The spider-like icosahedral shape is an abstract depiction of the Shiga toxin bacteriophage with a red genome, stylized as the puppet master of a potentially deadly EHEC infection.[UNSW Sydney]

Researchers at the University of New South Wales (UNSW) studying an E. coli strain (enterohemorrhagic E. coli or EHEC) that causes a severe intestinal infection in humans, say they discovered a new molecular pathway that controls Shiga toxin production. EHEC is a food-borne pathogen that releases Shiga toxins during infection, resulting in kidney and neurological damage.

The team adds that their finding “Early termination of the Shiga toxin transcript generates a regulatory small RNA,” published in PNAS, is important because there are no commercially available treatment for EHEC infections.

“Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage,” write the investigators.

“During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript.”

“StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions.”

“Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.”

“Antibiotic treatment of these infections is generally not recommended because antibiotics stimulate production of the Shiga toxin, leading to an increased risk of kidney failure, neurological damage, and death,” said Jai Tree, PhD, senior author of the study.

“The new pathway that we have found reduces toxin production and is not expected to be stimulated by antibiotic treatment. So, our results identify a potential new target for the development of drugs that can suppress Shiga toxin production during EHEC infection.”

Tree added that the group needs to conduct a lot more research to understand if their findings apply to a broad range of clinical EHEC isolates and to both types of Shiga toxins produced by human EHEC isolates. He pointed out that globally, Shiga toxin-producing E. coli was still a major food safety concern after a large outbreak in Germany in 2011.

The UNSW research was the first discovery of a new pathway that controls the Shiga toxins in almost 20 years, according to Tree.

“In 2001, researchers at Tufts and Harvard universities first showed how production of the Shiga toxin was controlled by a bacterial virus, known as a bacteriophage, within the genome. This has been the only known pathway that controls Shiga toxin production for almost two decades,” he explained.

“We have extended that work to show a new mechanism of toxin control that is, surprisingly, buried within the start of the DNA sequence that encodes the Shiga-toxin messenger RNA, a working copy of the gene.”

“We discovered a very short piece of the toxin messenger RNA is made into a regulatory non-coding RNA that silences the toxin and promotes growth of the pathogen.”

Tree said their findings were a surprise because Shiga toxin genes have been well studied, with almost 7000 published studies in the past 40 years.

“Only recently have we been able use advances in RNA sequencing technology to detect the presence of the new regulatory non-coding RNA embedded within the Shiga toxin messenger RNA,” he said. “This new regulatory non-coding RNA had been hiding in plain sight for almost 20 years.”

Regarding how researchers’ findings may open up new possibilities for the treatment of EHEC infections, Tree noted that patients largely receive supportive care to manage disease symptoms and to reduce the effects of the toxin on the kidneys.

“Our work shows a new mechanism for controlling toxin production that may be amenable to new RNA-based therapeutics to inhibit toxin production during an infection,” he continued. “We anticipate this would expand intervention options and potentially allow use of antibiotics that are currently not recommended because they stimulate Shiga toxin production.”

“New treatments could therefore reduce the risk of kidney damage, neurological complications and death. We look forward to testing these new interventions in the next stage of our research.”

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