Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria.

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7.

Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterial-growth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms.

Different approaches for using bacteriophages against antibiotic-resistant bacteria.

Bacterial resistance to antibiotics is an emerging threat requiring urgent solutions. Ever since their discovery, lytic bacteriophages have been suggested as therapeutic agents, but their application faces various obstacles: sequestration of the phage by the spleen and liver, antibodies against the phage, narrow host range, poor accessibility to the infected tissue, and bacterial resistance. Variations on bacteriophage use have been suggested, such as temperate phages as gene-delivery vehicles into pathogens. This approach, which is proposed to sensitize pathogens residing on hospital surfaces and medical personnel's skin, and its prospects are described in this addendum. Furthermore, phage-encoded products have been proposed as weapons against antibiotic resistance in bacteria. We describe a new phage protein which was identified during basic research into T7 bacteriophages. This protein may serendipitously prove useful for treating antibiotic-resistant pathogens. We believe that further basic research will lead to novel strategies in the fight against antibiotic-resistant bacteria.

Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division.

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.

CRISPR adaptation in Escherichia coli subtypeI-E system.

The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.

Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning.

Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using more than 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an "antidefense" protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.