Cholera, caused by the bacterium Vibrio cholerae, remains a major global health threat. Like most bacteria, Vibrio cholerae lives under constant attack from viruses. To survive, bacteria equip themselves with antiviral immune systems. Previous work has shown that V. cholerae carries a large genetic element called a sedentary chromosomal integron (SCI). This structure contains hundreds of small mobile DNA units known as “gene cassettes” arranged in a long array, like a chain of pearls. A new study by researchers at the School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), has now shown that V. cholerae can efficiently acquire new SCI gene cassettes from extracellular DNA released by other V. cholerae strains, and non-cholera vibrio species, potentially adding to their own defense arsenals.
Research lead Melanie Blokesch, PhD, and colleagues reported on their findings in Science, in a paper titled “Competence-mediated DNA uptake diversifies Vibrio cholerae sedentary chromosomal integrons,” in which they concluded “Given the widespread presence of SCIs and the conservation of natural competence across the genus, we propose that SCIs function as genus-wide reservoirs of exchangeable protective genes.”
Bacteria often survive viral attack and environmental stress by sharing genes that enhance their defenses, the authors wrote. A “defining genomic feature” of V. cholerae is its sedentary chromosomal integron (SCI), a genetic element containing hundreds of mostly promoterless gene cassettes. While the function of many cassettes remains unknown, some do encode antiviral immune systems. “Although most cassettes encode proteins of unknown function, ~10% encode phage defense systems, suggesting that SCIs as well as mobile integrons function as reservoirs, or “biobanks,” of defense genes,” the authors continued.
However, most of these genes are located far from the start of the array and remain silent. Prevailing models proposed that cassettes could be internally reshuffled to activate them, yet no such rearrangements have been observed in the pandemic lineage of V. cholerae for decades. “Cassettes are thought to reshuffle under stress to the favorable first array position, yet the SCI in pandemic V. cholerae has remained static for more than 60 years.”
This raises a key question: if internal reshuffling is rare, how are cassette-encoded immune systems activated, and how do new cassettes enter the array at all? To address this question, a team led by Blokesch at the Laboratory of Molecular Microbiology at EPFL investigated whether the SCI might capture gene cassettes from genetic material entering the cell from the outside. “We asked whether SCI cassettes move horizontally rather than by intracellular reshuffling.”
A key feature of this process is natural competence, the ability of bacteria to take up free DNA from their surroundings. V. cholerae becomes naturally competent when it grows on chitinous surfaces, a polymer found in the shells of crustaceans that is abundant in aquatic environments.
In the laboratory, the team mimicked these conditions by growing bacteria on chitin and supplying DNA from different Vibrio cholerae strains or from other Vibrio species. They then tracked whether newly acquired gene cassettes were inserted into the first position of the SCI array.
Through their studies the team confirmed that that V. cholerae can acquire new SCI gene cassettes from extracellular DNA. Collective experimental results, the authors stated, “We show that SCI cassettes are efficiently acquired by naturally competent V. cholerae and inserted at the first SCI array position in an integrase-dependent manner. This process incorporates cassettes not only from other V. cholerae strains but also from diverse Vibrio species.”
In aquatic habitats, DNA is released when bacterial cells are killed by viruses, antimicrobial compounds, or bacterial weapons. Nearby competent bacteria can take up this DNA and incorporate selected fragments into their own SCI. “A loose comparison would be the following,” said Blokesch. “Imagine your grandmother passes away and, as a farewell gift, hands over the immunity she built up against the Spanish flu a century ago, immediately protecting you from that same virus. Wouldn’t that be amazing? This is essentially what we show that V. cholerae can do.”
The team also showed that cassettes inserted in this position are functional. Several defense systems provided protection against viruses that infect Vibrio species, known as vibriophages. They stated, “In this study, we show that SCI diversification efficiently occurs by horizontal transfer linked to the genus’s aquatic lifestyle: DNA released from lysed cells is taken up by naturally competent vibrios and integrated into the first position of the SCI array, the primary site of strong expression, where it confers resistance to phage and potentially other threat,” the wrote in summary. “Together, these results demonstrate that SCI cassettes can cross species boundaries, supporting a model in which SCIs may function as genus-wide reservoirs of exchangeable genes, including defense genes, that confer selective advantages under certain conditions.”
An important exception emerged. In the pandemic 7PET lineage of V. cholerae, the SCI appears largely static. “The SCI of 7PET V. cholerae is large but remarkably stable,” the authors noted. They propose that this reflects adaptation to a human-associated niche. “We propose that this reflects adaptation to a human-associated niche, where chitin is less abundant and competence induction—requiring growth on chitin to high cell density plus relief from catabolite repression is unlikely to occur,” they suggested. “As a result, SCI-mediated diversification may be largely inactive in pandemic strains.”
However, if pandemic strains were to encounter environmental conditions that enable SCI cassette acquisition, they could expand their antiviral defenses. Blokesch commented. “This possibility matters because vibriophage-based approaches are currently being explored to prevent cholera in endemic regions, and such evolutionary flexibility could ultimately affect how effective these strategies remain.”
In their paper the team concluded that since onset of the seventh pandemic, “… acquisition of novel and diverse, large defense-related genomic regions by 7PET strains appears to have been relatively limited. Consequently, reduced SCI-mediated diversification may lower the capacity of this lineage to rapidly evolve new defenses—an important consideration for ongoing efforts to deploy phage-based prophylaxis against cholera in endemic settings such as Bangladesh.”
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