CRISPR-MEDIATED ANTIVIRAL DEFENSE IN SULFOLOBUS: A VERSATILE TOOL AND A DANGEROUS WEAPON FOR SETTING UP SAFEGUARDED INDUSTRIAL BIOPROCESSES
Creators: name: Fusco, Salvatore
Publisher: name: Università degli Studi di Napoli Federico II
Year: 2015

description: Since centuries microorganisms have been used to produce fermented products. Nowadays, the consciousness about the importance of these bags to set up eco-friendly industrial workflows is pronounced more than ever. Nevertheless, biobased processes are much more 'delicate' than chemistry-based counterparts. For instance, microbes are susceptible to infections by viruses that are naturally present in raw matrices, such as the milk for diary industries. Moreover, viruses are the most abundant biological entities on Earth, thus the probability that a give microorganism could encounter such 'predators' is very high. In this regard, huge losses of fermenting biomasses have been already documented, thus leading to open the way to search for new solutions to meet this issue. One of the antiviral mechanisms that could be exploited to obtain resistant strains is the CRISPR-Cas system. This latter relies on the incorporation, upon infection, of sequences from the foreign nucleic acid into the host chromosome at specific CRISPR loci. A locus is an array repeats (similar in length and sequence) interspaced by spacers (dissimilar in sequence) that derive from foreign genetic elements, such as viruses and plasmids. Transcription of these loci generates long precursor pre-crRNAs that are subsequent processed to short crRNAs. Thereafter, these mature RNAs provide specificity to a ribonucleoprotein complex that degrades the invading nucleic acid. To test the possibility of exploiting the CRISPR-Cas system to produce Virus Insensitive Thermophilic Strains (VITSs), it was resolved to analyse host-virus relationships between the thermophilic archaeon Sulfolobus solfataricus and two related member of the Fuselloviridae viral family, i.e. SSV1 and SSV2. Intriguingly, although these two viruses show strong similarities, such as virion morphology, genome sequence, gene synteny and expression patterns, it was shown that they are capable of inducing distinct hot reactions. Indeed, whereas SSV1 did not significantly affect the host gene expression, a strong host response was reported upon infection by SSV2. In particular, more than a hundred genes were differentially expressed during the growth of the SSV2-infected strain and, even more interesting, was the up-regulation of both CRISPR loci and cas genes. Data reported herein strongly suggest that the host response is influenced by the ability of the virus to control its own gene expression and genome replication during lysogenic growth. In this regard, it was found that SSV1 is a temperate virus that regulates the expression of its genes towards the activity of the transcription repressor F55. As a result, the SSV1 copy number is kept quite low and constant in the lysogenic cells. On the other hand, SSV2 lacks such a regulator and it is incapable of self-regulating its replication; therefore, the infection by SSV2 represents a mush more stressing load for the host. Moreover, this study highlighted an interesting feature of the host-viral interaction in the frame of the CRISPR-response, i.e. host cells containing an integrated copy of the viral genome (provirus) are forced to develop a surviving strategy in order to avoid self-attack by the CRISPR-system. This finding has cast doubt on the feasibility of using this system to challenge viral infections, at least as regards integrative viruses. Further implication and possible solutions to this potential drawback will be herein discussed.