Multifunctional Thioredoxin-Like Protein from the Gastrointestinal Parasitic Nematodes Strongyloides ratti and Trichuris suis Affects Mucosal Homeostasis

Abstract

The cellular redox state is important for the regulation of multiple functions and is essential for the maintenance of cellular homeostasis and antioxidant defense. In the excretory/secretory (E/S) products of Strongyloides ratti and Trichuris suis sequences for thioredoxin (Trx) and Trx-like protein (Trx-lp) were identified. To characterize the antioxidant Trx-lp and its interaction with the parasite's mucosal habitat, S. ratti and T. suis Trx-lps were cloned and recombinantly expressed. The primary antioxidative activity was assured by reduction of insulin and IgM. Further analysis applying an in vitro mucosal 3D-cell culture model revealed that the secreted Trx-lps were able to bind to monocytic and intestinal epithelial cells and induce the time-dependent release of cytokines such as TNF-α, IL-22, and TSLP. In addition, the redox proteins also possessed chemotactic activity for monocytic THP-1 cells and fostered epithelial wound healing activity. These results confirm that the parasite-secreted Trx-lps are multifunctional proteins that can affect the host intestinal mucosa.

Figure 1

Multiple alignment of the Trx-lps from different organisms. S. ratti (CEF66761.1); T. suis (KHJ44020.1); T. trichiura (CDW52389.1); Necator americanus (XP_013304103.1); Ascaris suum (ERG80831.1); Brugia malayi (XP_001892562.1); C. elegans (NP_491127.1); Schistosoma mansoni (CD80891.1); H. sapiens (NP_004777.1). Green box represents the Trx-like domain; orange box represents the PITH domain; red circle shows the active site.

Figure 2

Predicted 3D-structure of parasite Trx-lp. The structure of SrTrx-lp is shown here. Both parasite Trx-lps have a Trx-like domain (left) as well as the PITH domain (right) (Phyre2: [55]).

Figure 3

SrTrx-lp catalyzed reduction of insulin by DTT. Here, the rate of precipitation was plotted against time. While after 40 min the reduction of the SrTrx-lp was near the equilibrium, only a minor reduction of insulin was detected in the negative control (NC) without the SrTrx-lp and the lowest concentration used of SrTrx-lp (1 μM).

Figure 4

IgM reduction by the Trx-lps from S. ratti (a) and T. suis (b). Prior to incubation, the Trx-lps from both organisms were reduced by DTT. IgM was split in its chains (25 kDa, 70 kDa, and 950 kDa).

Figure 5

Binding of the SrTrx-lp (a) and TsTrx-lp (b) to different cell types. 2 × 10⁵ cells were incubated at 37°C for 30 min with Alexa Flour®-labeled SrTrx-lp or TsTrx-lp. Here, peripheral blood cells (monocytes, granulocytes, and lymphocytes) as well as cell culture cells (Caco-2 cells, THP-1 cells, and THP-1-derived DCs) were tested with 0.4 μg (purple (a), red (b) line) and 0.6 μg (blue (a), green (b) line) of labeled protein determining the median fluorescent intensity (MFI). The intensity of surface fluorescence (FI, x-axis) is plotted against cell counts. (The counts in the figures represent the median fluorescence index values.) Representative results of five independent experiments are shown.

Figure 6

(a) Exposure of 3D-cocultures to Trichuris suis (Ts) extract. Culture supernatants were harvested after 24 h, 48 h, and 72 h. The release of inflammatory (TNF-α), anti-inflammatory (IL-10), and T_H2-related cytokines (IL-22, TSLP) was analyzed in a 3D-cell culture model. Representative results of at least three independent experiments are shown as median. (b) Exposure of 3D-cocultures to SrTrx- and TsTrx-lp or medium (NC). Culture supernatants were harvested after 24 h (A), 48 h (B), and 72 h (C). The release of inflammatory (TNF-α), anti-inflammatory (IL-10), and T_H2-related cytokines (IL-22, TSLP) was analyzed in a 3D-cell culture model. Representative results of at least three independent experiments are shown. Significant increase of all measured cytokines compared to NC (^∗∗ P < 0.01). ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 7

Chemotactic activity of the Trx-lp from S. ratti (a) and T. suis (b) for monocytic THP-1 cells. The chemotactic activity of both proteins for THP-1 cells was investigated by using Boyden chambers. Different concentrations of the Trx-lps were added to the lower compartment of the chemotactic chambers. Protein concentrations per 100 μL of 3 ng, 30 ng, 300 ng, and 1 μg showed SrTrx-lp and TsTrx-lp have the greatest chemotactic activity at 3 ng. Chemotaxis buffer (NC) and THP-1 media (RPMI + FCS) were included as negative control (random cell migration), while LPS was used as positive control. All used Trx-lp concentrations led to significant higher cell migration than the negative controls (^∗∗ P < 0.01).

Figure 8

Percentage closure of the Caco-2 wound gap after 24 h (a), 48 h (b), 72 h (c), and 96 h (d). In general, the gap was narrowed by approx. 10–15% every day adding no stimulus. As a negative control, cells were observed without any stimulus (NC) and with LPS (1 μg). As a positive control, epidermal growth factor (EGF) was used, whereat 10 ng fostered wound healing the best. SrTrx-lp and TsTrx-lp were tested at different concentrations (3 ng, 30 ng, 300 ng, 1 μg, 10 μg, and 25 μg–each per 500 μL), here at 300 ng represented as the best wound healing promoting concentration. The wound healing process was highly significantly promoted by EGF and TsTrx-lp (^∗∗ P < 0.01) as well as significantly promoted by SrTrx-lp (^∗ P < 0.05).

Figure 9

Wound healing assay with Caco-2 cells and the S. ratti as well as the T. suis Trx-lp. The CytoSelect™ 24-Well Wound healing assay was performed. Here, two examples are described as representatives for different tested concentrations. Protein concentrations of 300 ng SrTrx-lp are given as example for best concentration for wound healing promotion and 25 μg of TsTrx-lp (each per 500 μL) indicated the less wound healing-promoting concentration. The wound healing process of a 0.9 mm wound field generated was observed over 96 h, whereby each 24 h a picture was taken. The size of the scale bar is 1000 μm and the dashed black lines indicate wound-like area [56].