Biology subjects

Du, L.

Publications and source records attributed to Du, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

Description, taxonomy, and comparative genomics of a novel Thermoleptolyngbya strain isolated from hot springs of Ganzi, Sichuan China.

Thermoleptolyngbya is a newly proposed genus of thermophilic cyanobacteria that are often abundant in thermal environments. However, a vast majority of Thermoleptolyngbya strains were not systematically identified, and genomic features of this genus are also sparse. Here, polyphasic approaches were employed to identify a thermophilic Thermoleptolyngbya A183 (FACHB-2491) isolated from hot spring Erdaoqiao, Ganzi prefecture, China. Morphological characterization was investigated by microscopy. Whilst the results of 16S rRNA were inconclusive, the phylogenetic reconstruction and secondary structures of 16S-23S ITS strongly suggested that A183 strain is a novel species within Thermoleptolyngbya. Moreover, genome-scale average nucleotide identity (ANI) confirmed the genetic divergence of A183 from Thermoleptolyngbya O-77. Comparative genome analysis revealed inconsistent genome structures of Thermoleptolyngbya strains. Further GO analysis showed that the specific genes were distributed in a wide range of functional categories. In addition, analysis of genes related to thermotolerance, signal transduction, and carbon/nitrogen/sulfur assimilation revealed the ability of this strain to adapt to inhospitable niches in hot springs.

microbiology↗

bioRxiv2020

Amphipathic Bax core dimer forms part of apoptotic pore wall in the mitochondrial membrane

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. They may embed in the cytosolic leaflet of the lipid bilayer generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, they may comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determine a high-resolution structure of the Bax core region that forms a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

biophysics↗

bioRxiv2020

Data-informed discovery of hydrolytic nanozymes

Nanozyme is a collection of nanomaterials with enzyme-like activity but exhibits higher environmental tolerance and long-term stability than their natural counterparts. Improving the catalytic activity and expanding the category of nanozymes are prerequisites to complement or even supersede natural enzymes. Specifically, a powerful hydrolytic nanozyme is demanded to degrade the unsustainable substance which natural enzymes hardly achieve. However, the development of hydrolytic nanozymes is still hindered by diverse hydrolytic substrates and following complicated mechanisms. Here, we apply two strategies which are informed by data to screen and predict catalytic active sites of MOF (metal-organic framework) based hydrolytic nanozymes. One is to increase the intrinsic activity by finely tuned Lewis acidity of the metal clusters. The other is to adjust the volume density of the active sites by shortening the length of ligands. Finally, we construct a Ce-FMA-MOF-based hydrolytic nanozyme with robust cleavage ability towards phosphate bonds, amide bonds, glycosidic bonds whose energy ascend in order; and even their mixture, biofilms. This work provides a rational methodology to design hydrolytic nanozyme, enriches the diversity of nanozymes, and potentially sheds a light on the evolution of enzyme engineering in the future.

biophysics↗

bioRxiv2020

Scavenger receptor A1 participates in the phagocytosis of Leptospira interrogans and leads to subsequent high inflammatory responses and bacterial dissemination in leptospirosis

Leptospirosis, caused by pathogenic Leptospira species, has emerged as a widespread zoonotic disease worldwide. Macrophages mediate the elimination of pathogens through phagocytosis and cytokine production. Scavenger receptor A1 (SR-A1), one of the critical receptors mediating this process, plays a complicated role in innate immunity. However, the role of SR-A1 in the immune response against pathogenic Leptospira invasion is unknown. In the present study, we found that SR-A1 is an important nonopsonic phagocytic receptor on murine macrophages for Leptospira. We also found that leptospiral LPS is the ligand of SR-A1. However, intraperitoneal injection of leptospires into WT mice presented with more severe jaundice, subcutaneous hemorrhaging, and higher bacteria burdens in blood and tissues than that of SR-A1-/- mice. Exacerbated cytokine and inflammatory mediator levels were also observed in WT mice and higher recruited macrophages in the liver than those of SR-A1-/- mice. Our findings collectively reveal that although beneficial in the uptake of Leptospira by macrophage, SR-A1 might be exploited by Leptospira to promote bacterial dissemination and modulate inflammatory activation, which causes a more severe infection in the host. These results provide our new insights into the innate immune response during early infection by L. interrogans.

microbiology↗

bioRxiv2020

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar ([~]3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter ([~]6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. Impact statementPotent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.

microbiology↗

bioRxiv2020

Persistence of SARS-CoV-2 specific B- and T-cell responses in convalescent COVID-19 patients 6-8 months after the infection

BackgroundThe longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. MethodsA cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. ResultsAnti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. ConclusionsAlthough the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection). FundingEuropean Unions Horizon 2020 research and innovation programme (ATAC), the Italian Ministry of Health, CIMED, the Swedish Research Council and the China Scholarship Council.

immunology↗

bioRxiv2020

The Biogeography of Fungal Communities Across Different Chinese Wine-producing Regions Associated with Environmental Factors and Spontaneous Fermentation Performance

Chinese Marselan grapes are believed to possess the potential to become a characteristic regional variety, whose quality is internationally recognized. The fermentation-related mycobiota from six climatically diverse Marselan-producing regions in China were analyzed via high-throughput sequencing (HTS), while the influence of environmental factors was evaluated as well. The results implied that the phyla Ascomycota and genus Aureobasidium dominated the fungal communities in 166 Marselan must and ferment samples. Significant differences were detected in the fungal microbiota from the regions, as well as the wineries, while these discrepancies decreased as the fermentation progressed. Moreover, the difference in fungal communities between the wineries exceeded the variation involving the regions, and higher diversity was identified in the types of species than in the compositions. Geoclimatic elements (Gc) and physicochemical indexes (Pi) exerted a significant effect on the fungal must consortium, explaining 58.17% of the taxonomic information. Therefore, a correlation was proposed between the spontaneous fermentation performance, as well as the polyphenol (PP) content, and their association with fungal taxonomic composition. In addition to enriching the knowledge regarding the wine microbiome and its influencing factors, this study may provide a new strategy for harnessing autochthonous "microbial terroir". ImportanceWine microbiome and their metabolism play a crucial role in wine quality. Chinese wine-producing regions are widely distributed with diverse geoclimatic conditions, this study reports a most comprehensive biogeography of fermentation-related fungal communities performed to date, taking the Chinese promising Marselan grape variety as the research object. It reveals significant differences in the fungal microbiota of Marselan must and ferment from different regions and wineries, and higher otherness between species type than that between species composition. The study also reports the main geoclimatic and physicochemical factors shaping fungal flora. Moreover, a correlation was proposed among the spontaneous fermentation performance with fungal taxonomic composition and polyphenol content. Taken together, the results shed light on the wine fungal communities and its influencing factors, which is crucial for vineyard and fermentation microorganism management, and may also provide a new strategy for harnessing autochthonous "microbial terroir" by utilizing spontaneous fermentation.

microbiology↗

bioRxiv2020

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

immunology↗

bioRxiv2020

A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

immunology↗

bioRxiv2020

Biomechanical Characterization of SARS-CoV-2 Spike RBD and Human ACE2 Protein-Protein Interaction

The current COVID-19 pandemic has led to a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry. STATEMENT OF SIGNIFICANCEThis study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics simulation approach to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 receptor-binding domain and human ACE2. The study reveals the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.

biophysics↗

bioRxiv2020

An enormous Paris polyphylla genome sheds light on genome size evolution and polyphyllin biogenesis

The monocot family Melanthiaceae with varying genome sizes in a range of 230-fold is an ideal model to study the genome size fluctuation in plants. Its family member Paris genus demonstrates an evolutionary trend of bearing huge genomes characterized by an average c-value of 49.22 pg. Here, we report a 70.18 Gb genome assembly out of the 82.55 Gb genome of Paris polyphylla var. yunnanensis (PPY), which represents the biggest sequenced genome to date. We annotate 69.53% repetitive sequences in this genome and 62.50% of which are long-terminal repeat (LTR) transposable elements. Further evolution analysis indicates that the giant genome likely results from the joint effect of common and species-specific expansion of different LTR superfamilies, which might contribute to the environment adaptation after speciation. Moreover, we identify the candidate pathway genes for the biogenesis of polyphyllins, the PPY-specific medicinal saponins, by complementary approaches including genome mining, comprehensive analysis of 31 next-generation RNA-seq data and 55.23 Gb single-molecule circular consensus sequencing (CCS) RNA-seq reads, and correlation of the transcriptome and phytochemical data of five different tissues at four growth stages. This study not only provides significant insights into plant genome size evolution, but also paves the way for the following polyphyllin synthetic biology.

plant biology↗

bioRxiv2020

Proper names from story recall are associated with beta-amyloid in cognitively unimpaired adults at risk for Alzheimer's disease

Due to advances in the early detection of Alzheimers disease (AD) biomarkers including beta-amyloid (A{beta}), neuropsychological measures that are sensitive to concurrent, subtle changes in cognition are critically needed. Story recall tasks have shown sensitivity to early memory declines in persons with Mild Cognitive Impairment and early stage dementia, as well as in persons with autosomal dominantly inherited AD up to 10 years prior to a dementia diagnosis. However, the evidence is inconclusive regarding relationships between evidence of A{beta} and story recall measures. Because story recall tasks require the encoding and delayed retrieval of several lexical-semantic categories, such as proper names, verbs, and numerical expressions, and because lexical categories have been shown to be differentially impaired in persons with MCI, we focused on item-level analyses of lexical-semantic retrieval from a quintessential story recall task, Logical Memory from the Wechsler Memory Scale. Our objective was to investigate whether delayed recall of lexical categories (proper names, verbs and/or numerical expressions), as well as the traditional total score measure, was associated with "preclinical AD," or cognitively unimpaired adults with positive A{beta} deposition on positron emission tomography (PET) neuroimaging using Pittsburgh Compound B. We developed an item-level scoring system, in which we parsed items into lexical categories and examined the immediate and delayed recall of these lexical categories from 217 cognitively unimpaired participants from the Wisconsin Registry for Alzheimers Prevention. We performed binary logistic regression models with story recall score as predictor and A{beta} status (positive/negative) as the outcome. Using baseline Logical Memory data, proper names from delayed story recall were significantly associated with A{beta} status, such that participants who recalled more proper names were less likely to be classified as PiB(+) (odds ratio = .58, p=.01). None of the other story recall variables, including total score, were associated with PiB status. Secondary analyses determined that immediate recall of proper names was not significantly associated with A{beta}, suggesting a retrieval deficit rather than that of encoding. The present findings suggest that lexical semantic retrieval measures from existing story recall tasks may be sensitive to A{beta} deposition, and may provide added utility to a widely-used, long-standing neuropsychological test for early detection of cognitive decline on the AD continuum.

neuroscience↗

bioRxiv2020

Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.

microbiology↗

bioRxiv2019

Essentiality of CREBBP in EP300 truncated B-cell lymphoma revealed by genome-wide CRISPR-Cas9 screen

Histone acetyltransferases (HATs), including CREBBP and EP300, are frequently mutated in B-cell malignancies and usually play a tumor-suppressive role. In this study, we performed whole genome and transcriptome sequencing and a genome-wide CRISPR-Cas9 knockout screen to study a germinal center B-cell like diffuse large B-cell lymphoma (DLBCL) cell line (RC-K8). Using a summarizing method that is optimized to address the complexity introduced by the time-course design, we identified a distinct pattern of genetic essentialities in RC-K8, including a dependency on CREBBP and MDM2, shown already at early time points and a gradually increased dependency on oxidative phosphorylation related genes. The dependency on CREBBP is associated with the corresponding genetic alterations identified in this cell line, i.e. a balanced translocation involves EP300, which resulted in a truncated form of protein that lacks the critical bromodomain and HAT domain. We further evaluated the previously published CRISPR-Cas9 screens and identified a genetic essentiality of CREBBP or EP300 gene in a small set of cancer cell lines, including several DLBCL cell lines that are highly sensitive for EP300 knockout and with CREBBP mutations or copy number loss. The dependency of the remaining HAT function in CREBBP and/or EP300-deficient genotype was validated by testing the HAT-domain inhibitor A-485. Our study suggests that integration of the unbiased, time-course-based functional screen results with the genomic and transcriptomic data can identify druggable vulnerability in individual or subgroups of cell lines/patients, which may help to develop more effective therapeutic strategies for cancers that are genetically highly heterogeneous, like DLBCL.

cancer biology↗

bioRxiv2019

Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domains involved in death receptor signaling through arginine-GlcNAcylation. This study applied a limited substrate screen from 12 death domain proteins with conserved arginines during EPEC and Salmonella infection and found that NleB from EPEC hijacked death receptor signaling tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNFR signaling through the modification of TRADD Arg235/245 and TNFR1 Arg376, respectively. SseK1 and SseK3 delivered by Salmonella inhibited TNF-- but not TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death, which was consistent with their host substrate recognition specificity. Taking advantage of the substrate specificity of SseK effectors, we found that only SseK1 fully rescued the bacteria colonization deficiency contributed by NleBc in Citrobacter rodentium infection animal model, indicating that TRADD was likely to be the preferred in vivo substrate corresponding to NleB/SseK1-induced bacterial virulence. Furthermore, novel auto-arginine-GlcNAcylation was observed in NleB and SseK1/3, which promoted the enzyme activity. These findings suggest that arginine-GlcNAcylation in death domains and auto-arginine-GlcNAcylation catalyzed by type III-translocated bacterial effector proteins NleB/SseKs are crucial for bacteria pathogenesis in regulating nuclear factor-{kappa}B (NF-{kappa}B) and death receptor signaling pathways. This study provides an insight into the mechanism by which EPEC and Salmonella manipulate death receptor signaling and evade host immune defense through T3SS effectors.\n\nAuthor SummaryEnteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are important food-borne pathogens infecting the intestine. They deliver type III secretion system effector NleB/SseKs to modify host death domain proteins by arginine GlcNAcylation. We screened the modification of 12 death domains containing conserved arginine in human genome by NleB, SseK1, SseK2, and SseK3 through ectopic co-expression and bacterial infection. Unlike multiple death receptor signaling inhibition by NleB, we found that SseK1 and SseK3 specifically hijacked tumor necrosis factor receptor 1 (TNFR1)-mediated death signaling through targeting TNFR1-associated death domain protein (TRADD) and receptor TNFR1, respectively. We identified the modification sites and suggested that TRADD was the in vivo target of NleB in mice infection model by utilizing the substrate specificity of SseK1 and SseK3, which highlighted anti-bacterial infection role of TRADD in death receptor signaling and non-death receptor signaling. In addition to the modification on host death domain substrates, we firstly elucidated the effect of auto-modification of the arginine GlcNAc transferases on the enzymatic activity, which widened our understanding of the newly discovered post translational modification in the process of pathogen-host interaction.

microbiology↗

bioRxiv2019

Clinical diagnosis of single-gene disorders and chromosomal abnormalities based on BGISEQ-500 platform

Most of the variation in the human genome is a single nucleotide variation (SNV) based on a single base or small fragment insertions and deletions and genomic copy number variation (CNV). Both types of mutations are involved in many human diseases. Such diseases often have complex clinical symptoms and difficult clinical diagnosis, so an effective detection method is needed to help clinical diagnosis and prevent birth defects. With the development of sequencing technology, the method of chip capture combined with high-throughput sequencing has been extensively used because of its high throughput, high accuracy, high speed and low cost. This study designed a chip that captures the coding region of 3043 genes associated with 4013 monogenic diseases. In addition, 148 chromosomal abnormalities can be identified by setting targets in specific regions. Compared with the whole exon chip, the chip can detect 4013 monogenic diseases and 148 chromosomal abnormalities at a lower cost, including SNV, intra-gene CNV and genomic copy number variation. This study utilized a strategy of combining the BGISEQ500 sequencing platform with the chip to identify 102 disease-associated mutations in 63 patients, 69 of which were new mutations. The evaluation test results also show that this combination complies with the requirements of clinical testing and has good clinical application value.

genomics↗

bioRxiv2019

Compartmentalized Biosynthesis of Mycophenolic Acid

Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic in human history and a first-line immunosuppressive drug for organ transplantations and autoimmune diseases. However, its biosynthetic mechanisms have remained a long-standing mystery. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and {beta}-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB as the long-sought key enzyme responsible for the oxidative cleavage of sesquiterpene side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC and O-methyltransferase MpaG, the Golgi apparatus-associated prenyltransferase MpaA, the endoplasmic reticulum-bound oxygenase MpaB and P450-hydrolase fusion enzyme MpaDE, and the peroxisomal acyl-CoA hydrolase MpaH. The whole pathway is elegantly co-mediated by these compartmentalized enzymes, together with the peroxisomal {beta}-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic pathway, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.\n\nSignificance StatementHere we elucidate the full biosynthetic pathway of the fungal natural product mycophenolic acid (MPA), which represents an unsolved mystery for decades. Besides the intriguing enzymatic mechanisms, we reveal that the MPA biosynthetic enzymes are elegantly compartmentalized; and the subcellular localization of the acyl-CoA hydrolase MpaH in peroxisomes is required for the unique cooperation between biosynthetic and {beta}-oxidation catabolism machineries. This work highlights the importance of a cell biology perspective for understanding the unexplored organelle-associated essential catalytic mechanisms in natural product biosynthesis of fungi and other higher organisms. The insights provided by our work will also benefit future efforts for both industrial strain improvement and novel drug development.

biochemistry↗