Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system.

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct 'anti-CRISPR' genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages.

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.

Myxoma virus oncolysis of primary and metastatic B16F10 mouse tumors in vivo.

Myxoma virus (MV) is a rabbit-specific poxvirus, whose unexpected tropism to human cancer cells has led to studies exploring its potential use in oncolytic therapy. MV infects a wide range of human cancer cells in vitro, in a manner intricately linked to the cellular activation of Akt kinase. MV has also been successfully used for treating human glioma xenografts in immunodeficient mice. This study examines the effectiveness of MV in treating primary and metastatic mouse tumors in immunocompetent C57BL6 mice. We have found that several mouse tumor cell lines, including B16 melanomas, are permissive to MV infection. B16F10 cells were used for assessing MV replication and efficacy in syngeneic primary tumor and metastatic models in vivo. Multiple intratumoral injections of MV resulted in dramatic inhibition of tumor growth. Systemic administration of MV in a lung metastasis model with B16F10LacZ cells was dramatically effective in reducing lung tumor burden. Combination therapy of MV with rapamycin reduced both size and number of lung metastases, and also reduced the induced antiviral neutralizing antibody titres, but did not affect tumor tropism. These results show MV to be a promising virotherapeutic agent in immunocompetent animal tumor models, with good efficacy in combination with rapamycin.

Efficacy of bacteriophage treatment on Pseudomonas aeruginosa biofilms.

INTRODUCTION: Bacterial viruses (phages) have been used successfully in the treatment of animal and human bacterial infections. This study examined the potential use of phage therapy against Pseudomonas aeruginosa strain PA14 biofilms in a root canal model. METHODS: Part 1: The 24-hour and 96-hour PA14 biofilms grown in microplates were treated with phages identified as possessing potential biofilm-degrading activities, and the post-treatment bacterial biomass was quantified by using crystal violet staining. Part 2: The 24-hour and 96-hour PA14 biofilms grown in prepared root canals of extracted human mandibular incisors were treated with phages identified with potential biofilm-degrading activities. Post-treatment intracanal samples were taken by using paper points and round burs to assess phage and bacterial counts. RESULTS: Part 1: We identified 2 phages (JBD4 and JBD44a) with putative biofilm-degrading activities. Treatment of PA14 biofilms with these phages produced a significant reduction in the mean percentage of biomass in 24-hour (P< .05) and 96-hour (P= .08) biofilms. Part 2: In 24-hour and 96-hour PA14 biofilms in a root canal model, no significant difference was found in the number of colony-forming units after phage treatment (P> .05). CONCLUSIONS: Phage application significantly reduced the biomass of 24-hour and 96-hour PA14 biofilms grown on microplates but did not produce significant reduction of 24-hour or 96-hour PA14 biofilms grown in the extracted tooth model.