Increased Protein Secretion Research Articles

Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

ObjectiveMost protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. MethodsIn this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. ResultsThe results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. ConclusionThese findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.

The role of endoplasmic reticulum stress in the pathogenesis of oral diseases.

The endoplasmic reticulum (ER) is crucial for protein synthesis, transport, and folding, as well as calcium storage, lipid and steroid synthesis, and carbohydrate metabolism. Endoplasmic reticulum stress (ERS) occurs when misfolded or unfolded proteins accumulate in the ER lumen due to increased protein secretion or impaired folding. While the role of ERS in disease pathogenesis has been widely studied, most research has focused on extraoral diseases, leaving the role of ERS in intraoral diseases unclear. This review examines the role of ERS in oral diseases and oral fibrosis pathogenesis. A systematic search of literature through July 2023 was conducted in the MEDLINE database (via PubMed) using specific terms related to ERS, oral diseases, and fibrosis. The findings were summarized in both table and narrative form. Emerging evidence indicates that ERS significantly contributes to the pathogenesis of oral diseases and fibrosis. ERS-induced dysregulation of protein folding and the unfolded protein response can lead to cellular dysfunction and inflammation in oral tissues. Understanding the relationship between ERS and oral disease pathogenesis could offer new therapeutic targets for managing oral health and fibrosis-related complications.

Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin.

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize β-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in β-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.

Indole-3-acetic acid regulating the initial adhesion of microalgae in biofilm formation

Regulating the microalgal initial adhesion in biofilm formation is a key approach to address the challenges of attached microalgae cultivation. As a type of phytohormone, Indole-3-acetic acid (IAA) can promote the growth and metabolism of microalgae. However, limited knowledge has been acquired of how IAA can change the initial adhesion of microalgae in biofilm formation. This study focused on investigating the initial adhesion of microalgae under different IAA concentrations exposure in biofilm formation. The results showed that IAA showed obvious hormesis-like effects on the initial adhesion ability of microalgae biofilm. Under exposure to the low concentration (0.1 mg/L) of IAA, the initial adhesion quantity of microalgae on the surface of the carrier reached the highest value of 7.2 g/m2. However, exposure to the excessively high concentration (10 mg/L) of IAA led to a decrease in the initial adhesion capability of microalgal biofilms. The enhanced adhesion of microalgal biofilms due to IAA was attributed to the upregulation of genes related to the Calvin Cycle, which promoted the synthesis of hydrophobic amino acids, leading to increased protein secretion and altering the surface electron donor characteristics of microalgal biofilms. This, in turn, reduced the energy barrier between the carriers and microalgae. The research findings would provide crucial support for the application of IAA in regulating the operation of microalgal biofilm systems.

Effects of Ficus capensis Leaves Aqueous Extracts (Lactogenic Medicinal Plant) on NMRI Mice Milk Secretion

Introduction: Ficus capensis is a plant used in traditional medicine to stimulate lactation in women and animals in Africa. However, the effects of their extracts on the mammary gland are poorly documented. The objective of the study was to evaluate the effects of Ficus capensis leaves aqueous extracts on NMRI mice milk secretion. Methodology: This was an experimental animal study using virgin female NMRI mice aged eight to ten weeks. The mice were grouped into for groups of eight mice each. Each group received one of the following products: distilled water, Galactogil™, sulpiride, aqueous extracts (AE) of capensis leaves. Data were analysed and processed using Microsoft Excel 2016 and Stata MP 16 with P ≤ .05 as the significance threshold. Results: Arborescence of the galactophorous ducts was more developed in the sulpiride lot. Galactogil™, and capensis leaves extracts treated groups showed almost equivalent arborescence with a higher tendency than the distilled water. With histological haematin- eosin staining, the ratio of galactophorous ducts containing secretions to total ducts was higher in the groups of Ficus capensis AE treated group than distilled water (P = .0001). Galactogil™, sulpiride and the group of Ficus capensis extracts each had higher levels of beta-casein in mammary tissue and average prolactinemia than distilled water (P < .01). Mammary tissue stained by immunohistochemistry with anti-prolactin receptor antibodies showed more intensely labelled mammary glands in the sulpiride and Ficus capensis extracts groups. There was no statistically significant difference between average progesteronemia among the different groups. Conclusion: F. capensis leaves AE administered to virgin female NMRI mice showed lactogenic and mammogenic effects. The extracts were able to increase the nutritional quality of the milk produced, as evidenced by the increase in protein secretion.

A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering.

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5-6 bases, corresponding to only 2-3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b-based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new-generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin-resistant E. coli strain (BL21) and a 6.49-fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

Edwardsiella piscicida requires SecY homeostasis facilitated by FtsH and YccA for stress resistance and virulence

Edwardsiella piscicida is a frequent fish pathogen that brings about huge economic losses in the aquaculture industry. Bacterial protein secretion is prominent for bacterial survival and virulence. SecY is the central subunit of Sec translocon and plays a vital role in protein secretion. However, the role of SecY in pathogenicity remains totally unknown in E. piscicida. In current study, we demonstrate that the decrease of SecY expression level in E. piscicida impairs bacterial stress tolerance including oxidative stress, high-temperature stress, antibiotic stress, and host serum stress, and weakens bacterial pathogenicity including adhesion to non-phagocyte, proliferation in phagocyte, dissemination in immune tissue, and the overall virulence. The SecY level increases in the absence of metalloproteinase FtsH and decrease in the absence of transmembrane protein YccA, indicating SecY level is controlled by FtsH and YccA. While the mutation of ftsH enhances SecY expression but diminishes bacterial stress resistance and virulence, which is similar to the phenotype caused by secY interference. Further works show that mutation of ftsH causes abnormal increase in secreted proteins and severely damaged cell membrane. We also find that secY overexpression leads to increased protein secretion and reduced survival in adversity, which is consistent with the result of secY interference. Taken together, we for the first time indicate that the homeostasis of SecY level is critical for normal physiological function of E. piscicida, which is conducive to understand the roles of SecY in bacterial stress resistance and pathogenicity.

The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa.

We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could notgrow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expressionin N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilizationin N. crassa.

Assessment of Joint Toxicity of Arsenate and Lead by Multiple Endpoints in Chlamydomonas reinhardtii.

In aquatic ecosystems, arsenate (As(V)) and lead (Pb(II)) frequently coexist but their joint toxicity on microalgae remains unknown. In this study, Chlamydomonas reinhardtii was exposed to various levels of combined As(V) and Pb(II) treatments. The cell growth, respiration, pigment synthesis, polysaccharides and protein secretion as well as As speciation of C. reinhardtii were analyzed. The low-level coexistence of As(V) and Pb(II) had a stimulatory effect, as indicated by enhanced cell proliferation. In the middle-level coexistence, the cells resisted the toxicity by significant increasing protein secretion. Under high-level coexistence, the presence of Pb(II) inhibited the efflux of As and caused the decline of cell numbers and occurrence of cell lysis, indicating that the interaction mode between As(V) and Pb(II) switched to synergistic. Taken together, the above findings may deepen the understanding of detoxification mechanisms of algae upon exposure to combined metal(loid)s in aqueous environments.

Prosthodontic implications of saliva and salivary gland dysfunction.

To provide a detailed overview of the fundamentals of saliva constituents and production. The review outlines the clinical manifestations as a consequence of salivary gland dysfunction and management strategies for patients with salivary gland dysfunction. Prosthodontic implications of saliva and salivary gland dysfunction are presented. English-language literature relating to saliva constituents, physiologic saliva production, clinical manifestations secondary to salivary gland dysfunction, salivary biomarkers, and management strategies were retrieved via electronic search. Relevant articles were summarized for this manuscript with a view toward providing pragmatic information. Saliva is produced by three pairs of major and minor salivary glands. The major salivary glands, namely, the parotid, submandibular, and sublingual glands, contribute approximately 90% of saliva production. Saliva contains serous and mucinous secretions produced by different types of cells within salivary glands. Parasympathetic and sympathetic fibers innervate the major salivary glands, and upon stimulation, the parasympathetic innervation increases serous secretions, while the sympathetic innervation increases protein secretion. Stimulated saliva is mainly derived from the parotid glands which are composed of serous acini, while unstimulated saliva is mainly derived from the submandibular glands which are composed of mixed seromucous acini. As major salivary glands contribute the most to salivary flow, local or systemic factors influencing those glands can disrupt saliva production resulting in clinically significant oral manifestations. This review provides a fundamental overview of saliva production. In addition, the review highlights the various clinical manifestations secondary to salivary gland dysfunction, explores salivary biomarkers for screening of systemic diseases, discusses management strategies for patients with salivary gland dysfunction, and outlines the prosthodontic implications of saliva and salivary gland dysfunction.

Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae.

Recombinant proteins produced by cell factories are now widely used in various fields. Many efforts have been made to improve the secretion capacity of cell factories to meet the increasing demand for recombinant proteins. Recombinant protein production usually causes cell stress in the endoplasmic reticulum (ER). The overexpression of key genes possibly removes limitations in protein secretion. However, inappropriate gene expression may have negative effects. There is a need for dynamic control of genes adapted to cellular status. In this study, we constructed and characterized synthetic promoters that were inducible under ER stress conditions in Saccharomyces cerevisiae. The unfolded protein response element UPRE2, responding to stress with a wide dynamic range, was assembled with various promoter core regions, resulting in UPR-responsive promoters. Synthetic responsive promoters regulated gene expression by responding to stress level, which reflected the cellular status. The engineered strain using synthetic responsive promoters P4UPRE2 - TDH3 and P4UPRE2 - TEF1 for co-expression of ERO1 and SLY1 had 95% higher α-amylase production compared with the strain using the native promoters PTDH3 and PTEF1. This work showed that UPR-responsive promoters were useful in the metabolic engineering of yeast strains for tuning genes to support efficient protein production.

Improved protein production in yeast using cell engineering with genes related to a key factor in the unfolded protein response

The yeast Saccharomyces cerevisiae is a widely used cell factory for protein production. Increasing the protein production capacity of a yeast strain may be beneficial for obtaining recombinant proteins as a product or exerting its competence in consolidated bioprocessing. However, heterologous protein expression usually imposes stress on cells. Improving the cell's ability to cope with stress enhances protein yield. HAC1 is a key transcription factor in the unfolded protein response (UPR). In this study, several genes related to the UPR signal pathway, including unfolded protein sensing, HAC1 mRNA splicing, mRNA ligation, mRNA decay, translation, and Hac1p degradation, were selected as targets to engineer yeast strains. The final engineered strain produced α-amylase 3.3-fold, and human serum albumin 15.3-fold, greater than that of the control strain. Key regulation and metabolic network changes in the engineered strains were identified by transcriptome analysis and physiological characterizations. This study demonstrated that cell engineering with genes relevant to the key node HAC1 in UPR increased protein secretion substantially. The verified genetic modifications of this study provide useful targets in the construction of yeast cell factories for efficient protein production.

ACTIVATION OF OSTEOBLASTS BY COBALT-CHROMIUM OCCURS VIA A TLR4-MEDIATED PATHWAY

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro.Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6).Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001).CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

PSVIII-21 Modulation of Goat Plasma Proteins Using Different Preparations of Garlic

Abstract The main objective of this study was to evaluate the effect of different garlic preparations on plasma protein concentration and Galectin secretion in goat blood. Inflammatory diseases caused by pathogens including gastrointestinal parasites impact goat gut health and productivity. Changes in plasma protein concentration are indicators of health status with application in inflammatory disease diagnosis and prognosis. Galectins (Gals) are small soluble proteins that contain one or two carbohydrate recognition domains (CRDS). ‘Prototype’ galectin-1, -2, -7, contain one CRD, Galectin 3 is chimeric, and galectin-4, -8, -9 and -12, are tandem repeat galectins with two CRDs. Galectins regulate biologic functions, immunity and disease pathogenesis and can serve as biomarkers. Garlic (Allium sativum) is a medicinal herb that has anti-parasitic and immuno-modulatory effects. Six (N=6) adult female clinically healthy Boer x Spanish goats were used. Blood was collected from the jugular vein and treated with different concentrations of fresh garlic, allicin, garlic barrier, or aged garlic (Sigma aldich). Total plasma protein secretion was evaluated using the BCA assay. Secretion of Gals 1(Prototype), Gal 3 (chimera type) and Gal 9 (tandem-repeat type) determined using commercial ELISA kits as recommended by the manufacturer. All variables were assessed using SAS analysis software (version 9.0, Sas Institute Cary, p≤0.05). Garlic treatment had a dose dependent effect on total plasma protein and Gals secretion. The protein secretion was affected by different types of garlic. Allicin (72.86μg/ml) reduced the concentration of plasma proteins. No change was observed with fresh garlic (156.334μg/ml) and garlic barrier (160.73μg/ml). Garlic barrier treatment increased protein secretion (6.3%) in goat blood. All Gals were detected (Gals 1 >9 >3), with a variable response to Garlic preparation (Garlic barrier > fresh garlic Gals secretion). Garlic modulates protein secretion. Studies are needed to determine the effect of different preparations on leukocytes and molecular targets in goat blood.

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst-mediated protein secretion.

Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age‐related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub‐proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age‐related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein‐loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst‐RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst‐RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst‐RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris.

SummaryIn our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro‐leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single‐amino‐acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis‐sorting of secretory protein into vacuoles among the wild‐type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.

A40 IDENTIFYING NOVEL ROLES FOR TLR2 SIGNALING IN THE INTESTINAL EPITHELIUM USING ORGANOIDS

Abstract Background Inflammatory bowel disease (IBD) is thought to result from an imbalance between protective and damaging immune responses to the resident microbiota and other luminal antigens. Increasing evidence suggests that toll-like-receptor (TLR)-mediated innate immune dysfunction contributes to the pathogenesis of IBD. Among the receptors in the TLR family, TLR2 and its signalling pathway appears to play an important protective role in the gastrointestinal (GI) tract. Notably, our group previously discovered that Tlr2 expression by nonhematopoietic cells played an important protective role in the Citrobacter rodentium model of infectious colitis. Aims To determine if intestinal epithelial cells (IECs) control Tlr2 dependent tissue protective responses, we sought to characterize the role of Tlr2 signalling in intestinal organoids. In addition, to better define the role of Tlr2 during C. rodentium infection, we developed a novel in vitro model of C. rodentium infection using organoid-derived monolayers. Methods Organoids were derived from the colonic tissue of wild type (C57BL/6J) and Tlr2 deficient (-/-) mice and then stimulated with Tlr2 agonists, and their responses were evaluated by qPCR, ELISA, Western blot and immunostaining. In addition, 2D monolayers were grown from organoids, and infected with C. rodentium to explore whether Tlr2 signaling regulates other protective IEC functions such as barrier proteins and cell death in response to noxious stimuli. Results Stimulation of WT, but not Tlr2-/- mouse orgnaoids led to increased transcription of chemokine and cytokine genes Ccl20, Mcp-1, Cxcl1 and Tnf-α. This was accompanied by increased protein secretion through a NF-κB and p38 MAP kinase dependent mechanism. Interestingly, organoids derived from the distal colon displayed stronger Tlr2 responses than organoids from the proximal colon. During C. rodentium infection of monolayers, Tlr2 signaling had no effect on the distribution of tight junction proteins such as ZO-1 or Claudin3, however, Tlr2 did regulate levels of IEC apoptosis upon C. rodentium infection. Conclusions Our study demonstrates that colonic organoids express functional Tlr2, which upon stimulation, leads to pro-inflammatory responses as well as control over cell death. Our novel C. rodentium infection model enables us to further study the role of IEC in promoting host defense during bacterial infection. Futher work will examine how interactions with Tlr2 dependent cytokines (ie. IL-6, IL-22) impact IEC responses to C. rodentium. Funding Agencies CCC, CIHRCH.I.L.D

CTHRC1 expressed in periodontitis and human periodontal fibroblasts exposed to inflammatory stimuli.

Collagen triple helix repeat containing-1 (CTHRC1) is a glycoprotein that can be secreted extracellularly and is involved in the regulation of collagen matrix in a variety of diseases. The expression level of CTHRC1 in periodontitis was detected in this study. The gingival tissues from clinically healthy subjects (15 cases) and those with periodontitis (30 cases) were taken for immunohistochemical staining. Lipopolysaccharide of the Porphyromonas gingivalis was added in the periodontal ligament fibroblast culture in vitro. Cells were collected, and the mRNA levels of the intracellular CTHRC1 and protein expression of the extracellular CTHRC1 were detected. The protein expression of CTHRC1 in the periodontitis group was higher than that of the clinically healthy group. The in vitro cell experiments showed that 10μg/ml of P.g LPS could induce a significant increase in protein secretion of CTHRC1, and 5μg/ml P.g LPS had a significant effect on promoting the mRNA expression of CTHRC1. Collagen triple helix repeat containing-1might be involved in the development of periodontitis, and the expression level might be significantly correlated with the stimulation of P.g LPS on fibroblasts. Different stimulation intensities of P.g LPS might result in different expression patterns of CTHRC1.