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Disruption of Epithelial Barrier of Caco-2 Cell Monolayers by Excretory Secretory Products of Trichinella spiralis Might Be Related to Serine Protease.

ABSTRACT: The physical barrier is composed of epithelial cells which are joined together through intercellular connections. It serves to prevent pathogenic microorganisms from departing the intestinal lumen to invade the host. The excretory secretory (ES) products of Trichinella spiralis are critical for invasion. However, whether ES products of T. spiralis can act on the intestinal barrier is still unknown. In this study, the role of ES products of T. spiralis muscle larvae (Ts-ML-ES) in host invasion was studied by establishing an in vitro cell monolayers model. Barrier integrity analysis by a transmembrane resistance test and a paracellular permeability assay revealed that the Ts-ML-ES was able to destroy barrier function. It occurred via a reduction in the expression of tight junction (TJ) proteins, which was induced by serine protease. Furthermore, Western bolt analysis indicated that Ts-ML-ES reduced the expression of TJ proteins via the MAPK signaling pathway. Based on these data, we conclude that serine protease are likely the main factors from Ts-ML-ES that affect host intestinal barrier integrity by reducing the expression of TJs via the P38-MAPK signaling pathway. Serine protease in Ts-ML-ES might be a key invasion factor in T. spiralis.

SUBMITTER: Li C

PROVIDER: S-EPMC8013981 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Disruption of Epithelial Barrier of Caco-2 Cell Monolayers by Excretory Secretory Products of Trichinella spiralis Might Be Related to Serine Protease.

Li Chengyao C Bai Xue X Liu Xiaolei X Zhang Yuanyuan Y Liu Lei L Zhang Lixiao L Xu Fengyan F Yang Yong Y Liu Mingyuan M

Frontiers in microbiology 20210317

The physical barrier is composed of epithelial cells which are joined together through intercellular connections. It serves to prevent pathogenic microorganisms from departing the intestinal lumen to invade the host. The excretory secretory (ES) products of Trichinella spiralis are critical for invasion. However, whether ES products of T. spiralis can act on the intestinal barrier is still unknown. In this study, the role of ES products of T. spiralis muscle larvae (Ts</i ...[more]

PMID: 33815318

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Project description:BackgroundIntestinal larva invasion is a crucial step of Trichinella spiralis infection. Intestinal infective larvae (IIL) and their excretory/secretory proteins (ESP) interact with gut epithelium, which often results in gut epithelium barrier injuries. Previous studies showed when T. spiralis invaded intestinal epithelium cells, the IIL ESP disrupted the tight junctions (TJs) of Caco-2 monolayer, but the mechanism is not clear. The IIL ESP might cause gut epithelial apoptosis, weaken the gut barrier and aid the larval invasion. The aim of this study was to investigate whether T. spiralis IIL ESP participate in enterocyte apoptosis and disrupt gut epithelial barrier to promote the larval invasion.Methodology/principal findingsCell viability was assessed by CCK-8 assay and the results showed that 200 μg/ml of IIL ESP incubated with Caco-2 cells for 18 h inhibited the Caco-2 cell viability. The results of trans-epithelial electrical resistance (TEER) and FITC-dextran showed that IIL ESP decreased the TEER, increased FITC-dextran flux in Caco-2 monolayer. qPCR, Western blot and immunofluorescence test (IFT) showed that IIL ESP decreased the mRNA and protein expression of TJs (ZO-1, E-cad, Occludin and Claudin-1). The IIL ESP-induced Caco-2 cell apoptosis was observed by DAPI, Hoechst 33358, TUNEL and Annexin V/PI staining. Besides, flow cytometry revealed an increasing apoptosis rate in Caco-2 cells after the IIL ESP treatment. qPCR and Western blot analysis indicated that IIL ESP activated caspases (Caspase 3, Caspase 9 and Caspase 8), up-regulated the pro-apoptotic factors (Bax and Cytochrome c) and down-regulated the anti-apoptosis molecule Bcl-2. Interestingly, pretreatment of Caco-2 cells with apoptosis inhibitor Z-VAD-FMK abrogated and recovered the barrier function of Caco-2 monolayer destroyed by IIL ESP. Furthermore, the Z-VAD-FMK pretreatment also impeded the in vitro larva invasion of Caco-2 monolayer.ConclusionsT. spiralis IIL ESP induced gut epithelial apoptosis, reduced the TJs expression, damaged gut epithelial integrity and barrier function, and promoted larval invasion. These findings provided a basis of further understanding the interaction mechanism between T. spiralis and host gut epithelium, and they were valuable to the development new prevention and therapeutic strategy of early T. spiralis infection.

Project description:The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Project description:BackgroundTrichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi.ResultsAccording to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase.ConclusionsBoth 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.

Dataset Information

Disruption of Epithelial Barrier of Caco-2 Cell Monolayers by Excretory Secretory Products of Trichinella spiralis Might Be Related to Serine Protease.

Publications

Disruption of Epithelial Barrier of Caco-2 Cell Monolayers by Excretory Secretory Products of <i>Trichinella spiralis</i> Might Be Related to Serine Protease.

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