Immune systems from an array of organisms have been intently studied by scientists for decades, often with the intent to understand how our own immune system keeps us safe from disease. However, it wasn’t until a few years ago that researchers even knew that bacteria even had an immune system, let alone how it can recognize foreign DNA from its own, but that’s precisely what a collaborative team of investigators from the Weizmann Institute of Science and Tel Aviv University have recently done.
The common threat to most bacteria come from viruses known as bacteriophages, or phage, that carry either an RNA or DNA genome, which they inject into the bacterium like a tiny hypodermic needle. Once inside, if not destroyed, the viral genetic material integrates itself into the bacterial genome. The researchers were very interested in the underlying mechanisms of how bacteria are able to determine the difference between its own DNA and the phage DNA to target for degradation.
“In most environments, phages are around ten times more abundant than bacteria. And, like all viruses, phages use the host cell's replication machinery to make copies of themselves,” explained Rotem Sorek, Ph.D., associate professor in the department of molecular genetics at the Weizmann Institute and senior author on the study. “And they are constantly evolving new ways to do this. So bacteria need a very active immune system to survive.”
The findings from this study were published recently in Nature through an article entitled “CRISPR adaptation biases explain preference for acquisition of foreign DNA.”
Several years ago scientists identified the clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins, or CRISPR-Cas system that was able to recognize foreign DNA and store smaller bits of it into a special section of the bacterial genome—encompassing the memory component of the bacterial immune system.
Yet, Dr. Sorek and his team were still curious to understand how the CRISPR/Cas system was able to distinguish the foreign bits of DNA to insert into its memory section, from its own endogenous DNA. In order to answer that question they devised a series of experiments using plasmid DNA as substitute for the viral phage DNA. The researchers were able to identify two proteins that are part of the CRISPR system, Cas1 and Cas2, which were responsible for acquiring the foreign stretches of genetic material.
Interestingly, Dr. Sorek’s team also observed that Cas 1 and 2 were able to identify which stretches of DNA came from phage, since it is able to be rapidly replicated by bacteria—a tactic employed by viruses to help ensure propagation.
“Still, this did not completely explain how the CRISPR system differentiates between self and non-self,” said Dr. Sorek.
After looking deeper into the DNA replication process the investigators found that during replication DNA double-strand breaks (DSB) often occur. DSB are a signal for the bacterial DNA repair protein RecBCD that unwinds and degrades the DNA until it hits properly oriented Chi sites, which are short DNA stretches in bacteria where homologous recombination occurs. Viral DNA has considerably fewer Chi sites and leads to more extensive processing, increasing the number of substrates for CRISPR to use as immune memory.
Put more simply, Chi sites serve as bacterial self-identifying markers. When present on DNA the CRISPR machinery avoids those regions, searching for stretches that are devoid of Chi sites, which typically represents the phage DNA.
“Solving the riddle of self versus non-self for the bacterial immune system and deciphering the exact mechanism of this step in the CRISPR process gives us important insight into the unseen confrontation that is taking place everywhere, all around us, all the time,” concluded Dr. Sorek.