Expression of a phage-encoded Gp21 protein protects Pseudomonas aeruginosa against phage infection

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection has been identified and characterized in detail. Here, we isolate a phage vB_Pae_QDWS capable of infecting Pseudomonas aeruginosa PAO1, and report its encoded Gp21 protein protects PAO1 against phage infection. Expressing of Gp21 regulate bacterial quorum sensing with an inhibitory effect in low cell density and activation effect in high cell density. By testing the TFPs-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found decreased the pilus synthesis. Further constructing the TFPs synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated Gp21 protein could block phage infection via decreasing the TFPs-mediated phage adsorption. Gp21 is a novel protein that inhibit phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. Importance The majority of the annotated phage genes are currently deposited as “hypothetical protein” with unknown function. Researches revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Differently, we report a phage encoded protein Gp21 that protect the host against phage infection. The pathways that Gp21 involved in anti-phage defense in Pseudomonas aeruginosa PAO1 are interfering with quorum sensing and decreasing the type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into the phage-host evolutionary dynamics.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Defense-associated sirtuins (DSR) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbor an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defense systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Friends or Foes? Rapid Determination of Dissimilar Colistin and Ciprofloxacin Antagonism of Pseudomonas aeruginosa Phages

Phage therapy is a century-old technique employing viruses (phages) to treat bacterial infections, and in the clinic it is often used in combination with antibiotics. Antibiotics, however, interfere with critical bacterial metabolic activities that can be required by phages. Explicit testing of antibiotic antagonism of phage infection activities, though, is not a common feature of phage therapy studies. Here we use optical density-based ‘lysis-profile’ assays to assess the impact of two antibiotics, colistin and ciprofloxacin, on the bactericidal, bacteriolytic, and new-virion-production activities of three Pseudomonas aeruginosa phages. Though phages and antibiotics in combination are more potent in killing P. aeruginosa than either acting alone, colistin nevertheless substantially interferes with phage bacteriolytic and virion-production activities even at its minimum inhibitory concentration (1× MIC). Ciprofloxacin, by contrast, has little anti-phage impact at 1× or 3× MIC. We corroborate these results with more traditional measures, particularly colony-forming units, plaque-forming units, and one-step growth experiments. Our results suggest that ciprofloxacin could be useful as a concurrent phage therapy co-treatment especially when phage replication is required for treatment success. Lysis-profile assays also appear to be useful, fast, and high-throughput means of assessing antibiotic antagonism of phage infection activities.

Impact of Phage CDHS-1 on the Transcription, Physiology and Pathogenicity of a Clostridioides difficile Ribotype 027 Strain, R20291

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection.

Genomic Changes and Genetic Divergence of Vibrio alginolyticus Under Phage Infection Stress Revealed by Whole-Genome Sequencing and Resequencing

Bacteriophages (phages) and their bacterial hosts were the most abundant and genetically highly diverse organisms on the earth. In this study, a series of phage-resistant mutant (PRM) strains derived from Vibrio alginolyticus were isolated and Infrequent-restriction-site PCR (IRS-PCR) was used to investigate the genetic diversity of the PRM strains. Phenotypic variations of eight PRM strains were analyzed using profiles of utilizing carbon sources and chemical sensitivity. Genetic variations of eight PRM strains and coevolved V. alginolyticus populations with phages were analyzed by whole-genome sequencing and resequencing, respectively. The results indicated that eight genetically discrepant PRM stains exhibited abundant and abundant phenotypic variations. Eight PRM strains and coevolved V. alginolyticus populations (VE1, VE2, and VE3) contained numerous single nucleotide variations (SNVs) and insertions/indels (InDels) and exhibited obvious genetic divergence. Most of the SNVs and InDels in coding genes were related to the synthesis of flagellar, extracellular polysaccharide (EPS), which often served as the receptors of phage invasion. The PRM strains and the coevolved cell populations also contained frequent mutations in tRNA and rRNA genes. Two out of three coevolved populations (VE1 and VE2) contained a large mutation segment severely deconstructing gene nrdA, which was predictably responsible for the booming of mutation rate in the genome. In summary, numerous mutations and genetic divergence were detected in the genomes of V. alginolyticus PRM strains and in coevolved cell populations of V. alginolyticus under phage infection stress. The phage infection stress may provide an important force driving genomic evolution of V. alginolyticus.

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Cell wall deficiency as an escape mechanism from phage infection

The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.