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When bacteria get the flu and sharpen their knives

Scientists identify a novel pathway to activate the bacterial immune system - It is true; also microbes get infected by viruses. Consequently, they have evolved immunity mechanisms to fend off attacks by the enemy. In bacteria and archaea CRISPR/Cas has recently been discovered as a novel immune system to fight alien genomes. The system, which is also known as prokaryotic RNA interference, consists of proteins and short RNA molecules (crRNAs) that target the intruders for destruction.

A key event in the activation of the system is the maturation of crRNAs. Joint research led by scientists in Umea, Sweden in collaboration with colleagues in Würzburg, Germany, revealed a new pathway, which allows bacteria to activate the crRNAs to block the entry of enemy genomes. The results are now published in the prestigious scientific journal Nature.

During their lifetime, microbes are constantly and heavily facing assaults by viruses known as phages or invading circular nucleic acid strands known as plasmids. On the one hand, these alien genomes can be detrimental for the microbial host; for example, lytic phages that destroy the microbes upon infection. On the other hand, foreign DNA such as plasmids can endow microbes with new genes to resist antibiotic action.

To protect themselves against infection by the enemy, microbes have invented a sophisticated defence mechanism. CRISPRs (for Clustered Regularly Interspaced Short Palindromic Repeats) are genetic islets in the microbial genome that consist of genes encoding the CRISPR-associated (Cas) proteins and an array of unique enemy-targeting elements, called spacers, interspaced by repeat sequences. The system is quite complex and has evolved into various subtypes that differ in the combination of Cas genes and in the number of spacers-repeats in the array.

All steps of the immune system require the activities of the Cas proteins that are thought to be sufficient for CRISPR activation and function. The latest research on CRISPR reported in the journal Nature in a paper titled "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III" demonstrates a fundamentally novel pathway that requires additional factors from the host machinery to activate CRISPR, evocative of the eukaryotic RNA interference system.

How does CRISPR/Cas work? When bacteria and archaea are exposed to viruses or plasmids, short pieces of DNA from the alien genomes are injected into the host cell and incorporated into the CRISPR spacer-repeat array on the chromosome. This genomic manipulation leads to reprogramming of the microbial host cell, which then uses the newly inserted DNA as a memory device of previously encountered alien genomes to provide specific immunity against future attacks by the same elements. In the following process, crRNA maturation, the host cell transcribes the CRISPR spacer-repeat array into an RNA molecule that gets diced into short mature RNAs (crRNAs) of unique truncated spacer-repeat sequences. In the last step, the silencing of the alien genome, the crRNAs target the invading genomes in a sequence-specific manner leading to destruction of the foreign genetic material.

The CRISPR/Cas pathway is only been studied for less than a decade and many details around its regulation and mechanisms are still unclear. New light is now shed on the activation of the crRNAs by the research team around Emmanuelle Charpentier at the Laboratory for Molecular Infection Medicine Sweden (MIMS) in Umea, Sweden with her former team at the Max F. Perutz Laboratories in Vienna, Austria in collaboration with the research group of Jörg Vogel at the Institute for Molecular Infection Biology (IMIB) at the University of Würzburg, Germany.

"We have analyzed the CRISPR/Cas pathway in our model organism Streptococcus pyogenes, a human pathogen", says Emmanuelle Charpentier, who led the study. "Unexpectedly, we have discovered a novel pathway of CRISPR activation involving the concerted action of three new factors to maturate the "crispy" crRNAs: a small RNA molecule, the endoribonuclease III from the bacterial host and a novel Cas protein (Csn1). The mechanism is particularly neat. The small RNA molecule, called tracrRNA, pairs specifically with the CRISPR precursor RNA at the level of each of the repeats. The duplexes formed are then recognized by an enzyme, the endoribonuclease III of the bacterial host, that dices the RNAs in the presence of the Cas protein Csn1, leading to the production of the shorter crRNAs".

In eukaryotes, Dicer and Drosha are enzymes of the endoribonuclease III family and are responsible for the production of small interfering RNAs and maturation of microRNAs. "As such, the requirement for the bacterial endoribonuclease III to mature the crRNAs is reminiscent of the RNA interference pathway in eukaryotes", Charpentier says. "Our findings of the first host factors recruited to CRISPR/Cas highlights the remarkable evolutionary diversification of CRISPR/Cas systems. The discovery raises also the exciting possibility that additional factors from the microbial host cell - outside CRISPR/Cas - might be involved in the other steps of the immune system."

"The pathway functions to protect bacteria from killing by lytic phages" Charpentier continues, "but in addition, we show that CRISPR can also target viruses of the so-called lysogenic phage family that are known to transfer virulence genes among streptococcal clinical isolates. Targeting of virulence genes highlights a novel biological function for CRISPR and underlines an alternative mechanism to modulate the pathogenic potential of bacterial clinical isolates."

Charpentier ends: "Finally, manipulation of the novel discovered pathway could lead to novel ways of specifically silencing targeted genes. Novel RNA-based interference approaches can have useful implications either to genetically engineer modified microbes for research studies or to generate bacterial strains resistant to destructive viruses. This could help maintain microbe stability in dairy food production".

This work was supported in part by funding from the Swedish Research Council, Umea University, the Austrian Science Fund, the German Research Council, the German Ministry for Education and Science, and the European Community.

The Laboratory for Molecular Infection Medicine Sweden, MIMS, is the Swedish node of the Nordic EMBL Partnership for Molecular Medicine. The institute is dedicated to research on the molecular mechanisms of infections and the development of new antimicrobial strategies. MIMS is part of the research consortium Umea Centre for Microbial Research, UCMR. Visit: <www.mims.umu.se> and <www.ucmr.umu.se>.

The Institute for Molecular Infection Biology (IMIB) is an interdisciplinary institution at the Medical Faculty of the University of Würzburg. Members of the institute are interested in biological problems of pathogens and infectious diseases, with an emphasis on molecular mechanisms. Visit: <www.infektionsforschung.uni-wuerzburg.de>

Additional Information: For more information, please contact:

Dr. Emmanuelle Charpentier The Laboratory for Molecular Infection Medicine Sweden, MIMS and the Umea Centre for Microbial Research UCMR Umea University, SE-90187 Umea emmanuelle.charpentier@mims.umu.se <www.mims.umu.se>; <www.ucmr.umu.se>

Original publication:

Elitza Deltcheva, Krzysztof Chylinski, Cynthia M. Sharma, Karine Gonzales, Yanjie Chao, Zaid A. Pirzada, Maria R. Eckert, Jörg Vogel & Emmanuelle Charpentier: CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 31 March 2011. (doi:10.1038/nature09886)

idw :: 30.03.2011