Haemonchus contortus: siRNA mediated knockdown of matrix metalloproteinase 12A (MMP-12) results in reduction of infectivity

Abstract

Background: RNA interference (RNAi) is an important tool to determine the role of genes. RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Matrix metalloprotease 12A (MMP-12) is known to have important roles during embryonic development, organ morphogenesis and pathological processes in animals. However, MMP-12 from Haemonchus contortus has not been characterized.Methods: Haemonchus contortus MMP-12 gene was cloned and recombinant protein of MMP-12 (rHc-MMP-12) was expressed. Binding activities of rHc-MMP-12 to goat peripheral blood mononuclear cells (PBMCs) were assessed by immunofluorescence assay (IFA) and the immuno-regulatory effects of rHc-MMP-12 on cell proliferation and nitric oxide production were observed by co-incubation of rHc-MMP-12 with goat PBMCs. Furthermore, a soaking method was used to knockdown the expression of Hc-MMP12 gene using three siRNA, targeting different regions of the gene and infectivity of effective siRNA on the development of H. contortus was evaluated in goat.Results: rHc-MMP-12 was successfully expressed in an expression vector as well as the tissues of the cuticle of adult H. contortus worms and a successful binding with PBMCs surface were observed. Increased cellular proliferation and nitric oxide production by goat PBMCs was observed in a dose-dependent manner. Quantitative real time PCR (qRT-PCR) results confirmed the successful silencing of Hc-MMP-12 gene in siRNA of 1, 2 and 3 treated third-stage larvae (L3) of H. contortus in vitro. The most efficient qRT-PCR-identified siRNA template was siRNA-2, with a 69% suppression rate compared to the control groups. Moreover, in an in vivo study, silencing of the Hc-MMP-12 gene by siRNA-2 reduced the number of eggs (54.02%), hatchability (16.84%) and worm burden (51.47%) as compared to snRNAtreated control group. In addition, a shorter length of worms in siRNA-2-treated group was observed as compared to control groups. Conclusions:Our results indicate that siRNA-mediated silencing of Hc-MMP-12 gene in H. contortus significantly reduce the egg counts, larval hatchability, and adult worm counts and sizes. The findings of the present study demonstrate important roles of Hc-MMP-12 in the development of H. contortus.

Metalloproteinases and Their Inhibitors: Potential for the Development of New Therapeutics

“…Upregulation of specific MMPs have been long associated with several diseases, and downregulation and inhibition of specific MMPs were proven to be effective in alleviating several diseases. Several MMP knockdown studies in mice were conducted to prove this point [73,88,[180][181][182][183]. Thus, inhibition of MMP catalytic activity provides a conventional strategy to inhibit progression of these diseases, and MMP inhibitors may offer potential therapy.…”

In vitro characterization of Haemonchus contortus trehalose-6-phosphate phosphatase and its immunomodulatory effects on peripheral blood mononuclear cells (PBMCs)

“…The rHcGOB protein was purified and expressed as previously described [ 36 , 37 ]. The recombinant plasmid [pET28a(+) ligated with GOB] was transformed into Escherichia coli BL21(DE3) and cultured into Luria–Bertani medium containing kanamycin at 37 °C.…”

“…Secretion of NO by cells was detected as described previously [ 36 ]. In short, the separated goat PBMCs (1 × 10 6 cells/ml) were first washed with PBS and then 100 μl of the cells in DMEM culture medium was poured into a 96-well plate, with wells containing different concentrations of rHcGOB (10, 20, 40, and 80 μg/ml) and PBS (negative control), and incubated at 37 °C in a humidified atmosphere (5% CO 2 for 24 h).…”

Genetic manipulations in helminth parasites

“…Unlike trematode parasites, RNAi in nematode is seen with a lack of visible phenotypic or morphological changes and variation in susceptibility of certain target genes, besides, some worm like Heligmosomoides polygyrus , murine intestinal nematode, are refractile to RNAi pathways (Lendner et al 2008 ). RNAi has been validated in Nippostrongylus braziliensis (Hussein et al 2002 ), Brugia malayi (Aboobaker and Blaxter 2003 ; Ford et al 2009 ; Song et al 2010 ; Misra et al 2017 ), Onchocercus volvulus (Lustigman et al 2004 ; Ford et al 2005 ), Trichostrongylus colubriformis (Issa et al 2005 ), Oestertagia ostertagi (Visser et al 2006 ), Haemonchus contortus (Geldhof et al 2006 ; Kotze and Bagnall 2006 ; Samarasinghe 2010 ; Naqvi et al 2020 ) and Ascaris suum (Xu et al 2010 ; Chen et al 2011 ; McCoy et al 2015 ) . Most of these studies used dsRNA as RNAi inducer in animal and human parasitic nematode in vitro and in vivo as well.…”

“…In the case of H. contortus , there is a discrepancy in the target gene silencing via RNAi where some transcripts are not successfully silenced as target silencing is mainly achieved in those which are accessible to the environment such as parasites alimentary canal, excretory cell and nervous system like amphids (Samarasinghe 2010 ; Naqvi et al 2020 ). It was observed that β-tubulin gene is consistently silenced where some gene failed to be silenced via RNAi in the case of H. contortus (Geldhof et al 2006 ) .…”