Inhibition of Trichinella spiralis Membrane-Associated Progesterone Receptor (MAPR) Results in a Reduction in Worm Burden

Abstract

Trichinella spiralis (T. spiralis), a nematode parasite, is the major cause of Trichinellosis, a zoonotic disease. A key role of MAPR in the reproductive system is to maintain pregnancy. Previous studies found that antihormone drug design and vaccine therapy of recombinant protein (rTs-MAPRC2) control T. spiralis infection. The current study investigates the inhibitory effects of different ratios of antibodies against Ts-MAPRC2 on the development of muscle larvae (ML) and newborn larvae (NBL). First, we performed indirect immunofluorescence assays and examined the effects of rTs-MAPRC2-Ab on ML and NBL in vitro as well as in vivo. Afterward, siRNA-Ts-MAPRC2 was transfected into T. spiralis muscle larvae. Following that, Ts-MAPRC2 protein was detected by Western Blotting, and mRNA levels were determined by qPCR. We also assessed whether siRNA-treated NBLs were infective by analyzing muscle larvae burden (MLs). Our results showed that rTs-MAPRC2-Ab greatly inhibited the activity of the Ts-MAPRC2 in ML and NBL of T. spiralis and rTs-MAPRC2-Ab reduced larval infectivity and survival in the host in a dose-dependent manner (1:50, 1:200, 1:800 dilutions). Furthermore, siRNA-Ts-MAPRC2 effectively silenced the Ts-MAPRC2 gene in muscle larvae (ML) in vitro, as well as in newborn larvae (NBL) of T. spiralis in vivo. In addition, siRNA-Ts-MAPRC2 (siRNA180, siRNA419, siRNA559) reduced host larval survival and infectivity significantly. This study, therefore, suggests that Ts-MAPRC2 might be a novel molecular target useful in the development of vaccines against T. spiralis infection.

Keywords: Trichinella spiralis; inhibitory effect; knockdown; rTs-MAPRC2-Ab; worm burden.

Figure 1

Identification of native Ts-MAPRC2 protein on the surface of T. spiralis muscle larvae (ML) analyzed by IIFT using rat sera against rTs-MAPRC2 at different concentrations (1:50, 1:200, and 1:800) with different time intervals (2 h, 4 h, 8 h, and 24 h) compared with pET32a rat serum (control group) and PBS (control group). Scale bar = 200 μm. White arrows point to positive results.

Figure 2

Identification of native Ts-MAPRC2 protein in T. spiralis newborn larvae (NBL) by IIFT using rat sera (at different concentrations) against rTs-MAPRC2. This was compared to pET32a rat serum (control group) and PBS (control group) after 24 h. Scale bar = 200 μm. White arrows point to positive results.

Figure 3

The selected concentration ratios of rTs-MAPRC2-Ab (1:50, 1:200, and 1:800) with varying time intervals, in conjunction with controls of pET32a rat serum (control group) and PBS (control group), were used at the ML stage to observe ecdysis (molting process) and motility at objective 10×. The green arrows point to ecdysis (molting process) and the black arrows represent motility.

Figure 4

Length of T. spiralis from mice treated with rTs-MAPRC2-Ab (1:50, 1:200, and 1:800 dilutions), pET32a rat serum (control group) or PBS (control group) at six days post-infection (6 dpi). (A) Length of female adult worms (µm). (B) Length of male adult worms (µm). Statistical data are presented as mean ± SD. p ≤ 0.05 was considered significant. Different letters mean significant and the same letters mean nonsignificant.

Figure 5

(A) number of adult worms collected after 6 dpi (n = 5), (B) the total number of newborn larvae collected after 24 h, and (C) the total number of muscle larvae collected after 35 days post-infection treated with rTs-MAPRC2-Ab (1:50, 1:200, and 1:800 dilutions), pET32a rat serum (control group), and PBS (control group) (n = 5). Statistical data are presented as mean ± SD. p ≤ 0.05 was considered significant. Different letters mean significant and the same letters mean nonsignificant.

Figure 6

The total number of muscle larvae collected after 35 days post-infection (35 dpi) treated NBL with rTs-MAPRC2-Ab (1:50, 1:200, and 1:800 dilutions), pET32a rat serum (control group), and PBS (control group) (n = 5). Statistical data are presented as mean ± SD. p ≤ 0.05 was considered significant. Different letters mean significant and the same letters mean nonsignificant.

Figure 7

Western blot analysis of Ts-MAPRC2 protein expression induced by siRNA. Western blot with anti-rTs-MAPRC2 antibodies showed the specific inhibition of Ts-MAPRC2 protein expression of T. spiralis muscle larvae (ML) with different siRNA-treated groups (siRNA-180, siRNA-419, siRNA-559, siRNA-Control, and PBS control) for 3 days compared with internal control (Ts-GADPH). Protein expression densities were measured by Image J software and expressed in percentages. The tests were conducted in triplicate. Data are presented as the mean ± SD. p ≤ 0.05 was considered significant. Different letters indicate significance, while the same letters indicate nonsignificance.

Figure 8

mRNA level of Ts-MAPRC2 gene induced by siRNAs. mRNA expression of the Ts-MAPRC2 gene of T. spiralis muscle larvae (ML) treated with different siRNAs (siRNA-180, siRNA-419, siRNA-559, siRNA-Control, and PBS control) for 3 days. Triplicates of the tests were performed. The data are presented as the mean ± SD. p ≤ 0.05 was considered significant. The same letter indicates insignificance, but a different letter indicates significance.

Figure 9

Various siRNAs targeting the Ts-MAPRC2 gene (siRNA-180, siRNA-419, siRNA-559) were used with varying time intervals (24 h, 48 h), in conjunction with siRNA-Control and PBS controls at the ML stage to observe motility and ecdysis (molting process) at objective 10×. The green arrows point to ecdysis (molting process) and the black arrows represent motility.

Figure 10

After 35 days post-infection (35 dpi), total muscle larvae were collected from NBLs treated with siRNAs (siRNA-180, siRNA-419, siRNA-559, siRNA-Control, and PBS). Statistical data are presented as mean ± SD. p ≤ 0.05 was considered significant. The same letters indicate significance, while different letters indicate nonsignificance.