Gut defence mechanism discovered

“The way cells in gut fight off toxins produced by a hospital bug has been discovered,” BBC News has reported.

In new research, scientists have shown that infection with the bacteria Clostridium difficile stimulates cells in the gut to modify the toxins produced by the bacteria. This modification, called nitrosylation, protects the body by making the toxins inactive. Researchers then found that a chemical called GSNO that encourages nitrosylation could be used to treat mice infected with Clostridium difficile, the bacteria behind a high proportion of hospital-acquired infectious diarrhoea and life-threatening colon inflammation.

This study’s exploration of nitrosylation has contributed to our understanding of how host organisms can protect themselves against toxins produced by organisms such as C. difficile. The researchers add that a larger number of microbial enzymes are similar to the C. difficile toxins, and that nitrosylation may represent a common form of defence mechanism against microbes. However, many of the body’s naturally-occurring proteins can also be nitrosylated, not just toxins from bacteria. Therefore, as the researchers conclude, before these finding can be used to develop a treatment against bacterial infections, scientists must find a way to target only those substances that are harmful to the body.

Where did the story come from?

The study was carried out by researchers from the University of Texas and a number of other American research institutes. It was funded by several organisations, including the Howard Hughes Medical Institute and various arms of the US National Institutes of Health. The study was published in the peer-reviewed journal, Nature Medicine.

The BBC reported the findings of this study well.

What kind of research was this?

This was animal and laboratory-based research, which used mouse model and cell culture-based techniques to examine cells’ response to infection with the bacteria Clostridium difficile. Infection with C. difficile is reported to be the most common cause of hospital-acquired infectious diarrhoea and life-threatening inflammation of the colon (colitis) worldwide.

The strains of C. difficile that cause disease produce several toxins, including two called TcdA and TcdB. These toxins inactivate enzymes in the infected person or animal (known as ‘the host’) and cause diarrhoea and inflammation once they have entered host cells. However, to become toxic, the toxin molecules have to ‘cleave’ or split themselves into smaller parts so that they can enter the cells of the gut. This paper identified a mechanism that operates in host organisms to reduce the cleavage of the toxins, and explored the potential of exploiting this mechanism to treat mice with C. difficile infections.

What did the research involve?

In this study the researchers performed a range of experiments to look at a range of biological and chemical mechanisms behind the body’s defences against the bacteria C. difficile.

The researchers started by creating an animal “model” of C. difficile infection that they could study. To do this they injected purified TcdA toxin into the small intestines of mice. Previous work has suggested that the body limits the toxic effects of C. difficile by using a process called nitrosylation, which chemically modifies proteins.

To further explore the role of nitrosylation the researchers looked at the levels of a chemical called S-nitrosogluthathione (GSNO), which is often required for nitrosylation to take place. To do so, they compared levels of GSNO areas of the gut of mice that had been injected with the toxin and in areas left uninfected. They also looked at the levels of modified (nitrosylated) proteins in infected and uninfected gut tissues. The researchers also identified which specific proteins had been nitrosylated.

The researchers then examined the levels of modified (nitrosylated) proteins in tissue samples from human colon tissue that was actively affected by inflammation. The researchers used their observations to construct a cell-based model to examine the potential role that toxin nitrosylation might play in protecting host cells from toxins. To confirm their findings, they injected nitrosylated TcdA toxin into mice to see whether it had the same effect as un-nitrosylated TcdA.

The researchers then examined and modelled the protein structure of the toxins TcdA and TcdB to identify the exact location on the protein molecule that nitrosylation modifies to bring about reduced toxicity. They then confirmed the sites of modification using a variety of experimental techniques.

Finally, the researchers used their findings to investigate whether GSNO (a chemical that causes nitrosylation) could be used to protect mice against C. difficile toxicity. They tested the effects of GSNO first on cells in the laboratory, and then on mice. To do this they injected the small intestines of mice with Tcd toxins, then injected some of the mice with GSNO as well. They then looked at whether the Tcd toxins had less effect in the mice injected with GSNO. They also tested the effects of GSNO given by mouth in another mouse model that closely resembles human C. difficile infection.

What were the basic results?

TcdA injection into the small intestine of mice caused damage to the lining of the intestine (called the intestinal mucosa). It could also cause fluid secretion into the intestine (which is what leads to diarrhoea) and the accumulation of white blood cells and other signs of inflammation.

There was a 12.1-fold increase in tissue levels of the chemical GSNO in tissues of animals injected with TcdA compared to animals injected with a “dummy” solution that lacked the toxin. There were also high levels of modified (nitrosylated) proteins in TcdA-exposed tissues, both in mice and humans. The researchers found that TcdA was itself a target for this modification.

The cell-based model showed that nitrosylation of the TcdA toxin protected the cells against the toxin’s effects. When nitrosylated TcdA was injected into mice it was less toxic than unmodified TcdA. The related toxin TcdB was also found to be nitrosylated. The researchers found that the nitrosylation occurred at the catalytic site that allows the toxins to be cleaved (a process which is necessary for toxicity), preventing it from occurring.

GSNO protected against Tcd toxicity in cells grown in the laboratory. Injection of GSNO into the intestine of mice reduced TcdA-induced symptoms, including inflammation and fluid secretion. Administering oral GSNO also increased survival in another mouse model of human C. difficile infection.

How did the researchers interpret the results?

The authors concluded that host organisms exhibit nitrosylation of C. difficile toxins, which reduces their harmful effects by preventing the toxin molecules from splitting and entering cells. They say that promotion of the nitrosylation process can be used to treat C. difficile infection in mice, and that this finding may suggest new treatment approaches for humans.

Conclusion

This study has contributed to our understanding of how host organisms defend themselves against toxins produced by C. difficile. It found that both mice and humans modify the toxins using a process called nitrosylation, and this decreases their toxicity. The researchers add that a large number of microbial proteins are similar to the C. difficile toxins, and that nitrosylation may be a common defence mechanism against microorganisms.

The study also found that the chemical GSNO, which is often required for nitrosylation, was effective in treating C. difficile infection in mice. However, it is not just these bacterial proteins that can nitrosylated - many other important proteins in the body can also undergo the process. Therefore, as the researchers conclude, the ability to selectively target the toxins or other proteins involved in disease (but not other proteins) remains a major challenge. This will have to be addressed before treatments based on this finding could be further investigated for C. difficile.