Non-classical Secretion Pathway Research Articles

Echinococcus granulosus ubiquitin-conjugating enzymes (E2D2 and E2N) promote the formation of liver fibrosis in TGFβ1-induced LX-2 cells

BackgroundCystic echinococcosis (CE) is a widespread zoonosis caused by the infection with Echinococcus granulosus sensu lato (E. granulosus s.l.). CE cysts mainly develop in the liver of intermediate hosts, characterized by the fibrotic tissue that separates host organ from parasite. However, precise mechanism underlying the formation of fibrotic tissue in CE remains unclear.MethodsTo investigate the potential impact of ubiquitin-conjugating enzymes on liver fibrosis formation in CE, two members of ubiquitin-conjugating (UBC) enzyme of Echinococcus granulosus (EgE2D2 and EgE2N) were recombinantly expressed in Escherichia coli and analyzed for bioinformatics, immunogenicity, localization, and enzyme activity. In addition, the secretory pathway and their effects on the formation of liver fibrosis were also explored.ResultsBoth rEgE2D2 and rEgE2N possess intact UBC domains and active sites, exhibiting classical ubiquitin binding activity and strong immunoreactivity. Additionally, EgE2D2 and EgE2N were widely distributed in protoscoleces and germinal layer, with differences observed in their distribution in 25-day strobilated worms. Further, these two enzymes were secreted to the hydatid fluid and CE-infected sheep liver tissues via a non-classical secretory pathway. Notably, TGFβ1-induced LX-2 cells exposed to rEgE2D2 and rEgE2N resulted in increasing expression of fibrosis-related genes, enhancing cell proliferation, and facilitating cell migration.ConclusionsOur findings suggest that EgE2D2 and EgE2N could secrete into the liver and may interact with hepatic stellate cells, thereby promoting the formation of liver fibrosis.Graphical

Efficient secreted expression of natural intracellular β-galactosidase from Bacillus aryabhattai via non-classical protein secretion pathway in Bacillus subtilis

In this study, the natural intracellular β-galactosidase (lacZBa) from Bacillus aryabhattai was expressed extracellularly in Bacillus subtilis. Sec and Tat signal peptides from different secretion pathways were incorporated to facilitate extracellular secretion of lacZBa, resulting in a yield of only 0.54 U/mL. Interestingly, it was discovered that lacZBa could be efficiently expressed and secreted in B. subtilis via a non-classical secretory pathway without the need for a signal peptide. The extracellular activity and secretion ratio were 5.3 U/mL and 65 %, respectively. Compared to E. coli, the expression of lacZBa in B. subtilis resulted in increased acid resistance and higher pH stability and thermostability, with a 1.7-fold increase in half-life at 50 °C and pH 6.0. Additionally, we combined single-factor experiments and response surface methodology to enhance extracellular expression of β-galactosidase in shake-flasks. The resulting optimal medium contained 4.46 % glucose, 1.47 % corn steep liquor, 1.5 % beef extract, 0.82 % CaCl2, and 0.1 % MgSO4. Under optimal conditions, the yield of extracellularly secreted β-galactosidase at the shake flask level was 17.41 U/mL, representing a 32.2-fold increase in initial extracellular enzyme activity. This study represents the first successful report of natural intracellular β-galactosidase being expressed through the non-classical secretory pathway in B. subtilis.

Metabolomic Analysis of Extracellular Vesicles from the Cereal Fungal Pathogen Fusarium graminearum.

Fusarium graminearum (F. graminearum) is a filamentous fungus that infects cereals such as corn, wheat, and barley, with serious impact on yield as well as quality when the grain is contaminated with mycotoxins. Despite the huge impact of F. graminearum on food security and mammalian health, the mechanisms used by F. graminearum to export virulence factors during infection are not fully understood and may involve non-classical secretory pathways. Extracellular vesicles (EVs) are lipid-bound compartments produced by cells of all kingdoms that transport several classes of macromolecules and are implicated in cell-cell communication. EVs produced by human fungal pathogens carry cargo that facilitate infection, leading us to ask whether plant fungal pathogens also deliver molecules that increase virulence via EVs. We examined the metabolome of the EVs produced by F. graminearum to determine whether they carry small molecules that could modulate plant-pathogen interactions. We discovered that EVs from F. graminearum were produced in liquid medium-containing inducers of trichothecene production, but in lower quantities compared to other media. Nanoparticle tracking analysis and cryo-electron microscopy revealed that the EVs were morphologically similar to EVs from other organisms; hence, the EVs were metabolically profiled using LC-ESI-MS/MS. This analysis revealed that EVs carry 2,4-dihydroxybenzophenone (BP-1) and metabolites that have been suggested by others to have a role in host-pathogen interactions. BP-1 reduced the growth of F. graminearum in an in vitro assay, suggesting that F. graminearum might use EVs to limit metabolite self-toxicity.

Regulation of Cell Membrane Permeability Enhanced the Non-Classical Secretion of γ-Cyclodextrin Glycosyltransferase in Bacillus subtilis

γ-Cyclodextrin glycosyltransferase (γ-CGTase, EC 2.4.1.19) is an essential enzyme required in the production of γ-cyclodextrin, which shows huge prospects in the food, medicine, materials, and chemical industries. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was successfully cloned and expressed in Bacillus subtilis WB600. The final extracellular activity of γ-CGTase can reach 45.34 U/mL with the deletion of the signal peptide, which was about 11.3 times of the initial level of γ-CGTase secreted by the general pathway. By monitoring the whole cultivation process, secretion was divided into two stages, which were dominated by cell membrane changes and apoptosis. The measurement of lactate dehydrogenase and the results of fluorescence microscopy demonstrated that the cell membrane permeability changed significantly in the middle stage of fermentation, proving that it played a crucial role in the non-classical secretion of γ-CGTase. Furthermore, the addition of Triton X-100, a non-ionic surfactant, remarkably enhanced the secretion level of γ-CGTase by 21.5%, which was caused by the increase in cell membrane permeability. This work is the first to obtain the extracellular expression of CGTase via the non-classical secretion pathway in B. subtilis and provides a new strategy to enhance extracellular expression by regulating the cell membranes.

Abstract 5919: Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer

Abstract Background: Secretory Leukocyte Peptidase Inhibitor (SLPI) functions in cancer progression, cellular invasion, metastasis, immune evasion, and chemoresistance. Our previous studies showed increased SLPI with overexpressed NFX1-123 and co-expressed high-risk (HR) human papillomavirus type 16 E6 oncogene (16E6) in primary keratinocytes (HFKs) as well as high SPLI levels in cervical cancers. The HR viral oncogenes E6 and E7 partner with host cell proteins to regulate cell cycle regulation and immortalization pathways. Understanding the mechanisms of SLPI upregulation in HPV-associated cancers is critical to validating SLPI as a non-invasive biomarker as well as to test its utility in future cancer treatments. This study was designed to quantify intracellular and secreted SLPI protein in cervical cancer and to determine the roles NFX1-123 and 16E6 have on SLPI expression and secretion. Methods: Using HPV- and HPV+ cervical cancer cell lines and HFKs transduced with overexpressed NFX1-123 and 16E6, or vector controls, we measured intracellular and secreted SLPI by Western blot and ELISA, respectively. In HPV+ cervical cancer cell lines, NFX1-123 was knocked down or out by shRNA or CRISPR-Cas9, respectively, and 16E6 and 16E7 were knocked down by siRNA. In those cells, intracellular and secreted SLPI was quantified and the relative role of NFX1-123, 16E6, and 16E7 were studied. Selected classical and non-classical secretory pathways markers were also measured to assess their effects on SLPI secretion. Finally, SLPI’s concentration was measured by ELISA in cervical cancer patient sera and compared to healthy controls. Results: Our results showed increased intracellular as well as secreted SLPI in HFKs with overexpressed NFX1-123 and 16E6. Similarly, we found increased SLPI secretion in HPV+ cervical cancer cell lines (SiHa, Caski and HeLa) when compared to HPV- C33A cells. A reduction in NFX1-123 and 16E6 led to decreased SLPI secretion, pointing to a regulation of SLPI by NFX1-123 and 16E6 on SLPI secretion. Analysis of classical and non-classical secretory pathway markers suggested that NFX1-123 regulated non-classical pathways, which may drive greater secretion of SLPI in HPV+ cervical cancers. Higher SLPI protein amounts were found cervical cancer patient sera when compared to healthy controls. Conclusions: Our results suggest a functional role for NFX1-123 and 16E6 in SLPI secretion in cervical cancer and that SLPI may be a potential biomarker in cervical cancer. Citation Format: Sreenivasulu Chintala, Kevin Quist, Rachel Katzenellenbogen. Regulation of SLPI secretion by NFX1-123 and HPV 16 E6 in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5919.

Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis. The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway. E. coli is a useful host for recombinant enzymes used in biocatalysis due to the ease of genetic manipulation in this organism, which allows tuning of enzymes for specific applications, by mutagenesis. When a truncated GST-tagged AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when GST-tagged AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in both the periplasmic space and the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (AmCut) was being recognised by the Gram-negative (E. coli) Sec transmembrane transport system. AmCut was transported into E. coli’s periplasmic space from where it was released into the culture medium. Surprisingly, the presence of a bulky GST tag at the N-terminus of the signal peptide did not hinder transmembrane targeting. Although the periplasmic targeting was unexpected, it is not unprecedented due to the conservation of the Sec pathway across species. It was more surprising that AmCut was secreted from the periplasmic space into the culture medium. This suggests that extracellular AmCut translocation across the E. coli outer membrane may involve non-classical secretion pathways. This tuneable recombinant E. coli expressing extracellular AmCut may be useful for degradation of polyester substrates in the environment; this and other applications are discussed.Graphical abstract

ASPIRER: a new computational approach for identifying non-classical secreted proteins based on deep learning.

Protein secretion has a pivotal role in many biological processes and is particularly important for intercellular communication, from the cytoplasm to the host or external environment. Gram-positive bacteria can secrete proteins through multiple secretion pathways. The non-classical secretion pathway has recently received increasing attention among these secretion pathways, but its exact mechanism remains unclear. Non-classical secreted proteins (NCSPs) are a class of secreted proteins lacking signal peptides and motifs. Several NCSP predictors have been proposed to identify NCSPs and most of them employed the whole amino acid sequence of NCSPs to construct the model. However, the sequence length of different proteins varies greatly. In addition, not all regions of the protein are equally important and some local regions are not relevant to the secretion. The functional regions of the protein, particularly in the N- and C-terminal regions, contain important determinants for secretion. In this study, we propose a new hybrid deep learning-based framework, referred to as ASPIRER, which improves the prediction of NCSPs from amino acid sequences. More specifically, it combines a whole sequence-based XGBoost model and an N-terminal sequence-based convolutional neural network model; 5-fold cross-validation and independent tests demonstrate that ASPIRER achieves superior performance than existing state-of-the-art approaches. The source code and curated datasets of ASPIRER are publicly available at https://github.com/yanwu20/ASPIRER/. ASPIRER is anticipated to be a useful tool for improved prediction of novel putative NCSPs from sequences information and prioritization of candidate proteins for follow-up experimental validation.

Regulate the hydrophobic motif to enhance the non-classical secretory expression of Pullulanase PulA in Bacillus subtilis

Bacillus subtilis has been widely used as a prokaryotic host for the secretory expression of heterologous proteins. In this study, a pullulanase (PulA) from Anoxybacillus sp. LM18-11 was firstly identified to be expressed in Bacillus subtilis 1A751 through non-classical secretion pathway. Results showed that both the N- and C-terminal regions of PulA were essential for its soluble expression. To explore its specific structural basis of secretion in B. subtilis, we revealed a hydrophobic motif A501-H507 which is vital for the secretion of the whole protein of PulA. Through a series of site-specific mutagenesis, the triple-sites mutants R503E/I506E/H507E and R503E/I506Y/H507E showed the highest extracellular activity (160.07 U/mL) and total activity (243.37 U/mL) which was 1.71 times and 1.55 times higher than those of PulA. The highest secretion rate of mutant I506E/H507E was more than 50% which was 34.72% higher comparing with that of PulA. The glutamic acid substitution on these three key surface sites which decreased the surface hydrophobicity of that region was confirmed to be beneficial to improve the secretory expression of PulA. This novel discovery for the secretory expression of PulA in B. subtilis would make a new perspective on regulating a kind of non-classical secretion in B. subtilis.

FtsH is required for protein secretion homeostasis and full bacterial virulence in Edwardsiella piscicida

Edwardsiella piscicida, as an important pathogen of fish, has caused huge losses in aquaculture. The virulence in E. piscicida has been increasingly concerned, but few studies have focused on the relationship between virulence and protein secretion homeostasis. FtsH, as a member of the AAA protease family, has important cellular functions, such as controlling the quality of membrane proteins. In this study, FtsH was demonstrated to be essential in maintaining protein secretion homeostasis, and its deletion could result in the secretion of massive cytoplasmic proteins by non-classical secretion pathway. Furthermore it was showed that FtsH is vital for E. piscicida to proliferate within host cells, and E. piscicida mutant ΔftsH will be obviously attenuated. After zebrafish was infected with the mutant ΔftsH, the lethality rate for zebrafish and the bacterial colonization in its organs were greatly reduced. These results suggested that FtsH, as a regulatory factor, closely linked protein secretory homeostasis with bacterial virulence, which provided clues for further exploring the involvement of protein secretion homeostasis in bacterial virulence.

In Vitro Secretome Analysis Suggests Differential Pathogenic Mechanisms between Fusarium oxysporum f. sp. cubense Race 1 and Race 4.

Banana Fusarium wilt, caused by the fungus pathogen Fusarium oxysporum f. sp. cubense (Foc), is a devastating disease that causes tremendous reductions in banana yield worldwide. Secreted proteins can act as pathogenicity factors and play important roles in the Foc–banana interactions. In this study, a shotgun-based proteomic approach was employed to characterize and compare the secretomes of Foc1 and Foc4 upon banana extract treatment, which detected 1183 Foc1 and 2450 Foc4 proteins. Comprehensive in silico analyses further identified 447 Foc1 and 433 Foc4 proteins in the classical and non-classical secretion pathways, while the remaining proteins might be secreted through currently unknown mechanisms. Further analyses showed that the secretomes of Foc1 and Foc4 are similar in their overall functional characteristics and share largely conserved repertoires of CAZymes and effectors. However, we also identified a number of potentially important pathogenicity factors that are differentially present in Foc1 and Foc4, which may contribute to their different pathogenicity against banana hosts. Furthermore, our quantitative PCR analysis revealed that genes encoding secreted pathogenicity factors differ significantly between Foc1 and Foc4 in their expression regulation in response to banana extract treatment. To our knowledge, this is the first experimental secretome analysis that focused on the pathogenicity mechanism in different Foc races. The results of this study provide useful resources for further exploration of the complicated pathogenicity mechanisms in Foc.

Monoamine Oxidase-B Inhibition Facilitates α-Synuclein Secretion In Vitro and Delays Its Aggregation in rAAV-Based Rat Models of Parkinson's Disease.

Cell-to-cell transmission of α-synuclein (α-syn) pathology is considered to underlie the spread of neurodegeneration in Parkinson's disease (PD). Previous studies have demonstrated that α-syn is secreted under physiological conditions in neuronal cell lines and primary neurons. However, the molecular mechanisms that regulate extracellular α-syn secretion remain unclear. In this study, we found that inhibition of monoamine oxidase-B (MAO-B) enzymatic activity facilitated α-syn secretion in human neuroblastoma SH-SY5Y cells. Both inhibition of MAO-B by selegiline or rasagiline and siRNA-mediated knock-down of MAO-B facilitated α-syn secretion. However, TVP-1022, the S-isomer of rasagiline that is 1000 times less active, failed to facilitate α-syn secretion. Additionally, the MAO-B inhibition-induced increase in α-syn secretion was unaffected by brefeldin A, which inhibits endoplasmic reticulum (ER)/Golgi transport, but was blocked by probenecid and glyburide, which inhibit ATP-binding cassette (ABC) transporter function. MAO-B inhibition preferentially facilitated the secretion of detergent-insoluble α-syn protein and decreased its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Moreover, in a rat model (male Sprague Dawley rats) generated by injecting recombinant adeno-associated virus (rAAV)-A53T α-syn, subcutaneous administration of selegiline delayed the striatal formation of Ser129-phosphorylated α-syn aggregates, and mitigated loss of nigrostriatal dopaminergic neurons. Selegiline also delayed α-syn aggregation and dopaminergic neuronal loss in a cell-to-cell transmission rat model (male Sprague Dawley rats) generated by injecting rAAV-wild-type α-syn and externally inoculating α-syn fibrils into the striatum. These findings suggest that MAO-B inhibition modulates the intracellular clearance of detergent-insoluble α-syn via the ABC transporter-mediated non-classical secretion pathway, and temporarily suppresses the formation and transmission of α-syn aggregates.SIGNIFICANCE STATEMENT The identification of a neuroprotective agent that slows or stops the progression of motor impairments is required to treat Parkinson's disease (PD). The process of α-synuclein (α-syn) aggregation is thought to underlie neurodegeneration in PD. Here, we demonstrated that pharmacological inhibition or knock-down of monoamine oxidase-B (MAO-B) in SH-SY5Y cells facilitated α-syn secretion via a non-classical pathway involving an ATP-binding cassette (ABC) transporter. MAO-B inhibition preferentially facilitated secretion of detergent-insoluble α-syn protein and reduced its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Additionally, MAO-B inhibition by selegiline protected A53T α-syn-induced nigrostriatal dopaminergic neuronal loss and suppressed the formation and cell-to-cell transmission of α-syn aggregates in rat models. We therefore propose a new function of MAO-B inhibition that modulates α-syn secretion and aggregation.

Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis

Phospholipids (PLs) are one of the main ingredients in food and nutraceutical, cosmetics, agriculture, and pharmaceutical products. Phospholipase D (PLD) is a crucial enzyme for the biocatalytic synthesis or modification of PLs. Here, to prepare PLD more efficiently, we constructed a PLD expression and secretion system in Bacillus subtilis and developed an environmentally friendly reaction system. A nonclassical secretory pathway where a super-folder green fluorescent protein plays as an N-terminal guide protein was introduced. This expression system can not only achieve rapid screening of high-level expression strains but can also achieve the secretion of the target proteins. Under optimal fermentation conditions, the enzyme activity of the culture medium was 0.35 U/mL, which was 2.05-fold that of the Sec secretion pathway strains. Meanwhile, the effects of several organic solvents in the biphasic reaction media were compared. The results showed that when using cyclopentyl methyl ether as the organic phase, the final conversion rate reached 96.9%. It has shown good application potential in the synthesis of phosphatidylserine, laid the foundation for the synthesis and application of other rare and high-value PLs, and provided a reference for the production of other biocatalysts.

Silico Functional Annotation and Molecular Characterization of an Uncharacterized Protein MBO_502153 of Mycobacterium Tuberculosis Variant Bovis

Background and aim: Bovine, zoonotic, and wildlife tuberculosis are caused by Mycobacterium Bovis, whose attenuated form is used in tuberculosis and cancer treatment. There are several proteins present in its genome considered as uncharacterized or hypothetical proteins. We aim to predict the structure and function of an uncharacterized protein present in the bacterial genome of this pathogenic bacteria, Mycobacterium Bovis, by using several bioinformatics tools. Materials and methods: Using in silico techniques, the secondary and tertiary structures of the selected uncharacterized proteins were predicted and further validated. Bioinformatics tools were used to explore physicochemical characteristics, homologous proteins, the active site, and the protein-protein interactions. Results: By in silico study, we found that though the bacterium is a pathogenic microorganism, its uncharacterized protein MBO_502153 is non–virulent. This protein is a metabolism molecule. As the protein does not contain any kind of signal peptide, it is not a secretory protein. The uncharacterized protein MBO_502153 also does not involve any non-classical secretory pathway. Conclusion: It can be concluded that the purposeful annotation of uncharacterized supermolecule showed some chemical activities, particularly degradation of the environmental waste product 4-chlorobenzoate and xenobiotics and synthesis of polyhydroxyalkanoate (PHA), which play an important role in environmental biodegradability and biocompatibility.

Non-classical secretion of a type I L-asparaginase in Bacillus subtilis

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

Comparative analysis of extracellular proteomes reveals putative effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata.

Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) are destructive fungal pathogens causing boxwood blight, a persistent threat to horticultural production, landscape industries, established gardens, and native ecosystems. Although extracellular proteins including effectors produced by fungal pathogens are known to play a fundamental role in pathogenesis, the composition of Che and Cps extracellular proteins has not been examined. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics prediction tools, 630 extracellular proteins and 251 cell membrane proteins of Che and Cps were identified in the classical secretion pathway in the present study. In the non-classical secretion pathway, 79 extracellular proteins were identified. The cohort of proteins belonged to 364 OrthoMCL clusters, with the majority (62%) present in both species, and a subset unique to Che (19%) and Cps (20%). These extracellular proteins were predicted to play important roles in cell structure, regulation, metabolism, and pathogenesis. A total of 124 proteins were identified as putative effectors. Many of them are orthologs of proteins with documented roles in suppressing host defense and facilitating infection processes in other pathosystems, such as SnodProt1-like proteins in the OrthoMCL cluster OG5_152723 and PhiA-like cell wall proteins in the cluster OG5_155754. This exploratory study provides a repository of secreted proteins and putative effectors that can provide insights into the virulence mechanisms of the boxwood blight pathogens.

A novel chorismate mutase from Erysiphe quercicola performs dual functions of synthesizing amino acids and inhibiting plant salicylic acid synthesis

Pathogens secrete effectors to establish a successful interaction with their host. It is well understood that plant pathogens recruit classically secreted chorismate mutase (Cmu) as an effector to disrupt plant salicylic acid (SA) synthesis. However, the identity and function of the Cmu effector from powdery mildew fungi remain unknown. Here, we identified a novel secreted Cmu effector, EqCmu, from rubber (Hevea brasiliensis Muell) powdery mildew fungus (Erysiphe quercicola). Unlike the classically secreted Cmu, EqCmu lack signal peptide, and exhibited characteristics of non-classically secreted proteins. EqCmu could fully complement a Saccharomyces cerevisiae ScAro7 mutant that was deficient in the synthesis of phenylalanine and tyrosine. In addition, transient expression of EqCmu could promote infection by Phytophthora capsici and reduce the levels of SA and the mRNA of PR1 gene in Nicotiana benthamiana in response to P. capsici infection, while confocal observations showed that EqCmu was localized within the cytoplasm and nucleus of transfected N. benthamiana leaf cells. These non-homologous systems assays provide evidences that EqCmu may serve as a “moonlighting” protein, which is not only a key enzyme in the synthesis of phenylalanine and tyrosine within fungal cells, but also has the function of regulating plant SA synthesis within plant cells. This is the first study to identify and functionally validate a candidate effector from E. quercicola. Overall, the non-classical secretion pathway is a novel mechanism for powdery mildew fungal effectors secretion and might play an important role in host–pathogen interactions.

CD44, a Predominant Protein in Methylglyoxal-Induced Secretome of Muscle Cells, is Elevated in Diabetic Plasma.

Methylglyoxal (MG), a glycolytic intermediate and reactive dicarbonyl, is responsible for exacerbation of insulin resistance and diabetic complication. In this study, MG-induced secretome of rat muscle cells was identified and relatively quantified by SWATH-MS. A total of 643 proteins were identified in MG-induced secretome, of which 82 proteins were upregulated and 99 proteins were downregulated by more than 1.3-fold in SWATH analysis. Further, secretory proteins from the classical secretory pathway and nonclassical secretory pathway were identified using SignalP and SecretomeP, respectively. A total of 180 proteins were identified with SignalP, and 113 proteins were identified with SecretomeP. The differentially expressed proteins were functionally annotated by KEGG pathway analysis using Cytoscape software with plugin clusterMaker. The differentially expressed proteins were found to be involved in various pathways like extracellular matrix (ECM)–receptor interaction, leukocyte transendothelial migration, fluid shear stress and atherosclerosis, complement and coagulation cascades, and lysosomal pathway. Since the MG levels are high in diabetic conditions, the presence of MG-induced secreted proteins was inspected by profiling human plasma of healthy and diabetic subjects (n = 10 each). CD44, a predominant MG-induced secreted protein, was found to be elevated in the diabetic plasma and to have a role in the development of insulin resistance.

Tumour Progression Stage-Dependent Secretion of YB-1 Stimulates Melanoma Cell Migration and Invasion.

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficient Bacillus subtilis

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC–MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

Principle and potential applications of the non-classical protein secretory pathway in bacteria.

In addition to the extracellular proteins secreted by known secretory pathways, a number of cytoplasmic proteins without predicable or known signal sequences or secretory motifs have been found in the extracellular milieu, and were consequently classified as non-classically secreted proteins. Non-classical protein secretion is considered to be a general, conserved cellular phenomenon in both eukaryotes and prokaryotes. There are several research hotspots on the non-classical protein secretory pathway, and the most important two of them are the recognition principle of substrate proteins and possible secretory mechanisms. To date, researchers have made some progress in understanding the characteristics of these proteins. For example, it was discovered that many non-classically secreted proteins exist and are secreted in multimeric form. Some of these proteins prefer to be clustered and exported at the poles and the septum of the cell. The majority of these proteins play different functions when they are in the intra- and extracellular environments, and several of their functions are related to survival and pathogenicity. Furthermore, non-classically secreted proteins can be used as leading proteins to guide a POI (protein of interest) out of the cells, which provides a novel strategy for protein secretion with potential applications in the industry. Summarizing these findings, this review emphasizes the hot spots related to non-classically secreted proteins in bacteria, lists the most important hypotheses on the selection and secretion mechanisms of non-classically secreted proteins, and put forward their potential applications.