved under cefotaxime exposure show preferential enrichment for blaCTX-M genes over all other beta-lactamase genes, as well as positive selection and co-selection for antibiotic resistant, opportunistic pathogens. These findings have far reaching implications for our understanding of the evolution of antibiotic resistance, by challenging the long-standing assumption that selection occurs in a dose-dependent manner.

microbiology↗

bioRxiv2018

Localization and protein-protein interaction of protein kinase CK2 suggest a chaperone-like activity is integral to its function in M. oryzae.

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures as septa and appressorial pores.

cell biology↗

bioRxiv2018

Deciphering the metabolic perturbation in hepatic alveolar echinococcosis: a 1H NMR-based metabolomics study

Hepatic alveolar echinococcosis (HAE) is a chronic and potentially lethal parasitic disease. It is caused by growth of Echinococcus multilocularis larvae in liver. To date, early-stage diagnosis for the disease is not mature due to its long asymptomatic incubation period. In this study, a proton nuclear magnetic resonance (1H NMR) -based metabolomics approach was applied in conjunction with multivariate statistical analysis to investigate the altered metabolic profiles in blood serum and urine samples from HAE patients and to identify characteristic metabolic markers associated with HAE. The current results identified 21 distinctive metabolic difference between the HAE patients and healthy individuals, which can be associated with perturbations in energy metabolism, amino acid metabolism, oxidative stress, and neurotransmitter imbalance. In addition, the Fischer ratio, which is the molar ratio of branched-chain amino acids to aromatic amino acids was found significantly lower (p<0.001) in blood serum from HAE patients. The ratio, together with changes in other metabolic pathways may provide new insight into mechanistic understanding of HAE pathogenesis, and may be useful for early-stage HAE diagnosis.\n\nAuthor SummaryHepatic alveolar echinococcosis (HAE) is a life-threatening disease caused by Echinococcus multilocularis infection. The disease has a long asymptomatic early stage (5~15 years), which complicates effective diagnosis of early-stage HAE even with advanced imaging techniques. Metabolomics is an emerging analytical platform that comprises of analysis of all small molecule metabolites that are present within an organism. The applications of metabolomics method on HAE may help to reveal the molecular biology mechanisms of HAE. In the current study, we had used 1H NMR-based metabolomics technique to investigate blood serum and urine samples from HAE patients. Altered metabolic responses and characteristic differential metabolites for HAE were identified. The metabolic profiling of human biofluids provided valuable information for early-stage HAE diagnosis and for therapeutic interventions, without having to extract HAE vesicles from patients. By featuring global and comprehensive metabolic status, the metabolomics approach holds considerable promise as a noninvasive, dynamic, and effective tool for probing the underlying mechanism of HAE.

systems biology↗

bioRxiv2018

Parallel Evolution of Key Genomic Features and Cellular Bioenergetics Across the Marine Radiation of a Bacterial Phylum

Diverse bacterial and archaeal lineages drive biogeochemical cycles in the global ocean, but the evolutionary processes that have shaped their genomic properties and physiological capabilities remain obscure. Here we track the genome evolution of the globally-abundant marine bacterial phylum Marinimicrobia across its diversification into modern marine environments and demonstrate that extant lineages have repeatedly switched between epipelagic and mesopelagic habitats. Moreover, we show that these habitat transitions have been accompanied by repeated and fundamental shifts in genomic organization, cellular bioenergetics, and metabolic modalities. Lineages present in epipelagic niches independently acquired genes necessary for phototrophy and environmental stress mitigation, and their genomes convergently evolved key features associated with genome streamlining. Conversely, lineages residing in mesopelagic waters independently acquired nitrate respiratory machinery and a variety of cytochromes, consistent with the use of alternative terminal electron acceptors in oxygen minimum zones (OMZs). Further, while surface water clades have retained an ancestral Na+-pumping respiratory complex, deep water lineages have largely replaced this complex with a canonical H+-pumping respiratory complex I, potentially due to the increased efficiency of the latter together with more energy-limiting environments deep in the oceans interior. These parallel evolutionary trends across disparate clades suggest that the evolution of key features of genomic organization and cellular bioenergetics in abundant marine lineages may in some ways be predictable and driven largely by environmental conditions and nutrient dynamics.

microbiology↗

bioRxiv2018

TSA-Seq Mapping of Nuclear Genome Organization

While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription \"hot-zones\". Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

genomics↗

bioRxiv2018

Early candidate biomarkers in urine of Walker-256 lung metastasis rat model.

Cancer metastasis accounts for the majority of deaths by cancer. Detection of cancer metastasis at its early stage is important for the management and prediction of cancer progression. Urine, which is not regulated by homeostatic mechanisms, reflects systemic changes in the whole body and can potentially be used for the early detection of cancer metastasis. In this study, a lung metastasis of a Walker-256 rat model was established by tail-vein injection of Walker-256 cells. Urine samples were collected at days 2, 4, 6 and 9 after injection, and the urinary proteomes were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The urinary protein patterns changed significantly with the development of Walker-256 lung metastasis. On the fourth day, lung metastasis nodules appeared. On the sixth day, clinical symptoms started. On days 2, 4, 6 and 9, 11, 25, 34 and 44 differential proteins were identified in 7 lung metastatic rats by LC-MS/MS. Seventeen of these 62 differential proteins were identified on the second day, and 18 of them were identified on the fourth day. The differential urinary proteins changed significantly two days before lung metastasis nodules appeared. Differential urinary proteins differed in Walker-256 lung metastasis rat models and Walker-256 subcutaneous rat models. A total of 9 differential proteins (NHRF1, CLIC1, EZRI, AMPN, ACY1A, HSP7C, BTD, NID2, and CFAD) were identified in 7 lung metastatic rats at one or more common time points, and these 9 differential proteins were not identified in the subcutaneous rat model. Seven of these 9 differential proteins were associated with both breast cancer and lung cancer, eight of the nine were identified on the second day, and 8 of the nine can be identified on the fourth day; these early changes in urine were also identified with differential abundances at late stages of lung metastasis. Our results indicate that (1) the urine proteome changed significantly, even on the second day after tail-vein injection of Walker-256 cells and that (2) the urinary differential proteins were different in Walker-256 lung metastatic tumors and Walker-256 subcutaneous tumors. Our results provide the potential to detect early breast cancer lung metastasis, monitor its progression and differentiate it from the same cancer cells grown at other locations.

biochemistry↗

bioRxiv2018

Expression levels of glycoprotein O (gO) vary between strains of human cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity.

Tropism of human cytomegalovirus (HCMV) is influenced by the envelope glycoprotein complexes gH/gL/gO and gH/gL/UL128-131. During virion assembly, gO and the UL128-131 proteins compete for binding to gH/gL in the ER. This assembly process clearly differs among strains since Merlin (ME) virions contain abundant gH/gL/UL128-131 and little gH/gL/gO, whereas TR contains much higher levels of total gH/gL, mostly in the form of gH/gL/gO, but much less gH/gL/UL128-131 than ME. Remaining questions include 1) what are the mechanisms behind these assembly differences, and 2) do differences reflect in vitro culture adaptations or natural genetic variations? Since the UL74(gO) ORF differs by 25% of amino acids between TR and ME, we analyzed recombinant viruses in which the UL74(gO) ORF was swapped. TR virions were >40-fold more infectious than ME. Transcriptional repression of UL128-131 enhanced infectivity of ME to the level of TR, despite still far lower levels of gH/gL/gO. Swapping the UL74(gO) ORF had no effect on either TR or ME. A quantitative immunoprecipitation approach revealed that gH/gL expression was within 4-fold between TR and ME, but gO expression was 20-fold less by ME, and suggested differences in mRNA transcription, translation or rapid ER-associated degradation of gO. Trans-complementation of gO expression during ME replication gave 6-fold enhancement of infectivity beyond the 40-fold effect of UL128-131 repression alone. Overall, strain variations in assembly of gH/gL complexes result from differences in expression of gO and UL128-131, and selective advantages for reduced UL128-131 expression during fibroblast propagation are much stronger than for higher gO expression.\n\nIMPORTANCESpecific genetic differences between independently isolated HCMV strains may result from purifying selection on de novo mutations arising during propagation in culture, or random sampling among the diversity of genotypes present in clinical specimens. Results presented indicate that while reduced UL128-131 expression may confer a powerful selective advantage during cell-free propagation of HCMV in fibroblast cultures, selective pressures for increased gO expression are much weaker. Thus, variation in gO expression among independent strains may represent natural genotype variability present in vivo. This may have important implications for virus-host interactions such as immune recognition, and underscores the value of studying molecular mechanisms of replication using multiple HCMV strains.

microbiology↗

bioRxiv2018

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Herein, high-throughput screening of an FDA-approved drug library was performed against LASV entry using a pseudo-type virus enveloping LASV glycoproteins. Two hit drugs, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both drugs inhibited LASV entry by blocking low-pH-induced membrane fusion. Moreover, lacidipine irreversibly bound to the LASV glycoprotein complex (GPC), resulting in virucidal activity. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine without apparent loss of the viral growth profile. Furthermore, lacidipine showed antiviral activity and specificity against both LASV and the Guanarito virus (GTOV), which is also a category A new world arenavirus. Drug-resistant variants indicate that the V36M in ectodomain of SSP mutant and V436A in the transmembrane domain of GP2 mutant conferred GTOV resistance to lacidipine, suggesting that lacidipine might act via a novel mechanism other than calcium inhibition. This study shows that both lacidipine and phenothrin are candidates for LASV therapy, and the membrane-proximal external region of the GPC might provide an entry-targeted platform for inhibitors.

microbiology↗

bioRxiv2018

Towards automation of germline variant curation inclinical cancer genetics

Cancer care professionals are confronted with interpreting results from multiplexed gene sequencing of patients at hereditary risk for cancer. Assessments for variant classification now require orthogonal data searches, requiring aggregation of multiple lines of evidence from diverse resources. The burden of evidence for each variant to meet thresholds for pathogenicity or actionability now poses a growing challenge for those seeking to counsel patients and families following germline genetic testing. A computational algorithm that automates, provides uniformity and significantly accelerates this interpretive process is needed. The tool described here, Pathogenicity of Mutation Analyzer (PathoMAN) automates germline genomic variant curation from clinical sequencing based on ACMG guidelines. PathoMAN aggregates multiple tracks of genomic, protein and disease specific information from public sources. We compared expert manually curated variant data from studies on (i) prostate cancer (ii) breast cancer and (iii) ClinVar to assess performance. PathoMAN achieves high concordance (83.1% pathogenic, 75.5% benign) and negligible discordance (0.04% pathogenic, 0.9% benign) when contrasted against expert curation. Some loss of resolution (8.6% pathogenic, 23.64% benign) and gain of resolution (6.6% pathogenic, 1.6% benign) was also observed. We highlight the advantages and weaknesses related to the programmable automation of variant classification. We also propose a new nosology for the five ACMG classes to facilitate more accurate reporting to ClinVar. The proposed refinements will enhance utility of ClinVar to allow further automation in cancer genetics. PathoMAN will reduce the manual workload of domain level experts. It provides a substantial advance in rapid classification of genetic variants by generating robust models using a knowledge-base of diverse genetic data https://pathoman.mskcc.org.

genetics↗

bioRxiv2018

Knockdown of GAS5 restores ox-LDL-induced impaired autophagy flux via upregulating miR-26a in human endothelial cells

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced endothelial cell (EC) injury and autophagy dysfunction play a vital role in the development of atherosclerosis. LncRNAs have been identified to participate in the regulation of pathogenesis of atherosclerosis. However, it remains largely undefined whether growth-arrest specific transcript 5 (GAS5) could influence ox-LDL-induced autophagy dysfunction in ECs.\n\nMethodsThe expressions of GAS5 and miR-26a in the plasma samples of patients with atherosclerosis and ox-LDL-treated human aortic endothelial cells (HAECs) were detected by qRT-PCR. Luciferase reporter assay, RNA immunoprecipitation (RIP), and RNA pull down were performed to validate whether GAS5 could directly interact with miR-26a. The effects of ox-LDL, GAS5 or combined with miR-26a on apoptosis and autophagy were evaluated by flow cytometry analysis and western blot, respectively.\n\nResultsGAS5 expression was upregulated and miR-26a was downregulated in the plasma samples of patients with atherosclerosis and ox-LDL-treated HAECs. There was reciprocal inhibition between GAS5 and miR-26a expressions in ox-LDL-treated HAECs. We further demonstrated that GAS5 directly interacted with miR-26a in ox-LDL-treated HAECs. Additionally, ox-LDL administration induced apoptosis and impaired autophagy flux in HAECs. Rescue experiments demonstrated that GAS5 knockdown restored ox-LDL-induced impaired autophagy flux by upregulating miR-26a in HAECs.\n\nConclusionKnockdown of GAS5 restores ox-LDL-induced impaired autophagy flux via upregulating miR-26a in human endothelial cells, revealing a novel regulatory mechanism for ox-LDL-induced impaired autophagy flux in ECs through ceRNA crosstalk.

biochemistry↗

bioRxiv2018

Hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.\n\nSignificanceSensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome activation and alerting host immune recognition, which provides insights into the more sophisticated biological functions of hemolysin upon infection.

immunology↗

bioRxiv2018

Opposing roles of endosomal innate immunity proteins IFITM3 and TLR7 in human metapneumovirus infection

Human metapneumovirus (hMPV) utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosome membrane. Whether cellular factors restrict or enhance either entry pathway is largely unknown. We found that the interferon-induced transmembrane protein 3 (IFITM3) inhibits hMPV infection to an extent similar to endocytosis-inhibiting drugs, and an IFITM3 variant that accumulates at the plasma membrane in addition to its endosome localization provided increased virus restriction. Mechanistically, IFITM3 blocks hMPV F protein-mediated membrane fusion, and inhibition of infection was reversed by the membrane destabilizing drug amphotericin B. Conversely, we unexpectedly found that infection by some hMPV strains is enhanced by Toll-like receptor 7 (TLR7), an endosomal protein, suggesting that cellular entry via endocytosis may be particularly advantageous for hMPV despite eventual restriction of this pathway upon induction of IFITM3. Overall, our results identify IFITM3 and TLR7 as endosomal factors differentially regulating hMPV infection.

immunology↗

bioRxiv2018

Bradyrhizobium diazoefficiens USDA 110-Glycine max interactome provides candidate proteins associated with symbiosis

Although the legume-rhizobium symbiosis is a most important biological process, there is a limited knowledge about the protein interaction network between host and symbiont. Using interolog and domain-based approaches, we constructed an inter-species protein interactome with 5115 protein-protein interactions between 2291 Glycine max and 290 Bradyrhizobium diazoefficiens USDA 110 proteins. The interactome was validated by expression pattern analysis in nodules, GO term semantic similarity, and co-expression analysis. One sub-network was further confirmed using luciferase complementation image assay. In the G. max-B. diazoefficiens interactome, bacterial proteins are mainly ion channel and transporters of carbohydrates and cations, while G. max proteins are mainly involved in the processes of metabolism, signal transduction, and transport. We also identified the top ten highly interacting proteins (hubs) for each of the two species. KEGG pathway analysis for each hub showed that two 14-3-3 proteins (SGF14g and SGF14k) and five heat shock proteins in G. max are possibly involved in symbiosis, and ten hubs in B. diazoefficiens may be important symbiotic effectors. Subnetwork analysis showed that 18 symbiosis-related SNARE proteins may play roles in regulating bacterial ion channels, and SGF14g and SGF14k possibly regulate the rhizobium dicarboxylate transport protein DctA. The predicted interactome and symbiosis proteins provide a valuable basis for understanding the molecular mechanism of root nodule symbiosis in soybean.

bioinformatics↗