RNA-Sequencing Reveals the Progression of Phage-Host Interactions between phi R1-37 and Yersinia enterocolitica

Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_phi R1-37 (phi R1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of phi R1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins.Peer reviewe

Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages

Pseudomonas virus vB_PaeM_PA5oct is proposed as a model jumbo bacteriophage to investigate phage-bacteria interactions and is a candidate for phage therapy applications. Combining hybrid sequencing, RNA-Seq and mass spectrometry allowed us to accurately annotate its 286,783 bp genome with 461 coding regions including four non-coding RNAs (ncRNAs) and 93 virion-associated proteins. PA5oct relies on the host RNA polymerase for the infection cycle and RNA-Seq revealed a gradual take-over of the total cell transcriptome from 21% in early infection to 93% in late infection. PA5oct is not organized into strictly contiguous regions of temporal transcription, but some genomic regions transcribed in early, middle and late phases of infection can be discriminated. Interestingly, we observe regions showing limited transcription activity throughout the infection cycle. We show that PA5oct upregulates specific bacterial operons during infection including operons pncA-pncB1-nadE involved in NAD biosynthesis, psl for exopolysaccharide biosynthesis and nap for periplasmic nitrate reductase production. We also observe a downregulation of T4P gene products suggesting mechanisms of superinfection exclusion. We used the proteome of PA5oct to position our isolate amongst other phages using a gene-sharing network. This integrative omics study illustrates the molecular diversity of jumbo viruses and raises new questions towards cellular regulation and phage-encoded hijacking mechanisms

In Vitro and In Vivo Assessments of Two Newly Isolated Bacteriophages against an ST13 Urinary Tract Infection Klebsiella pneumoniae.

peer reviewedAntibiotic resistance represents a major public health concern requiring new alternatives including phage therapy. Klebsiella pneumoniae belongs to the ESKAPE bacteria and can cause urinary tract infections (UTIs). The aims of this study were to isolate and characterize new bacteriophages against a K. pneumoniae strain isolated from UTIs and to assess their efficacy in vitro and in vivo in a Galleria (G.) mellonella larvae model. For this purpose, two bacteriophages were newly isolated against an ST13 K. pneumoniae strain isolated from a UTI and identified as K3 capsular types by wzi gene PCR. Genomic analysis showed that these bacteriophages, named vB_KpnP_K3-ULINTkp1 and vB_KpnP_K3-ULINTkp2, belong to the Drulisvirus genus. Bacteriophage vB_KpnP_K3-ULINTkp1 had the narrowest host spectrum (targeting only K3), while vB_KpnP_K3-ULINTkp2 also infected other Klebsiella types. Short adsorption times and latent periods were observed for both bacteriophages. In vivo experiments showed their ability to replicate in G. mellonella larvae and to decrease host bacterial titers. Moreover, both bacteriophages improved the survival of the infected larvae. In conclusion, these two bacteriophages had different in vitro properties and showed in vivo efficacy in a G. mellonella model with a better efficiency for vB_KpnP_K3-ULINTkp2

A suggested new bacteriophage genus: “Viunalikevirus”

We suggest a bacteriophage genus, “Viunalikevirus”, as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-012-1360-5) contains supplementary material, which is available to authorized users

Whole transcriptome analyses of Pseudomonas aeruginosa after infection by representatives of different phage clades

Detailed knowledge of the progression of bacteriophage infection has been limited to few model bacteriophages, mostly infecting Escherichia coli, as well as the results of low throughput and increasingly antiquated methods. However, state-of-the-art approaches now make it possible to explore decades of interest in the transcriptional strategies of phage as well as how they affect host transcription and metabolism, now re-sparked by modern therapeutic and biotechnological potential, as well as the ability to ask deeper more comprehensive questions. By performing RNA-Seq on the non-ribosomal RNA of synchronously infected cells we are able to compare host transcript data for early, middle and late transcription with six fundamentally different lytic phages. These include PhiKZ, PEV2, 14-1, LUZ19, YuA, and the novel F7 infecting P. aeruginosa PAO1. We also collaborated with colleagues at the Pasteur Institute to collect similar data for PAK_P3 and PAK_P4 infections of P. aeruginosa PAK. With this data we now have experimental evidence to support or revise in silico predictions of open reading frames, operons, promoters, 5’ untranslated regions, regulatory elements, and terminators as well as posit the presence of non-coding and small RNA species for the whole phage at once. With experimental evidence identifying and defining these features at each of the various stages of infection, we are able to construct a transcription level narrative for each phage that powerfully describes them on a fundamental level. Indeed, we have found a number of significant surprises including a large number of antisense RNA molecules that dominate middle transcription of structural and lysis coding sequences in PEV2 presumably silencing all of late translation until late protein expression is triggered. At the same time, unlike any other tailed virus that we are aware of, phage 14-1 appears to not differentially express its genome on a transcriptional level, and instead uses a translation level system to express gene features associated with converting the cell to a phage metabolism during early infection while expressing structural coding sequences in late infection. Similarly, this technology enables looking at the impact of phage infection on the relative abundance of host transcripts during infection. Having data for so many fundamentally different phage types, all infecting the same host, also provides the unique ability to distinguish phage-mediated manipulations of host transcription (by looking for effects that are specific to each phage), from host mediated responses to phage infection(establishing effects that are common to each studied infection). Chapters 3-9 detail and discuss the distinct ways in which the individual phages manipulate between 7% and 11% of the gene features of their hosts to their benefit. demonstrating for the first time that not only does this phenomenon exist but that it is diverse and a key feature of phage infection. At the same time, we have also found that 5.5% of gene features in P. aeruginosa PAO1 are differentially expressed by the host in response to each of the phage infections studied here. Interestingly, this includes the pqsABCDE-phnAB operons of P. aeruginosa PAO1 as well as their PhrS sRNA regulator. As the PQS quorum sensing system has been previously demonstrated to control a stress response encouraging metabolic dormancy, and as we have found phiKMV-like phage LUZ2 is only able to infect PA14 when PQS is knocked out, this may indicate the discovery of a novel micro-colony wide phage defense system. This research not only contributes significantly to our fundamental understanding of Pseudomonas phages (doubling the number of known transcription regulation mechanisms of the host), it also impacts bacteriophage biology and biotechnology in general. It also has direct implications for the safety of phage therapy as it is currently being assessed in medically vulnerable patients. Indeed, our finding that phage LUZ19 upregulates virulence factors responsible for dehydrogenating hydrogen cyanide from glycine not only suggests that the production of HCN might be a direct concern, but that host virulence factors in general have the potential to interfere.status: publishe

RNA-Sequencing Reveals the Progression of Phage-Host Interactions between φR1-37 and Yersinia enterocolitica

Despite the expanding interest in bacterial viruses (bacteriophages), insights into the intracellular development of bacteriophage and its impact on bacterial physiology are still scarce. Here we investigate during lytic infection the whole-genome transcription of the giant phage vB_YecM_φR1-37 (φR1-37) and its host, the gastroenteritis causing bacterium Yersinia enterocolitica. RNA sequencing reveals that the gene expression of φR1-37 does not follow a pattern typical observed in other lytic bacteriophages, as only selected genes could be classified as typically early, middle or late genes. The majority of the genes appear to be expressed constitutively throughout infection. Additionally, our study demonstrates that transcription occurs mainly from the positive strand, while the negative strand encodes only genes with low to medium expression levels. Interestingly, we also detected the presence of antisense RNA species, as well as one non-coding intragenic RNA species. Gene expression in the phage-infected cell is characterized by the broad replacement of host transcripts with phage transcripts. However, the host response in the late phase of infection was also characterized by up-regulation of several specific bacterial gene products known to be involved in stress response and membrane stability, including the Cpx pathway regulators, ATP-binding cassette (ABC) transporters, phage- and cold-shock proteins.status: publishe

Comparative transcriptomics analyses reveal the conservation of an ancestral infectious strategy in two bacteriophage genera

Although the evolution of tailed bacteriophages has increasingly been better understood through comparisons of their DNA sequences, the functional consequences of this evolution on phage infectious strategies have remained unresolved. In this study, we comprehensively compared the transcriptional strategies of two related myoviruses, PAK_P3 and PAK_P4, infecting the same Pseudomonas aeruginosa host strain. Outside of the conservation of their structural clusters, their highly syntenic genomes display only limited DNA similarity. Despite this apparent divergence, we found that both viruses follow a similar infection scheme, relying on a temporal regulation of their gene expression, likely involving the use of antisense transcripts, as well as a rapid degradation of 90% of the host non-ribosomal mRNA, as previously reported for PAK_P3. However, the kinetics of the mRNA degradation is remarkably faster during PAK_P4 infection. Moreover, we found that each virus has evolved specific adaptations, as exemplified by the distinct patterns of their core genes expression as well as the specific manipulation of the expression of iron-related host genes by PAK_P4. This study enhances our understanding of the evolutionary process of virulent phages, which relies on adjusting globally conserved ancestral infection mechanisms.The ISME Journal advance online publication, 12 May 2017; doi:10.1038/ismej.2017.63.status: publishe

Pseudomonas predators: understanding and exploiting phage-host interactions

Species in the genus Pseudomonas thrive in a diverse set of ecological niches and include crucial pathogens, such as the human pathogen Pseudomonas aeruginosa and the plant pathogen Pseudomonas syringae. The bacteriophages that infect Pseudomonas spp. mirror the widespread and diverse nature of their hosts. Therefore, Pseudomonas spp. and their phages are an ideal system to study the molecular mechanisms that govern virus-host interactions. Furthermore, phages are principal catalysts of host evolution and diversity, which directly affects the ecological roles of environmental and pathogenic Pseudomonas spp. Understanding these interactions not only provides novel insights into phage biology but also advances the development of phage therapy, phage-derived antimicrobial strategies and innovative biotechnological tools that may be derived from phage-bacteria interactions.status: publishe

K1 Capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

peer reviewed[en] AIMS: Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. METHODS AND RESULTS: Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production and virulence in the in-vivo Galleria mellonella larvae model. One out of the 6 resistant isolates exhibited a significantly slower growth rate suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. CONCLUSIONS: A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production, and a decreased virulence in vivo