Unlocking the Mechanism of a Deadly Infection in Pigs

Clostridium perfringens is a major cause of food poisoning, enteritis, wound infection, and septicaemia. While it infects humans, infections in livestock can have a devastating economic impact due to high mortality rates in young animals; infection with C. perfringens type C causes necrotic enteritis (fatal intestinal bleeding) in piglets with up to 100% mortality rate (Posthaus et al. 2020). 

This blog discusses a paper published in Cell Host and Microbe by Bruggisser et al. (2020) that shows that CD31 is crucial for C. perfringens pathogenesis in pigs.

The C. perfringens Problem

While it is possible to vaccinate pregnant sows to provide protection to piglets via antibodies secreted into their colostrum, sometimes a proportion of piglets will not obtain sufficient passive immunity, leaving them susceptible to infection (Richard et al. 2019). Additionally, C. perfringens can persist in the environment for a long time resulting in new infections many years after an initial outbreak, unless there is a thorough vaccine strategy in place. Understanding the mechanism of the disease could help develop new strategies to prevent infection and new treatment approaches.

However, while the pore-forming toxin responsible for destruction of intestinal endothelial cells has been identified, until now the receptor that it acted through was still a mystery — hindering progress in this field.

Like other pathogens, C. perfringens secretes several different toxins that cause its pathogenesis, with various combinations of up to 16 toxins mediating disease (Uzal et al. 2010). The bacteria are classified into five taxinotypes (A-E) depending on the production of four major toxins, alpha (CPA), beta (CPB), epsilon (ETX), and iota (ITX) (Uzal et al. 2010). Beta toxin (CPB) is essential for the pathogenesis in C. perfringens type C that is responsible for necrotic enteritis in pigs (Sayeed et al. 2008).

Intriguingly, studies have shown that not all cell types are susceptible to infection and damage by CPB; fibroblasts and epithelial cells are among the cell types that are resistant. These findings were replicated by Bruggisser et al. (2020). They infected 13 human and mouse cell lines with up to 10 µg/ml CPB. Fibroblasts and epithelial cells were not affected by CBP, and only a subset of the tested cell lines were susceptible to infection including endothelial cells, platelets, and the monocytic cell line THP-1. This suggested that there is a key molecule common to these cell types that confers susceptibility, and that this molecule is not present in other cell types like epithelial cells. The researchers set out to identify this susceptibility molecule.

CD31 Is Key to Infection

The initial idea that CD31 could be key came from an observation that CD31 and beta toxin are found in a similar distribution on endothelial cells (University of Bern 2021). According to an article on The Pig Site, a pig knowledge hub, it was this observation by researchers that sowed the seeds for the larger study.

By comparing data on protein expression from the Human Protein Atlas and the PlateletWeb, the scientists were able to identify two molecules that are highly expressed in endothelial cell lines, THP-1 and platelets. These were CD31 (PECAM-1) and CD102 (ICAM-2) (Bruggisser et al.2020). To discover whether one of these could be the susceptibility factor they were looking for, they infected wildtype, CD31 knockout (KO) and CD102 KO endothelial cell lines with C. perfringens. Confirming their initial hypothesis, they found that loss of CD31 resulted in a loss of susceptibility to infection. Moreover, expressing CD31-GFP in epithelial cell lines (usually resistant to C. perfringens infection) led to them becoming susceptible — demonstrating that CD31 was key to CBP toxicity.

They then confirmed this finding in vivo by comparing infected wildtype and CD31 KO mice.  While the wildtype mice died from infection, the CD31KO mice were resistant to the lethal effects of CBP (Bruggisser et al. 2020).

CD31 Interacts with CPB

Now that they had identified a susceptibility factor, they needed to prove that CD31 directly interacts with CBP to cause this toxicity. CD31 is a protein found on platelets, endothelial cells, and some immune cells, like macrophages and lymphocytes. It is a type 1 transmembrane protein with six Ig domains, and its expression is concentrated at cell junctions with a variety of roles in vascular biology, including angiogenesis, platelet function, and thrombosis.

CPB is a pore forming toxin that causes perforation of the cell membrane, damaging cells leading to their destruction. In intestinal endothelial cells, it results in damage to blood vessels, bleeding into the intestine, and usually death.

They were able to show that CD31 and CPB interact through a combination of co-immunofluorescence, co-immunoprecipitation, and in situ proximity ligation assays. CPB was found at the borders of wildtype endothelial cells, but not in CD31 KO endothelial cells, and both CPB and CD31 demonstrated reciprocal co-immunoprecipitation (Bruggisser et al. 2020).

They identified the Ig6 domain of CD31 as important for this interaction, as cells expressing mutant CD31, lacking this domain, were resistant to CBP infection, and identified conserved amino acids that form the CBP binding epitope in this region (amino acids 527–536 in mouse and 537–546 in human and pigs) (Bruggisser et al. 2020). To further demonstrate that binding of CBP to CD31 resulted in toxicity, they showed that CD31 reconstituted into liposomes, was sufficient for CPB to form functional pores, like those that lead the destruction of endothelial cells.

Summary

Prior to this study, it was unknown why CBP only affected some cell types but not others. The identification of CD31, as a molecule key to the attachment of beta toxin to endothelial cells, explains how infection with C. perfringens can lead to fatal intestinal bleeding in pigs. It is hoped that this knowledge will now help inform future vaccine strategies.