Abstract
The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.
Highlights
Bacteriophages are abundant and ubiquitous viruses in most natural environments and play an important role in the ecology of their bacterial hosts
One of these strategies involves the Clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system, in which acquired immunity is achieved against invading nucleic acids, providing resistance that can be passed on to future generations [1,2,3,4]
Clustered regularly interspaced short palindromic repeats (CRISPRs) are loci found in approximately 46% and 87% of bacteria and archaea, respectively [5]
Summary
Bacteriophages (phages) are abundant and ubiquitous viruses in most natural environments and play an important role in the ecology of their bacterial hosts. Bacteria have evolved various mechanisms to defend themselves against viral predation One of these strategies involves the CRISPR/Cas system, in which acquired immunity is achieved against invading nucleic acids, providing resistance that can be passed on to future generations [1,2,3,4]. Clustered regularly interspaced short palindromic repeats (CRISPRs) are loci found in approximately 46% and 87% of bacteria and archaea, respectively [5]. These hypervariable regions consist of a leader sequence followed by an array of direct nucleotide repeats interspersed with non-repetitive DNA regions called spacer sequences. The sequence and function variability across the Cas proteins of the three CRISPR/Cas types [17], along with the functional idiosyncrasies of the various core Cas proteins, have compounded the difficulty of Cas proteins characterization
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