Author: Margaret Woodruff Majors: Biology and Chemistry

A senior premedical student majoring in biology and chemistry, I spent my second semester of research funded by the Honors College research grant in Dr. Mack Ivey’s molecular biology lab doing online modelling of a regulatory protein in Clostridium difficile and the potential effects of introducing mutations.

My sophomore year, I wanted to find a laboratory to do honors research in, so I began to read about the areas of research that various professors in the biological sciences department were interested in. I interviewed with multiple of these professors and decided to join Dr. Ivey’s lab because his research with Clostridium difficile seemed extremely relevant to my future career aspirations of becoming a doctor.

Clostridium difficile is the leading cause of hospital-acquired diarrhea and spreads through sporulation. Infecting the intestines, this type of bacteria is usually unproblematic until antibiotic treatment disrupts the flora of bacteria that normally exists in and defends the human gut. This weakened state allows C. difficile to attach to the intestinal linings and become toxic, potentially leading to inflammation of the intestines, diarrhea, and sometimes death. Prior to starting my research, I knew little to nothing about C. difficile. Dr. Ivey has helped me learn about the bacteria and its mechanisms, as well as give me a specific protein to focus my research on, examining its structure and function in sporulation and toxicity.

My research specifically examines the SpoIIE protein of C. difficile, which has been found to control the transfer of peptides across the cell membrane with a repressor for peptide permease genes. Other research in the Ivey laboratory suggested that the bacteria prefers peptide nutrients—which are usually not readily available in the intestines—because it has an impact on the release of C. difficile spores and toxins. The SpoIIE protein is also present in Bacillus subtilis, where it plays a role in sporulation. The transmembrane portion and the C-terminal domain control the localization and timing of the release of spores, respectively. The dimerization of this protein in the body is likely to also have an effect on the timing of sporulation by regulating the removal of a phosphate group from SpoIIAA.

Prior to undergraduate research laboratories closing in Spring 2020 due to COVID-19, I was planning to introduce mutations into the amino acid sequence of the SpoIIE protein of C. difficile as has been previously done in B. subtilis. These two mutations would change a glutamine at residue 260 to an alanine and an aspartate at residue 274 to a lysine. Located in a specific region of a large alpha-helix near the C-terminal, these mutations should not prevent dimer formation, but should interfere with phosphatase activity. This should inhibit the ability for peptides to be transferred into the bacteria and therefore have an effect on sporulation and toxicity.

This semester, I had to take a step back from research in the laboratory, since it would have been difficult to do so safely during the pandemic. Instead, Dr. Ivey directed me towards a few online protein modeling software where I could analyze the expected structure of the normal SpoIIE protein and the potential structural effects of the two mutations. Using Swiss-Model and I-TASSER, multiple models of the wild-type of the protein were produced, as well as models for the protein with the two previously mentioned mutations. The models produced through Swiss-Model shows that the most likely structure of both the wild type and mutant proteins will be homodimers, which is expected as the selected mutations were chosen so as to not interfere with dimer formation. The two mutations are also unlikely to interfere with the alpha-helical structure that they are located in. This software also provides a QMEAN value, which is an indicator of how native the structures are, with lower values being less native. The QMEAN value for the mutant protein (-4.08) is lower than the value for the wild-type protein (-3.73), which suggests that these mutations could interfere with the protein’s activity as expected.

Going forward this next semester, I hope to return to the lab so that I can create these mutated proteins and study the changes that I observed in the models. Hopefully my research, alongside the rest of the research being done on C. difficile in Dr. Ivey’s laboratory, can eventually be useful in the improved treatment of this antibiotic resistant bacteria.