Biology subjects

Liu, D.

Publications and source records attributed to Liu, D..

At least 397 records · Page 22Linked to original sources

bioRxiv2020

Methylation Data Processing Protocol & Comparison of Blood and Cerebral Spinal Fluid Following Aneurysmal Subarachnoid Hemorrhage

One challenge in conducting DNA methylation-based epigenome-wide association studies (EWAS) is the appropriate cleaning and quality-checking of the methylation values to minimize biases and experimental artifacts, while simultaneously retaining potential biological signals. These issues are compounded in studies that include multiple tissue types, and/or tissues for which reference data are unavailable to assist in adjusting for cell-type mixture, for example cerebral spinal fluid (CSF). For our study that evaluated blood and CSF taken from aneurysmal subarachnoid hemorrhage (aSAH) patients, we developed a protocol to clean and quality-check genome-wide methylation levels and compared the methylomic profiles of the two tissues to determine whether blood is a suitable surrogate for CSF. CSF samples were collected from 279 aSAH patients longitudinally during the first 14 days of hospitalization, and a subset of 88 of these patients also provided blood samples within the first two days. Quality control (QC) procedures included identification and exclusion of poor performing samples and low-quality probes, functional normalization, and correction for cell-type heterogeneity via surrogate variable analysis (SVA). Significant differences in rates of poor sample performance was observed between blood (1.1% failing QC) and CSF (9.12% failing QC; p = 0.003). Functional normalization increased the concordance of methylation values among technical replicates in both CSF and blood. Likewise, SVA improved the asymptotic behavior of the test of association in a simulated EWAS under the null hypothesis. To determine the suitability of blood as a surrogate for CSF, we calculated the correlation of adjusted methylation values between blood and CSF globally and by genomic regions. Overall, mean correlation (r < 0.26) was low, suggesting that blood is not a suitable surrogate for global methylation in CSF. However, differences in the magnitude of the correlation were observed by genomic region (CpG island, shore, shelf, open sea; p < 0.001 for all) and orientation with respect to nearby genes (3 UTR, transcription start site, exon, body, 5 UTR; p < 0.01 for all). In conclusion, the correlation analysis and QC pipelines indicated that DNA extracted from blood was not, overall, a suitable surrogate for DNA extracted from CSF in aSAH methylomic studies.

genomics↗

bioRxiv2020

A reversibly induced CRISPRi system targeting Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited Photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased Photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.

synthetic biology↗

bioRxiv2020

High-Throughput 5' UTR Engineering for Enhanced Protein Production in Non-Viral Gene Therapies

Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major challenge. One approach to increase protein expression is by optimizing translation through the engineering of 5 untranslated regions (5 UTRs). Here, we developed a high-throughput strategy to design, screen, and optimize novel 5UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identified naturally occurring 5 UTRs with high translation efficiencies and used this information with in silico genetic algorithms to generate synthetic 5 UTRs. A total of [~]12,000 5 UTRs were then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identified three synthetic 5 UTRs that outperformed commonly used non-viral gene therapy plasmids in expressing protein payloads. Furthermore, combinatorial assembly of these 5 UTRs enabled even higher protein expression than obtained with each individual 5 UTR. In summary, we demonstrate that high-throughput screening of 5 UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.

synthetic biology↗

bioRxiv2020

Transcriptional mediators of treatment resistance in lethal prostate cancer

Metastatic castration resistant prostate cancer (mCRPC) is primarily treated with therapies that prevent transcriptional activity of the androgen receptor (AR), cause DNA damage, or prevent cell division. Clinical resistance to these therapies, including second-generation androgen-targeting compounds such as enzalutamide and abiraterone, is nearly universal. Other treatment modalities, including immune checkpoint inhibitors, have provided minimal benefit except in rare subsets of patients1,2. Both tumour intrinsic and extrinsic cellular programs contributing to therapeutic resistance remain areas of active investigation. Here we use full-length single-cell RNA-sequencing (scRNA-seq) to identify the transcriptional states of cancer and immune cells in the mCRPC microenvironment. Within cancer cells, we identified transcriptional patterns that mediate a significant proportion of inherited risk for prostate cancer, extensive heterogeneity in AR splicing within and between tumours, and vastly divergent regulatory programs between adenocarcinoma and small cell carcinoma. Moreover, upregulation of TGF-{beta} signalling and epithelial-mesenchymal transition (EMT) were both associated with resistance to enzalutamide. We found that some lymph node metastases, but no bone metastases, were heavily infiltrated by dysfunctional CD8+ T cells, including cells undergoing dramatic clonal expansion during enzalutamide treatment. Our findings suggest avenues for rational therapeutic approaches targeting both tumour-intrinsic and immunological pathways to combat resistance to current treatment options.

genomics↗

bioRxiv2020

Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds

We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf phage delays wound re-epithelialization in the absence of live Pa, indicative that Pf effects on wound healing are independent of Pa pathogenesis. Pf phage directly inhibits autocrine signaling of CXCL1 (KC) to impede keratinocyte migration and wound re-epithelization. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.

microbiology↗

bioRxiv2020

AI-aided design of novel targeted covalent inhibitors against SARS-CoV-2

The focused drug repurposing of known approved drugs (such as lopinavir/ritonavir) has been reported failed for curing SARS-CoV-2 infected patients. It is urgent to generate new chemical entities against this virus. As a key enzyme in the life-cycle of coronavirus, the 3C-like main protease (3CLpro or Mpro) is the most attractive for antiviral drug design. Based on a recently solved structure (PDB ID: 6LU7), we developed a novel advanced deep Q-learning network with the fragment-based drug design (ADQN-FBDD) for generating potential lead compounds targeting SARS-CoV-2 3CLpro. We obtained a series of derivatives from those lead compounds by our structure-based optimization policy (SBOP). All the 47 lead compounds directly from our AI-model and related derivatives based on SBOP are accessible in our molecular library at https://github.com/tbwxmu/2019-nCov. These compounds can be used as potential candidates for researchers in their development of drugs against SARS-CoV-2.

pharmacology and toxicology↗

bioRxiv2020

A novel bat coronavirus reveals natural insertions at the S1/S2 cleavage site of the Spike protein and a possible recombinant origin of HCoV-19

The unprecedented epidemic of pneumonia caused by a novel coronavirus, HCoV-19, in China and beyond has caused public health concern at a global scale. Although bats are regarded as the most likely natural hosts for HCoV-191,2, the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats collected from Yunnan Province in China between May and October, 2019. RmYN02 shared 93.3% nucleotide identity with HCoV-19 at the scale of the complete virus genome and 97.2% identity in the 1ab gene in which it was the closest relative of HCoV-19. In contrast, RmYN02 showed low sequence identity (61.3%) to HCoV-19 in the receptor binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, however, and in a similar manner to HCoV-19, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the Spike (S) protein. This provides strong evidence that such insertion events can occur in nature. Together, these data suggest that HCoV-19 originated from multiple naturally occurring recombination events among those viruses present in bats and other wildlife species.

microbiology↗

bioRxiv2020

Altered B cells homeostasis in child-onset immunoglobulin A vasculitis

BackgroundImmunoglobulin A vasculitis (IgAV), also called Henoch-Schonlein purpura, is a systemic small vessels vasculitis with immunoglobulin A1-dominant immune deposits. B-cells are a heterogeneous population with unique subsets distinguished by their phenotypes and cytokine production. Here, we explored the status of B cell subsets in patients with IgAV. MethodsThirty IgAV patients and fifteen age- and sex-matched healthy individuals were enrolled in this study. Fresh blood samples were collected from both healthy and IgAV patients. Upon the distinct expressions of CD3, CD19, CD20, CD38, CD27 and IgD, peripheral blood mononuclear cells (PBMCs) were initially categorized into plasmablasts and memory B cells. Subsequently, using surface markers including CD138 and IgM, and intracellular markers containing IgM and IgG, plasmablasts and memory B cells were further divided into distinct subgroups. A total of eleven populations were detected using multiple flow cytometry. ResultsCD3-CD19+IgD+CD27-, CD3-CD19+CD20-CD38+, CD3-CD19+CD20-CD38+IgM+, and CD3-CD19+CD20-CD38+CD138+ B cells were larger in patients with IgAV than in the HCs. Only CD3-CD19+IgD-CD27+IgM+ B cell counts were reduced in IgAV. The elevated B cell numbers returned to normal after treatment. Plasma and plasmablast B cell numbers correlated with plasma IgA levels. On the contrary, CD3-CD19+IgD-CD27+IgM+ B cell numbers were negatively proportional to the plasma IgA levels while naive B cell numbers correlated with plasma and plasmablast B cell counts. ConclusionsWe hypothesized that immunoglobulin production was abnormally elevated in IgAV and could be explained by altered B-cell subset homeostasis.

immunology↗

bioRxiv2020

Generation of multi-transgenic pigs using PiggyBac transposons co-expressing pectinase, xylanase, cellulase?β-1.3-1.4-glucanase and phytase

The current challenges facing the pork industry are to maximize feed efficiency and minimize fecal emissions. Unlike ruminants, pigs lack a number of digestive enzymes like pectinase, xylanase, cellulase, {beta}-1.3-1.4-glucanase and phytase to hydrolyze the cell walls of grains to release endocellular nutrients into their digestive tracts. Herein, we synthesized multiple cellulase and pectinase genes derived from lower organisms and then codon optimized these genes to be expressed in pigs. These genes were then cloned into our previously optimized XynB (xylanase)- EsAPPA (phytase) bicistronic construct. We then successfully generated transgenic pigs that expressed four enzymes (Pg7fn (pectinase), XynB (xylanase), EsAPPA (phytase) and TeEGI (cellulase and {beta}-glucanase)) using somatic cell cloning. Expression of these genes was parotid gland specific. Enzymatic assays using the saliva of these founders demonstrated high levels of phytase (2.0~3.4 U/mL) and xylanase (0.25~0.42 U/mL) activity, but low levels of pectinase (0.06~0.08 U/mL) activity. These multi-transgenic pigs are expected to contribute to enhance feed utilization and reduce environmental impact.

genetics↗

bioRxiv2020

β-L-1- uracil (L-BHDU) effectiveness against varicella-zoster virus and herpes simplex virus type 1 depends on thymidine kinase activity

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 {micro}M in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TKG22R-GST and TKR130Q- GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDUR virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.

microbiology↗

bioRxiv2020

β-L-1- Uracil (L-BHDU) Inhibits Varicella Zoster Virus Replication by Depleting the Cellular dTTP Pool

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) inhibits varicella zoster virus (VZV) replication in cultured cells, human skin, and in SCID-Hu mice with skin xenografts. VZV thymidine kinase converts L-BHDU to monophosphate (MP) and diphosphate (DP) forms, but the triphosphate form was not detected in infected cells and the antiviral mechanism was unknown. Given its similar structure to uridine, we asked if L-BHDU interfered with viral DNA replication via inhibition of the purine and/or pyrimidine biosynthesis pathways. Addition of purines to the medium was unable to restore VZV replication in the presence of L-BHDU. In contrast, excess thymidine and uridine in proportion to L-BHDU restored VZV replication, suggesting that the active form of L-BHDU interfered with pyrimidine biosynthesis. However, addition of thymidine and uridine failed to restore VZV replication in non-dividing cells treated with L-BHDU. Like other herpesviruses, VZV infection increased thymidine triphosphate (dTTP) in confluent cells while L-BHDU treatment decreased the dTTP pool by nearly 4-fold. The active form(s) of L-BHDU did not interfere with cellular metabolism, suggesting viral target(s).

microbiology↗

bioRxiv2020

Molecular and cellular heterogeneity of gastric cancer explained by methylation-driven key regulators

Gastric cancer (GC) is a heterogeneous disease of diverse genetic, genomic, and epigenetic alterations. Tumor microenvironment (TME) also contributes to the heterogeneity of GC. To investigate GC heterogeneity, we developed an Integrative Sequential Causality Test (ISCT) to identify key regulators of GC by integrating DNA methylation, copy number variation, and transcriptomic data. Applying ISCT to three GC cohorts containing methylation, CNV and transcriptomic data, 11 common methylation-driven key regulators (ADHFE1, CDO1, CRYAB, FSTL1, GPT, PKP3, PTPRCAP, RAB25, RHOH, SFN, and SORD) were identified. Based on these 11 genes, gastric tumors were clustered into 3 clusters which were associated with known molecular subtypes, Lauren classification, tumor stage, and patient survival, suggesting significance of the methylation-driven key regulators in molecular and histological heterogeneity of GC. We further showed that chemotherapy benefit was different in the 3 GC clusters and varied depending on the tumor stage. Both immune/stromal proportions in TME and tumor cell genomic variations contributed to expression variations of the 11 methylation-driven key regulators and to the GC heterogeneity.

genomics↗

bioRxiv2020

Selective germline genome edited pigs and their long immune tolerance in Non Human Primates

Organ transplantation is the only curative treatment for patients with terminal organ failure, however, there is a worldwide organ shortage. Genetically modified pig organs and tissues have become an attractive and practical alternative solution for the severe organ shortage, which has been made possible by significant progress in xenotransplantation in recent years. The past several decades witnessed an expanding list of genetically engineered pigs due to technology advancements, however, the necessary combination of genetic modifications in pig for human organ xenotransplantation has not been determined. In the current study, we created a selective germline genome edited pig (SGGEP). The first triple xenoantigens (GGTA, B4GAL, and CAMH) knockout somatic cells were generated to serve as a prototype cells and then human proteins were expressed in the xenoantigen knockout cells, which include human complement system negative regulatory proteins (CD46, CD55, and CD59); human coagulation system negative regulatory proteins thrombomodulin (THBD); tissue factor pathway inhibitor (TFPI); CD39; macrophage negative regulatory proteins (human CD47); and natural killer cell negative regulatory human HLA-E. After the successful establishment of SGGEP by the nuclear tranfer, we engrafted SGGEP skin to NHP, up to 25 days graft survival without immunosuppressive drugs was observed. Because a pig skin graft does not impact the success of a subsequent allograft or autograft or vice versa, thus our SGGEP could have a great potential for clinical value to save severe and large area burn patients and the other human organ failure. Therefore, this combination of specific gene modifications is a major milestone and provides proof of concept to initiate investigator-initiated clinical trials (IITs) in severe burn patients with defined processes and governance measures in place and the other clinical application.

synthetic biology↗

bioRxiv2020

Spatial and functional links between cellular virological state and progression of liver fibrosis in chronic hepatitis B

Chronic Hepatitis B Virus (HBV) infection is strongly associated with the progression of liver fibrosis, cirrhosis and hepatocellular carcinoma. Despite intensive study, the detailed mechanisms leading to HBV induced liver disease have not been fully elucidated. Previously, we reported a mosaic distribution of viral antigens and nucleic acids at single-cell level in liver tissues of chronic hepatitis B (CHB) patients and proposed a three-stage model of HBV infection in vivo. Here, we explored whether the different stages at cellular level is functionally linked with fibrogenesis. We observed a tight spatial relationship between the invasion of collagen fibers and transitions from S-rich to DNA-rich stage. While S-rich cells mainly localized within minimally fibrotic tissue, DNA-rich cells were often closely surrounded by a milieu of stiffened extracellular matrix (ECM). cDNA microarray and subsequent validation analyses revealed that S-rich cells manifested elevated ribosomal proteins and oxidative phosphorylation genes in a disease phase-dependent manner. On the other hand, DNA-rich cells exhibited gradually deteriorated expression of hepatocyte-specific antigen and transcriptional regulator in parallel with the progression of hepatic fibrosis. Finally, during fibrogenesis, inflammatory genes such as IP-10 were found to be expressed in both portal infiltrated cells and surrounding parenchymal cells which resulted in suppressed antigen expression. Taken together, we propose that liver inflammation and accompanying fibrogenesis is spatially and functionally linked with the transition of virological stages at cellular level. These transitions occur possibly due to an altered hepatocyte transcription profile in response to a transformed ECM environment. The collective viral and host activities shape the histological alterations and progression of liver disease during CHB infection.

microbiology↗

bioRxiv2020

Single-cell RNA-seq reveals endoimmune cells in zebrafish

Endothelial cells (ECs) constitute a monolayer that covers the interior surface of blood vessels and participates in various processes. Although vascular ECs share certain common properties, they differ in both structure and function. So far, the transcriptome profile and heterogeneity of the full repertoire of ECs in vertebrates remain poorly understood. The relatively small size of zebrafish embryos and larvae allows a feasible analysis of the broad spectrum of ECs within every tissue and organ of a whole organism. ECs have been suggested to be conditional innate immune cells. Whether ECs possess the comparable capacity of involvement in immune response is so far undetermined. Currently, through single-cell RNA sequencing analysis of total ECs of zebrafish we identified a fraction of endothelial cells expressing the marker genes of innate immune cells, named "endoimmune cells". We found the percentage of these cells gradually increased along with the embryonic development. Then, we observed the patrolling mCherry+ cells displayed the morphology alike to the macrophages and neutrophils. Furthermore, we revealed that some of the kdrl:ras-mCherry ECs were labelled with coro1a:EGFP as well. In addition, we demonstrated that the mCherry+ EC from intersegmental vessel could gradually present the expression of GFP in Tg(kdrl:ras-mCherry::coro1a:GFP) line, suggesting the endoimmune cells are derived from ECs. Importantly, we showed the endoimmune cells are responsive to the inflammation in zebrafish. Taken together, these data suggested the existence of endoimmune cells, a novel type of subpopulations of ECs. It will provide novel insights for understanding endothelial roles in both normal physiological function and human diseases and enable endoimmune cells-based target therapies.

cell biology↗

bioRxiv2019

Vineyard ecosystems are structured and distinguished by fungal communities impacting the flavour and quality of wine

The flavours of foods and beverages are formed by the agricultural environment where the plants are grown. In the case of wine, the location and environmental features of the vineyard site imprint the wine with distinctive aromas and flavours. Microbial growth and metabolism play an integral role in wine production from the vineyard to the winery, by influencing grapevine health, wine fermentation, and the flavour, aroma and quality of finished wines. The mechanism by which microbial distribution patterns drive wine metabolites is unclear and while flavour has been correlated with bacterial composition for red wines, bacterial activity provides a minor biochemical conversion in wine fermentation. Here, we collected samples across six distinct winegrowing areas in southern Australia to investigate regional distribution patterns of both fungi and bacteria and how this corresponds with wine aroma compounds. Results show that soil and must microbiota distinguish winegrowing regions and are related to wine chemical profiles. We found a strong relationship between microbial and wine metabolic profiles, and this relationship was maintained despite differing abiotic drivers (soil properties and weather/ climatic measures). Notably, fungal communities played the principal role in shaping wine aroma profiles and regional distinctiveness. We found that the soil microbiome is a potential source of grape- and must-associated fungi, and therefore the weather and soil conditions could influence the wine characteristics via shaping the soil fungal community compositions. Our study describes a comprehensive scenario of wine microbial biogeography in which microbial diversity responds to surrounding environments and ultimately sculpts wine aromatic characteristics. These findings provide perspectives for thoughtful human practices to optimise food and beverage flavour and composition through understanding of fungal activity and abundance.

microbiology↗

bioRxiv2019

Redox-mediated regulation of a labile, evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain

An evolutionarily conserved low complexity (LC) domain is found within a 152 residue segment localized to the carboxyl-terminal region of the TDP43 RNA-binding protein. This TDP43 LC domain contains ten conserved methionine residues. Self-association of this domain leads to the formation of liquid-like droplets composed of labile, cross-{beta} polymers. Exposure of polymers to low concentrations of H2O2 leads to a phenomenon of droplet melting that can be reversed upon exposure of the oxidized protein to the MsrA and MsrB methionine sulfoxide reductase enzymes, thioredoxin, thioredoxin reductase and NADPH. Morphological features of the cross-{beta} polymers were revealed by a method of H2O2-mediated footprinting. Similar TDP43 LC domain footprints were observed in highly polymerized, hydrogel samples, liquid-like droplet samples, and living cells. The ability of H2O2 to impede cross-{beta} polymerization was abrogated by a prominent ALS-causing mutation that changes methionine residue 337 to valine. These observations offer potentially useful insight into the biological role of TDP43 in facilitating synapse-localized translation, as well as aberrant aggregation of the protein in neurodegenerative disease.

biochemistry↗

bioRxiv2019

Characterization of the novel clinical isolate X-4 containing a new tp0548 sequence-type

BackgroundA novel tp0548 sequence-type was identified in one clinical isolate (X-4) from a patient diagnosed with primary syphilis in Xiamen, China. To precisely define and characterize this new clinical isolate, we performed further genome-scale molecular analysis. Methodology/Principal findingsThe alignment of all published tp0548 genotypes revealed that this new genotype had a unique nucleotide substitution G->T at position 167, and the letter "ao" was assigned to the genotype. Phylogenetic analysis showed that the "ao" genotype belonged to the SS14-like clade of Treponema pallidum (TPA) strains. The genome of the X-4 isolate was then sequenced and analyzed, and the result of a multi-locus sequence analysis using a set of nine chromosomal loci showed that the X-4 isolate was clustered with a monophyletic group of TPA strains, which clearly identified the isolate as a TPA strain. Whole-genome phylogenetic analysis was subsequently conducted to corroborate the TPA strain classification of the X-4 isolate. And the isolate was genetically related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, high intrastrain heterogeneity in the length of the poly G/C tracts was found in the TPAChi_0347 locus, which indicated that this gene was most likely involved in phase variation events. The first investigation of the length heterogeneity of the poly A/T tracts showed the variability of the ploy A/T was lower, and all the observed intrastrain variations fell within coding regions. Conclusions/SignificanceThe study demonstrated the X-4 isolate was a TPA isolate containing a novel tp0548 sequence-type. The identification of intrastrain genetic heterogeneity at poly G/C tracts and poly A/T tracts of the isolate could provide a snapshot of the genes that potentially involved in genotype-phenotype variations. These findings provide an unequivocal characterization for better understanding the molecular variation of this emerging isolate. Author summaryThree subspecies of Treponema pallidum (pallidum, pertenue, and endemicum) are increasingly showing overlap in terms of transmission and clinical manifestations. We recently identified a novel tp0548 genotype in the X-4 isolate, which was obtained from an adult male with genital lesions. The novel genotype contained a unique nucleotide substitution G->T at position 167 and belonged to the SS14-like clade of TPA strains, as determined by phylogenetic analysis. We conducted an in-depth exploration of the genome of the X-4 isolate using the pooled segment genome sequencing method followed by Illumina sequencing. Multi-locus sequence analysis of nine chromosomal loci demonstrated that the X-4 isolate was clustered within a monophyletic group of TPA strains, which identified the isolate as a TPA strain. Whole-genome phylogenetic analysis subsequently corroborated the TPA strain classification of the X-4 isolate and revealed that the isolate was very closely related to the SS14 strain, with 42 single nucleotide variations and 12 insertions/deletions. In addition, characterization of the intrastrain heterogeneity in the lengths of homopolymeric tracts in the X-4 isolate showed that the heterogeneity of the poly G/C tracts was greater than that of the poly A/T tracts, and high poly G/C tract diversity was observed in the TPAChi_0347 locus.

genetics↗