Secretion Signal Research Articles

Mechanical Stretch-Induced ATP Release from Osteocytes Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells.

Mandibular distraction osteogenesis (MDO) is a highly effective method for bone regeneration, commonly employed in treating craniofacial defects and deformities. Osteocytes sense mechanical forces in the pericellular space, relay external stimuli to biochemical changes, and send signals to other effector cells, including bone marrow mesenchymal stem cells (BM-MSCs), to regulate bone resorption and formation. Piezo1 potentially affects the secretion signal molecules of bone cells under mechanical stretch. The primary aim of this study was to enhance our comprehension of the molecular biology underlying this therapeutic approach and to identify specific signaling molecules that facilitate bone formation in response to stretch forces. Mechanical stretching was applied to negative controls and Piezo1 knockdown osteocyte-like MLO-Y4 cells. Alkaline phosphatase and Alizarin Red S staining were used to survey the osteogenic potential of BM-MSCs. The production and secretion content of adenosine triphosphate (ATP) was measured using ATP content determination analysis. Pathway-related and osteo-specific genes and proteins were evaluated using real-time polymerase chain reaction (RT-PCR), Western blots, and immunofluorescence. Mitochondrial organization was examined with a transmission electron microscope. The conditioned medium of stretch-exposed MLO-Y4s significantly upregulated osteogenesis-related indicators of BM-MSCs (p < 0.001). The upregulation of BM-MSC osteogenesis was associated with ATP release from osteocytes. Mechanically induced calcium transfer and transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation mediated by Piezo1 could promote mitochondrial fission and ATP release. Osteocytes detected stretch forces through Piezo1, triggering calcium influx, TAZ nuclear translocation, and ATP production. The stretch stimulation of Piezo1 induces calcium influx, which in turn promotes calcium-related TAZ nuclear translocation, changes in mitochondrial dynamics, and the release of ATP in osteocytes. This signaling cascade leads to an up-regulation in the osteogenic capacity of BM-MSCs. Mitochondrial energy metabolism of mechanosensitive protein Piezo1-dependent and ATP release may provide a new effective intervention method for mechanically related bone remodeling.

Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia

BackgroundIn addition to being secreted into the intercellular spaces by exocytosis, insulin-like growth factor binding protein 5 (IGFBP5) may also remain in the cytosol or be transported to the nucleus. Depending on the different cellular context and subcellular distribution, IGFBP5 can act as a tumor suppressor or promoter through insulin-like growth factor -dependent or -independent mechanisms. Yet, little is known about the impacts of IGFBP5 on acute myeloid leukemia (AML) and its underlying mechanism. MethodsHere we investigated the roles of IGFBP5 in human AML by using recombinant human IGFBP5 (rhIGFBP5) protein and U937 and THP1 cell lines which stably and ectopically expressed IGFBP5 or mutant IGFBP5 (mtIGFBP5) with the lack of secretory signal peptide. Cell counting kit-8 and flow cytometry assay were conducted to assess the cell viability, cell apoptosis and cell cycle distribution. Cytotoxicity assay was used to detect the chemosensitivity. Leukemia xenograft model and hematoxylin-eosin staining were performed to evaluate AML progression and extramedullary infiltration in vivo. ResultsIn silico analysis demonstrated a positive association between IGFBP5 expression and overall survival of the AML patients. Both IGFBP5 overexpression and extrinsic rhIGFBP5 suppressed the growth of THP1 and U937 cells by inducing cell apoptosis and arresting G1/S transition and promoted the chemosensitivity of U937 and THP1 cells to daunorubicin and cytarabine. However, overexpression of mtIGFBP5 failed to demonstrate these properties. An in vivo xenograft mouse model of U937 cells also indicated that overexpression of IGFBP5 rather than mtIGFBP5 alleviated AML progression and extramedullary infiltration. Mechanistically, these biological consequences depended on the inactivation of insulin-like growth factor 1 receptor -mediated phosphatidylinositol-3-kinase/protein kinase B pathway. ConclusionsOur findings revealed secreted rather than intracellular IGFBP5 as a tumor-suppressor and chemosensitizer in AML. Upregulation of serum IGFBP5 by overexpression or addition of extrinsic rhIGFBP5 may serve as a suitable therapeutic approach for AML.

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris

Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using PAOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1β, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

A type VII-secreted lipase toxin with reverse domain arrangement

The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a class of T7 substrate, TslA, that has a reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the capacity to recognise a secretion signal present at either end of a substrate.

Endogenous SIRT6 in platelets negatively regulates platelet activation and thrombosis.

Thromboembolism resulting from platelet dysfunction constitutes a significant contributor to the development of cardiovascular disease. Sirtuin 6 (SIRT6), an essential NAD+-dependent enzyme, has been linked to arterial thrombosis when absent in endothelial cells. In the present study, we have confirmed the presence of SIRT6 protein in anucleated platelets. However, the precise regulatory role of platelet endogenous SIRT6 in platelet activation and thrombotic processes has remained uncertain. Herein, we present compelling evidence demonstrating that platelets isolated from SIRT6-knockout mice (SIRT6-/-) exhibit a notable augmentation in thrombin-induced platelet activation, aggregation, and clot retraction. In contrast, activation of SIRT6 through specific agonist treatment (UBCS039) confers a pronounced protective effect on platelet activation and arterial thrombosis. Moreover, in platelet adoptive transfer experiments between wild-type (WT) and SIRT6-/- mice, the loss of SIRT6 in platelets significantly prolongs the mean thrombus occlusion time in a FeCl3-induced arterial thrombosis mouse model. Mechanistically, we have identified that SIRT6 deficiency in platelets leads to the enhanced expression and release of proprotein convertase subtilisin/kexin type 9 (PCSK9), subsequently activating the platelet activation-associated mitogen-activated protein kinase (MAPK) signaling pathway. These findings collectively unveil a novel protective role of platelet endogenous SIRT6 in platelet activation and thrombosis. This protective effect is, at least in part, attributed to the inhibition of platelet PCSK9 secretion and mitogen-activated protein kinase signaling transduction. Our study provides valuable insights into the intricate interplay between SIRT6 and platelet function, shedding light on potential therapeutic avenues for managing thrombotic disorders.

Comprehensive analysis of the gene expression profile of the male and female BTBR mice with diabetic nephropathy

Comprehensive insight into the gender-based gene expression-related omics data in a rodent model of diabetic nephropathy (DN) is scarce. In the present study, the gender-based genes regulating different pathways involved in the progression of DN were explored through an unbiased RNA sequence of kidneys from BTBR mice with DN. We identified 17,739 and 17,981 genes in male and female DN mice; 1121 and 655 genes were expressed differentially (DEGs, differentially expressed genes) in male and female DN mice; both genders displayed only 195 DEGs. In the male DN mice, the number of upregulated genes was nearly the same as that of the down-regulated genes. In contrast, the number of upregulated genes was lesser than that of the down-regulated genes in the female DN mice, manifesting a remarkable gender disparity during the progression of DN in this animal model. Gene Ontology (GO) and KEGG-enriched results showed that most of these DEGs were related to the critical biological processes, including metabolic pathways, natural oxidation, bile secretion, and PPAR signaling; all are highly associated with DN. Notably, the DEGs significantly enriched for steroid hormone biosynthesis pathway were identified in both genders; the number of DEGs increased was 22 in male DN mice and 14 in female DN mice. Specifically, the Ugt1a10, Akr1c12, and Akr1c14 were upregulated in both genders. Interestingly, the Hsd11b1 gene was upregulated in female DN mice but downregulated in male DN mice. These results suggest that a significant gender-based variance in the gene expression occurs during the progression of DN and may be playing a role in the advancement of DN in the BTBR mouse model.

Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 μg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.

The Pneumococcal Protein SufC Binds to Host Plasminogen and Promotes Its Conversion into Plasmin.

Streptococcus pneumoniae causes otitis media, sinusitis, and serious diseases such as pneumonia and bacteremia. However, the in vivo dynamics of S. pneumoniae infections and disease severity are not fully understood. In this study, we investigated pneumococcal proteins detected in the bronchoalveolar lavage fluid of an S. pneumoniae-infected mouse, which were assumed to be expressed during infection. Analysis of three proteins with unknown infection-related functions revealed that recombinant Fe-S cluster assembly ATP-binding protein (SufC) binds to the host plasminogen and promotes its conversion into plasmin. SufC was detected in the bacterial cell-surface protein fraction, but it had no extracellular secretory signal. This study suggests that S. pneumoniae releases SufC extracellularly through LytA-dependent autolysis, binding to the bacterial cell surface and host plasminogen and promoting its conversion into plasmin. The recruitment of plasmin by S. pneumoniae is considered useful for bacterial survival and spread, and SufC is suggested to facilitate this process.

Overexpression of T3SS translocation signals in Salmonella causes delayed attenuation.

Engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We previously engineered Salmonella enterica serovar Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. Here, we show that after 48 hours, the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFN-γ-deficient mice, this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell's physiology, which makes host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Production and characterization of basic fibroblast growth factor protein in rice suspension cultures

The basic fibroblast growth factor (bFGF) plays a major role in cell growth, survival, and other mitogenic activities. General applications of this protein involve tissue repair, morphogenesis, embryonic development, cellular growth, etc. Due to its importance and high demand, research has been done on producing recombinant bFGF in different expression platforms. We have produced bFGF in transgenic rice (Oryza sativa) cells growing in liquid suspension culture. The gene construct consists of the rice alpha-amylase (RAmy) 3D sugar-inducible promotor and rice glutelin secretion signal peptide for controlled production and release of the bFGF protein by manipulating the sugar concentration for batch or semi-continuous bioreactor runs. A thermal shift assay was performed on the purified rice cell produced bFGF (rbFGF) and the results were compared with E. coli derived bFGF. The purified rbFGF protein from rice cells was tested for propagating human-induced pluripotent stem cells (hiPSCs) up to eight passages. Furthermore, the biological activity measured in both crude lysate and purified rbFGF showed a similar pattern of the extracellular signal-regulated kinase (ERK) activity compared to E. coli derived bFGF (control), indicating that rbFGF is active before purification and the protein activity is not inhibited by other components of the lysate. From the current results, the rice-produced rbFGF protein is biologically active and could serve as a viable product for various applications.

Bioinformatics Methods for Prediction of Gene Families Encoding Extracellular Peptides.

Genes encoding small secreted peptides are widely distributed among plant genomes but their detection and annotation remains challenging. The bioinformatics protocol described here aims to identify as exhaustively as possible secreted peptide precursors belonging to a family of interest. First, homology searches are performed at the protein and genome levels. Next, multiple sequence alignments and predictions of a secretion signal are used to define a set of homologous proteins sharing features of secreted peptide precursors. These protein sequences are then used as input of motif detection and profile-based tools to build representative matrices and profiles that are used iteratively as guides to scan again the proteome and genome until family completion.

Using Inducible Signaling Receptors to Increase Erythropoietic Output from Genome-Edited Hematopoietic Stem Cells

Introduction: Oxygen is delivered to all tissues in the body through the hemoglobin molecule uniquely present in red blood cells (RBCs). Proper production and function of RBCs is therefore critical to human health. However, genetic defects of the hematologic system such as anemias and hemoglobin disorders (broadly termed “hemoglobinopathies”) are among the most common genetic disorders in the world. Therefore, developing strategies to regulate RBC development may allow researchers and clinicians to increase therapeutic potential of hematologic cell and gene therapies. Erythropoietin (EPO) signaling is indispensable to RBC development. In its native form, two EPO receptor (EPOR) monomers dimerize in the presence of EPO cytokine to initiate a downstream signaling cascade. Prior work has shown that conformational requirements of EPOR dimerization are rather “loose” and signaling may also be initiated by agonistic diabodies or in the context of chimeric receptors. While these chimeric receptors have limited clinical applicability, inducible dimerization domains such as engineered FKBPs are currently being used in clinical trials in the form of inducible safety switches. Due to the modular nature of cytokine receptors, we hypothesized that inducible dimerization domains (such as the FKBP domain) could be paired with endogenous receptors and signaling pathways to generate chimeric receptors that place RBC development under the control of a bioavailable small molecule - and therefore in the control researchers and clinicians. This study presents a set of chimeric, synthetic receptors that support HSPC output towards the erythroid lineage via a non-immunogenic, non-toxic small molecule (AP30187, hereafter termed “BB”). Methods: In-vitro editing was performed in primary human CD34+ HSPCs by electroporating Cas9 protein complexed with sgRNA along with an AAV6 DNA repair template to facilitate gene insertion via homology-directed repair (HDR). Bulk edited CD34+ HSPCs were then differentiated into RBCs using established differentiation protocols in the presence and absence of both erythropoietin and BB. Differentiation was determined by staining for RBC surface markers (CD71 and GPA) and subsequent flow cytometry analysis. Erythropoietic output was determined by cell density counts. Allele frequency of the edited cell population was determined through droplet digital PCR. Results: We generated seven FKBP-EPOR chimeric receptors - with the basic structure of SFFV-[FKBP-EPOR]-2A-YFP-bGH - that placed the FKBP binding domain at different regions of the endogenous receptor (Figure 1) and analyzed their ability to support RBC differentiation in the absence of EPO but with BB. Out of the seven constructs we found a set of receptors - 1.4 and 1.5 - that demonstrated erythropoietin-independent, BB-mediated erythroid differentiation at 108% and 94% mock RBC differentiation respectively. Construct 1.5 - dubbed inducible EPOR (iEPOR) - was chosen over 1.4 for further optimization because it was also no longer EPO responsive. We further optimized the design of iEPOR by 1) introducing a secretory signal peptide to improve trafficking of the receptor to the cell surface; 2) inclusion of a naturally occurring EPOR truncation that results in supraphysiological RBC differentiation; and 3) modification of the genomic insertion site and promoter of the iEPOR cassette to generate a toolbox of expression profiles. Our results suggest that iEPOR mimics the native EPOR signaling cascade with high fidelity and sensitivity according to bulk RNA-Seq data. We also observe a significant increase in erythropoietic output for iEPOR version 1.4 (6.7-fold by day 11 of RBC differentiation in +BB over -BB conditions, P=0.003), which was further enhanced by the above modifications for vector iEPOR version 3.5.2 (12.9-fold by day 11 of RBC differentiation in +BB over -BB conditions, P&amp;lt;0.0001). Additionally, iEPOR is demonstrated to be erythropoietin-independent, dose-responsive, and tunable via expression from different loci and promoters. Conclusion:iEPORs represent a powerful new tool to alter the cellular dynamics of red blood cell development. The work demonstrated in this study has the potential to dramatically improve the efficacies of existing gene therapies, offer a route to EPO-free ex-vivo RBC production, as well as serve as a new tool by which to better understand stem cell biology and differentiation.

FGF12: biology and function

Fibroblast growth factor 12 (FGF12) belongs to the fibroblast growth factor homologous factors (FHF) subfamily, which is also known as the FGF11 subfamily. The human FGF12 gene is located on chromosome 3 and consists of four introns and five coding exons. Their alternative splicing results in two FGF12 isoforms – the shorter ‘b’ isoform and the longer ’a’ isoform. Structurally, the core domain of FGF12, is highly homologous to that of the other FGF proteins, providing the classical tertiary structure of β-trefoil. FGF12 is expressed in various tissues, most abundantly in excitable cells such as neurons and cardiomyocytes. For many years, FGF12 was thought to be exclusively an intracellular protein, but recent studies have shown that it can be secreted despite the absence of a canonical signal for secretion. The best-studied function of FGF12 relates to its interaction with sodium channels. In addition, FGF12 forms complexes with signaling proteins, regulates the cytoskeletal system, binds to the FGF receptors activating signaling cascades to prevent apoptosis and interacts with the ribosome biogenesis complex. Importantly, FGF12 has been linked to nervous system disorders, cancers and cardiac diseases such as epileptic encephalopathy, pulmonary hypertension and cardiac arrhythmias, making it a potential target for gene therapy as well as a therapeutic agent.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a − Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1–40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion

ObjectivesChronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of...

Draft genome sequencing of Tilletia caries inciting common bunt of wheat provides pathogenicity-related genes.

Common bunt of wheat caused by Tilletia caries is an important disease worldwide. The T. caries TC1_MSG genome was sequenced using the Illumina HiSeq 2500 and Nanopore ONT platforms. The Nanopore library was prepared using the ligation sequencing kit SQK-LSK110 to generate approximately 24 GB for sequencing. The assembly size of 38.18 Mb was generated with a GC content of 56.10%. The whole genome shotgun project was deposited at DDBJ/ENA/GenBank under the accession number JALUTQ000000000. Forty-six contigs were obtained with N50 of 1,798,756 bp. In total, 10,698 genes were predicted in the assembled genome. Out of 10,698 genes, 10,255 genes were predicted significantly in the genome. The repeat sequences made up approximately 1.57% of the genome. Molecular function, cellular components, and biological processes for predicted genes were mapped into the genome. In addition, repeat elements in the genome were assessed. In all, 0.89% of retroelements were observed, followed by long terminal repeat elements (0.86%) in the genome. In simple sequence repeat (SSR) analysis, 8,582 SSRs were found in the genome assembly. The trinucleotide SSR type (3,703) was the most abundant. Few putative secretory signal peptides and pathogenicity-related genes were predicted. The genomic information of T. caries will be valuable in understanding the pathogenesis mechanism as well as developing new methods for the management of the common bunt disease of wheat.

Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

Molecular basis of Wnt biogenesis, secretion, and Wnt7-specific signaling

Wnt proteins are enzymatically lipidated by Porcupine (PORCN) in the ER and bind to Wntless (WLS) for intracellular transport and secretion. Mechanisms governing the transfer of these low-solubility Wnts from the ER to the extracellular space remain unclear. Through structural and functional analyses of Wnt7a, a crucial Wnt involved in central nervous system angiogenesis and blood-brain barrier maintenance, we have elucidated the principles of Wnt biogenesis and Wnt7-specific signaling. The Wnt7a-WLS complex binds to calreticulin (CALR), revealing that CALR functions as a chaperone to facilitate Wnt transfer from PORCN to WLS during Wnt biogenesis. Our structures, functional analyses, and molecular dynamics simulations demonstrate that a phospholipid in the core of Wnt-bound WLS regulates the association and dissociation between Wnt and WLS, suggesting a lipid-mediated Wnt secretion mechanism. Finally, the structure of Wnt7a bound to RECK, a cell-surface Wnt7 co-receptor, reveals how RECKCC4 engages the N-terminal domain of Wnt7a to activate Wnt7-specific signaling.