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PirABVP Toxin Binds to Epithelial Cells of the Digestive Tract and Produce Pathognomonic AHPND Lesions in Germ-Free Brine Shrimp - PubMed

  • ️Tue Jan 01 2019

PirABVP Toxin Binds to Epithelial Cells of the Digestive Tract and Produce Pathognomonic AHPND Lesions in Germ-Free Brine Shrimp

Vikash Kumar et al. Toxins (Basel). 2019.

Abstract

Acute hepatopancreatic necrosis disease (AHPND), a newly emergent farmed penaeid shrimp bacterial disease originally known as early mortality syndrome (EMS), is causing havoc in the shrimp industry. The causative agent of AHPND was found to be a specific strain of bacteria, e.g., Vibrio and Shewanella sps., that contains pVA1 plasmid (63-70 kb) encoding the binary PirAVP and PirBVP toxins. The PirABVP and toxins are the primary virulence factors of AHPND-causing bacteria that mediates AHPND and mortality in shrimp. Hence, in this study using a germ-free brine shrimp model system, we evaluated the PirABVP toxin-mediated infection process at cellular level, including toxin attachment and subsequent toxin-induced damage to the digestive tract. The results showed that, PirABVP toxin binds to epithelial cells of the digestive tract of brine shrimp larvae and produces characteristic symptoms of AHPND. In the PirABVP-challenged brine shrimp larvae, shedding or sloughing of enterocytes in the midgut and hindgut regions was regularly visualized, and the intestinal lumen was filled with moderately electron-dense cells of variable shapes and sizes. In addition, the observed cellular debris in the intestinal lumen of the digestive tract was found to be of epithelial cell origin. The detailed morphology of the digestive tract demonstrates further that the PirABVP toxin challenge produces focal to extensive necrosis and damages epithelial cells in the midgut and hindgut regions, resulting in pyknosis, cell vacuolisation, and mitochondrial and rough endoplasmic reticulum (RER) damage to different degrees. Taken together, our study provides substantial evidence that PirABVP toxins bind to the digestive tract of brine shrimp larvae and seem to be responsible for generating characteristic AHPND lesions and damaging enterocytes in the midgut and hindgut regions.

Keywords: PirABVP toxins; brine shrimp; digestive tract; epithelial cells; necrosis; sloughing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

PirABVP toxins bind to the digestive tract and induce sloughing of epithelial cells in brine shrimp (Artemia franciscana) larvae. Immunohistochemistry of brine shrimp (A. franciscana) larvae after 0, 12, 24, 36, 48, and 60 h post PirABVP toxin challenge. The paraffin sections were treated with anti-his Mab (monoclonal antibody) specific to His-tagged PirAB toxins, then counterstained with haematoxylin. Legend: (1) Gut cells; (2) brush border; (3) gut lumen (A,B): 0 h post-challenge; (C,D): 12 h post-challenge; (E,F): 24 h post-challenge (G,H): 36 h post-challenge; (I,J): 48 h post-challenge; (K,L): 60 h post-challenge. (C,L): PirABVP toxin binds with digestive tract epithelium and induce shedding or sloughing of enterocytes in the midgut and hindgut regions (arrowhead). At 48 h and 60 h post-challenge, epithelium was severely damaged and the remaining cellular components including nuclei were further detached into the lumen and showed signs of degeneration such as pyknotic nuclei and lysed cellular membrane (arrowhead).

Figure 1
Figure 1

PirABVP toxins bind to the digestive tract and induce sloughing of epithelial cells in brine shrimp (Artemia franciscana) larvae. Immunohistochemistry of brine shrimp (A. franciscana) larvae after 0, 12, 24, 36, 48, and 60 h post PirABVP toxin challenge. The paraffin sections were treated with anti-his Mab (monoclonal antibody) specific to His-tagged PirAB toxins, then counterstained with haematoxylin. Legend: (1) Gut cells; (2) brush border; (3) gut lumen (A,B): 0 h post-challenge; (C,D): 12 h post-challenge; (E,F): 24 h post-challenge (G,H): 36 h post-challenge; (I,J): 48 h post-challenge; (K,L): 60 h post-challenge. (C,L): PirABVP toxin binds with digestive tract epithelium and induce shedding or sloughing of enterocytes in the midgut and hindgut regions (arrowhead). At 48 h and 60 h post-challenge, epithelium was severely damaged and the remaining cellular components including nuclei were further detached into the lumen and showed signs of degeneration such as pyknotic nuclei and lysed cellular membrane (arrowhead).

Figure 2
Figure 2

PirAB toxin-induced accumulation of cellular debris in the digestive tract are of epithelial cells origin. Immunohistochemistry of brine shrimp (A. franciscana) larvae after 12 and 24 h post PirABVP toxin challenge. The paraffin sections were treated with anti-cytokeratin polyclonal antibody (Pab), specific to keratin group of fibrous proteins in epithelial cells, then counterstained with haematoxylin. (A): 12 h post- experiment, negative control (no PirABVP toxin); (B): 24 h post- experiment, negative control (no PirABVP toxin); (C–E): 12 h post-challenge; (F–H): 24 h post-challenge. (C–H): PirABVP challenge-induced sloughing that leads to accumulation of cellular debris in the intestinal lumen (arrowhead) is of epithelial cell origin.

Figure 3
Figure 3

PirABVP toxin-binding leads to necrosis and damages the digestive tracts of brine shrimp larvae. Transmission electron microscopy (TEM) analysis of brine shrimp (A. franciscana) larvae after 48 h post-PirABVP toxin challenge. (A–D): PirABVP toxin challenge damages epithelial enterocytes and induces focal to extensive necrosis, resulting in the movement of bacterial cells (autoclaved feed) outside of the intestinal lumen. Legend: (1) Autoclaved bacteria feed for brine shrimp larvae; (2) damaged enterocytes with different degrees of focal to extensive necrosis. *Because brine shrimp larvae were too small, before tissue processing and sectioning, the larvae were transferred to 2% agarose which resulted in development of black lines in some TEM figures (A,B).

Figure 4
Figure 4

Digestive tract in control brine shrimp larvae appeared normal, with intact cellular membranes. Transmission electron microscopy (TEM) analysis of control group brine shrimp (A. franciscana) larvae after 48 h. (A–F): The digestive tract epithelial enterocytes appeared normal with an intact mitochondrion, nucleus, rough endoplasmic reticulum (RER), and intercellular junctions. Legend: (1) nucleus; (2) midgut lumen; (3) midgut enterocytes; (4) tight junction; (5) microvilli; (6) mitochondria; (7) rough endoplasmic reticulum (RER).

Figure 5
Figure 5

SDS-PAGE analysis of purified V. parahaemolyticus PirAVP and PirBVP toxins. Molecular mass standards (M) in kilodaltons (Protein ladder), Lane 1-PirAVP at 13 kDa, Lane 2-PirBVP at 50 kDa.

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