Phage-antibiotic synergy: Cell filamentation is a key driver of successful phage predation.

Phages are promising tools to fight antibiotic-resistant bacteria, and as for now, phage therapy is essentially performed in combination with antibiotics. Interestingly, combined treatments including phages and a wide range of antibiotics lead to an increased bacterial killing, a phenomenon called phage-antibiotic synergy (PAS), suggesting that antibiotic-induced changes in bacterial physiology alter the dynamics of phage propagation. Using single-phage and single-cell techniques, each step of the lytic cycle of phage HK620 was studied in E. coli cultures treated with either ceftazidime, cephalexin or ciprofloxacin, three filamentation-inducing antibiotics. In the presence of sublethal doses of antibiotics, multiple stress tolerance and DNA repair pathways are triggered following activation of the SOS response. One of the most notable effects is the inhibition of bacterial division. As a result, a significant fraction of cells forms filaments that stop dividing but have higher rates of mutagenesis. Antibiotic-induced filaments become easy targets for phages due to their enlarged surface areas, as demonstrated by fluorescence microscopy and flow cytometry techniques. Adsorption, infection and lysis occur more often in filamentous cells compared to regular-sized bacteria. In addition, the reduction in bacterial numbers caused by impaired cell division may account for the faster elimination of bacteria during PAS. We developed a mathematical model to capture the interaction between sublethal doses of antibiotics and exposition to phages. This model shows that the induction of filamentation by sublethal doses of antibiotics can amplify the replication of phages and therefore yield PAS. We also use this model to study the consequences of PAS on the emergence of antibiotic resistance. A significant percentage of hyper-mutagenic filamentous bacteria are effectively killed by phages due to their increased susceptibility to infection. As a result, the addition of even a very low number of bacteriophages produced a strong reduction of the mutagenesis rate of the entire bacterial population. We confirm this prediction experimentally using reporters for bacterial DNA repair. Our work highlights the multiple benefits associated with the combination of sublethal doses of antibiotics with bacteriophages.

The prophage-encoded transcriptional regulator AppY has pleiotropic effects on E. coli physiology.

Bacterial genome diversity is influenced by prophages, which are viral genomes integrated into the bacterial chromosome. Most prophage genes are silent but those that are expressed can provide unexpected properties to their host. Using as a model E. coli K-12 that carries 9 defective prophages in its genome, we aimed at highlighting the impact of genes encoded by prophages on host physiology. We focused our work on AppY, a transcriptional regulator encoded on the DLP12 prophage. By performing RNA-Seq experiments, we showed that AppY production modulates the expression of more than 200 genes. Among them, 11 were identified by ChIP-Seq as direct AppY targets. AppY directly and positively regulates several genes involved in the acid stress response including the master regulator gene gadE but also nhaR and gadY, two genes important for biofilm formation. Moreover, AppY indirectly and negatively impacts bacterial motility by favoring the degradation of FlhDC, the master regulator of the flagella biosynthesis. As a consequence of these regulatory effects, AppY increases acid stress resistance and biofilm formation while also causing a strong defect in motility. Our research shed light on the importance to consider the genetic interactions occurring between prophages and bacteria to fully understand bacterial physiology. It also highlights how a prophage-encoded transcriptional regulator integrates in a complex manner into the host regulatory network and how it benefits its host, allowing it to cope with changing environmental conditions.

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.

Genetic Mining of Newly Isolated Salmophages for Phage Therapy.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans.

Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area.

Epigenetic biosensors for bacteriophage detection and phage receptor discrimination.

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvABON subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvABON subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed biosensors able to detect LPS, as well as protein-binding coliphages. Moreover, the combination of these tools allowed to identify the bacterial receptor triggering phage infection. The epigenetic opvAB::gfp biosensor thus comes in different flavours to detect a wide range of bacteriophages and identify the type of receptor they recognize.

[Antibacterial applications of bacteriophages]. / Les applications antibactériennes des bactériophages.

In the 1917 article in which Félix d'Hérelle describes his first observations and proposes the name of bacteriophage, he also reports the first use of these viruses to treat bacterial infections, thus giving birth to phage therapy. Soon after antibiotics supplanted bacteriophages. Today, bacteria resistant to multiple antibiotics become a growing public health issue worldwide. This situation has revived research aiming at developing the antibacterial activity of bacteriophages to treat patients as well as diseases in animals and plants. In fact, the areas of applications of bacteriophages as antibacterial are widening as current solutions of chemical nature are questioned. This review summarizes the basic principles of therapeutic applications of bacteriophages and presents recent data in areas where commercial exploitation is occurring or about to emerge.

[A century of research on bacteriophages]. / Un siècle de recherche sur les bactériophages.

Bacteriophages have a prominent place in the living world. They participate to our understanding of the living world through three main aspects : (i) the dissection of the most intimist aspects of viral infection molecular mechanisms (molecular biology), (ii) the description and functioning mechanisms of ecosystems (ecology), and (iii) the adaptive dynamics of integrated viral and host-cell populations (evolution). This review looks back at the genesis of these fundamental findings and draws a picture of the most active fields of current research.

Prophages in Salmonella enterica: a driving force in reshaping the genome and physiology of their bacterial host?

Thanks to the exponentially increasing number of publicly available bacterial genome sequences, one can now estimate the important contribution of integrated viral sequences to the diversity of bacterial genomes. Indeed, temperate bacteriophages are able to stably integrate the genome of their host through site-specific recombination and transmit vertically to the host siblings. Lysogenic conversion has been long acknowledged to provide additional functions to the host, and particularly to bacterial pathogen genomes where prophages contribute important virulence factors. This review aims particularly at highlighting the current knowledge and questions about lysogeny in Salmonella genomes where functional prophages are abundant, and where genetic interactions between host and prophages are of particular importance for human health considerations.

"French Phage Network"-Third Meeting Report.

In its third year of existence, the French Phage Network (Phages.fr) is pursuing its expansion. With more than 25 groups, mostly based in France, working on the various aspects of phage research, the network has increased its visibility, interactivity, and activity. The third meeting of the Phages.fr network, held on November 2017 at the Gif-sur-Yvette Centre National de la Recherche Scientifique (CNRS) campus, was a great opportunity for many young scientists to present their work and interact with more senior scientists, amongst which several were invited from abroad. Here we provide a summary of the work presented at this occasion during the oral presentations and poster sessions.

"French Phage Network"-Second Meeting Report.

The study of bacteriophages (viruses of bacteria) includes a variety of approaches, such as structural biology, genetics, ecology, and evolution, with increasingly important implications for therapeutic and industrial uses. Researchers working with phages in France have recently established a network to facilitate the exchange on complementary approaches, but also to engage new collaborations. Here, we provide a summary of the topics presented during the second meeting of the French Phage Network that took place in Marseille in November 2016.

Substrate-independent luminescent phage-based biosensor to specifically detect enteric bacteria such as E. coli.

Water quality is a major safety consideration in environments that are impacted by human activity. The key challenge of the COMBITOX project is to develop a unique instrument that can accommodate several biodetector systems (see the accompanying COMBITOX papers) able to detect different pollutants such as bacteria, toxins, and heavy metals. The output signal chosen by our consortium is based on luminescence detection. Our group recently developed phage-based biosensors using gfp as a reporter gene to detect enteric bacteria in complex environments such as sea water, and the main challenge we faced was to adapt our biodetector to a luminescent signal that could fit the COMBITOX project requirements. Another key point was to use a substrate-independent reporter system in order to avoid substrate addition in the detection prototype. This paper describes the development of a phage-based biodetector using a luminescent and substrate-independent output to detect some enteric bacteria, such as Escherichia coli, in water samples. We have successfully engineered various prototypes using the HK620 and HK97 bacteriophages that use different packaging systems, and both proved functional for the integration of the full luxCDABE operon controlled by two different bacterial promoters. We show that the luxCDABE operon controlled by the PrplU bacterial promoter is the most efficient in terms of signal emission. The emission of luminescence is specific and allows the detection of 104 bacteria per milliliter in 1.5 h post-infection with neither a concentration nor enrichment step.

Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors.

The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium.

Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

Bacterial genome remodeling through bacteriophage recombination.

Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes.

Transcription termination controls prophage maintenance in Escherichia coli genomes.

Prophages represent a large fraction of prokaryotic genomes and often provide new functions to their hosts, in particular virulence and fitness. How prokaryotic cells maintain such gene providers is central for understanding bacterial genome evolution by horizontal transfer. Prophage excision occurs through site-specific recombination mediated by a prophage-encoded integrase. In addition, a recombination directionality factor (or excisionase) directs the reaction toward excision and prevents the phage genome from being reintegrated. In this work, we describe the role of the transcription termination factor Rho in prophage maintenance through control of the synthesis of transcripts that mediate recombination directionality factor expression and, thus, excisive recombination. We show that Rho inhibition by bicyclomycin allows for the expression of prophage genes that lead to excisive recombination. Thus, besides its role in the silencing of horizontally acquired genes, Rho also maintains lysogeny of defective and functional prophages.

Insights into the functions of a prophage recombination directionality factor.

Recombination directionality factors (RDFs), or excisionases, are essential players of prophage excisive recombination. Despite the essentially catalytic role of the integrase in both integrative and excisive recombination, RDFs are required to direct the reaction towards excision and to prevent re-integration of the prophage genome when entering a lytic cycle. KplE1, HK620 and numerous (pro)phages that integrate at the same site in enterobacteria genomes (such as the argW tRNA gene) all share a highly conserved recombination module. This module comprises the attL and attR recombination sites and the RDF and integrase genes. The KplE1 RDF was named TorI after its initial identification as a negative regulator of the tor operon. However, it was characterized as an essential factor of excisive recombination. In this study, we designed an extensive random mutagenesis protocol of the torI gene and identified key residues involved in both functions of the TorI protein. We show that, in addition to TorI-TorR protein-protein interaction, TorI interacts in solution with the IntS integrase. Moreover, in vitro, TorR and IntS appear to compete for TorI binding. Finally, our mutagenesis results suggest that the C-terminal part of the TorI protein is dedicated to protein-protein interactions with both proteins TorR and IntS.

DnaJ (Hsp40 protein) binding to folded substrate impacts KplE1 prophage excision efficiency.

Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.