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C. Difficile Epigenomic Analysis Uncovers Highly Conserved Methyltransferase

NEW YORK – A new epigenomic study of Clostridioides difficile has identified a highly conserved methyltransferase that may enable sporulation and could be targeted to treat infections.

C. difficile causes about half a million infections and 30,000 deaths each year and is one of the leading causes of hospital-acquired infections in the developed world. It spreads as spores, which can be resistant to many cleaning and disinfecting routines.

By characterizing the methylome of three dozen C. difficile isolates, researchers led by Mount Sinai School of Medicine's Gang Fang homed in on a highly conserved methyltransferase within not only their sample set, but within a global array of C. difficile isolates, as they reported this week in Nature Microbiology. They further reported that inhibiting this methyltransferase affects the ability of C. difficile to form spores and spread.

Methyltransferases like this are found in a number of bacteria, the researchers noted. "We can envisage..., similarly to what we did in C. difficile, inhibit[ing] many of these MTases to lower the virulence and pathogenicity of several other bacterial species," they added in a statement. "Hence, under the current global scenario of increasing resistance to antibiotics, this strategy is definitely a promising plan B to fight bacterial infection."

Using SMRT-seq, the researchers analyzed 36 C. difficile isolates collected from fecal samples of infected patients from the Pathogen Surveillance Program at Mount Sinai. These isolates belonged to 15 different multilocus sequence typing groups, representing clades 1 and 2. Through this, they uncovered 17 unique methylation motifs, the majority of which were the 6mA type. About 95 percent of these motifs were methylated, which the researchers noted is consistent with most bacterial methylomes.

One 6mA motif, CAAAAA, was present across the genomes the researchers analyzed, suggesting to them that it might have a key, conserved function.

This led the researchers to investigate the methyltransferase genes present in the C. difficile genome and they found 139 MTase genes. But one — a type II 6mA solitary DNA MTase — was present across all the isolates, and it methylates the conserved CAAAAA motif. They further found that this DNA MTase, which was encoded by CD2758 in the C. difficile reference strain, is found throughout a global collection of 300 C. difficile strains.

They named CD2758 CamA for C. difficile adenine methyltransferase A.

Because sporulation is a key part of C. difficile's ability to spread, the researchers suspected camA might be involved in that process. When they inactivated camA, the researchers found sporulation formation dropped in the inactivated strain, as compared to wildtype C. difficile. Through further functional analyses, the researchers found that the loss of camA leads to a drop in the number of cells that can form functional spores.

In animal models, they found that the loss of camA didn't affect the ability of C. difficile to infect and cause disease, but did influence their ability to generate the spores to persist and further spread.

MTases like this are found in other bacteria, which suggested to the researchers that targeting them could be an avenue to pursue to address bacterial infections in light of microbes' increasing resistance to disease.