Analysis of C Diff Outer Structure Gives Insight on Resilience

A chainmail-like, flexible outer layer on Clostridium difficile may explain its success at defending itself against antibiotics.

Clostridium difficile infection (CDI) is the primary cause of hospital-acquired, antibiotic-associated disease both in the United States and throughout the rest of the world.

The burden of CDI on the health care system is mainly caused by the superbug’s resilience. To address this challenge, scientists from Newcastle, Sheffield, and Glasgow Universities partnered with Imperial College and Diamond Light Source have worked to show that this resilience is largely attributable to a chainmail-like, flexible outer layer on the bacteria.

To outline the external structure of C diff, the research team used x-rays and electron crystallography to evaluate the protein structure, which showed that the links of the chainmail are primarily made of the protein SlpA, which is arranged in a pattern that creates its flexible armor.

Most bacteria and archaea have a proteinaceous coat, called the surface or S-layer. Whereas the S-layers of other pathogens have pores of approximately 30-100 Å in diameter, C diff’s array is approximately 10 Å, according to the study.

Prior to this research, a complete, intact, atomic-level rendering of the assembly and anchoring domains had not been created for any major S-layer protein.

The study found that SlpA is post-traditionally cleaved into 2 S-layer proteins, one of which has a high molecular weight and one of which has a low molecular weight. These subunits combine to form the S-layer complex.

C diff’s anchoring domain ties the S-layer to the cell wall and assembly domain to form the 2D paracrystalline array, as opposed to other known S-layer structures, which are the same.

“Surprisingly, we found that the protein forming the outer layer, SlpA, packs very tightly, with very narrow openings that allow very few molecules to enter the cells. S-layer from other bacteria studied so far tend to have wider gaps, allowing bigger molecules to penetrate. This may explain the success of C diff at defending itself against the antibiotics and immune system molecules sent to attack it,” said study author Dr. Paula Salgado, a senior lecturer in Macromolecular Crystallography at Newcastle University.

Treating CDI is difficult, because recurrent infections are common and can lead to death; however, antibiotics can exacerbate the problem. C diff is able to evade antibiotics as a result of antimicrobial resistance, therefore administering antibiotics can eliminate the healthy bacteria in the gut and enable CDI to flourish.

The research team noted that determining the structure of the bacteria will help investigators to create specific drugs that can create holes in the S-layer to enter and kill the bacterium cell.