Phage-encoded cationic antimicrobial peptide required for lysis

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp 28 in OM disruption. Additionally, gp 28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp 28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp 28 protein prior to lysis. Gp 28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp 28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp 28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

The resident TP712 prophage of Lactococcus lactis MG1363 provides extra holin functions to the new P335 phage CAP for effective host lysis

Prophages are widely present in Lactococcus lactis , a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid- and prophage-free, MG1363 carries two complete prophages TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the “lytic TP712 lysogens”, which had remained unnoticed. CAP is able to excise after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression i n trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work to generate the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor L. lactis NCDO712 and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the “pre-sequencing” era. Importance Prophages are bacterial viruses that integrate in the chromosome of bacteria until an environmental trigger induces their lytic cycle ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we have discovered that two temperate phages TP712 and CAP infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the formerly thought to be single TP712 lysogens that had remained a mystery for many years.

Phenotypic and Genotypic Characterization of the New Bacillus Cereus Phage SWEP1

A new Bacillus cereus phage SWEP1 was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins where 103 were assigned functionally. No tRNA gene was found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggesting that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.

Host-dependent differences in replication and virion release strategy of the Sulfolobus Spindle-shaped Virus strain SSV9 (a.k.a., SSVK1): Lytic replication in hosts of the family Sulfolobaceae

The Sulfolobus Spindle-shaped Virus (SSV) system is a model for studying thermophilic archaeal virus biology. Several factors make the SSV system amenable to studying archaeal genetics and virus-host interactions in extreme environments. It has been shown that populations of Sulfolobus, the natural host, exhibit biogeographic structure. The acidic (pH<4.5) high temperature (65-88°C) habitats have low biodiversity, which diminishes prospects for host switch. SSVs and their hosts are readily cultured in liquid media and on plates. Given the wide geographic separation between various SSV-Sulfolobus habitats, the system is also amenable to studying allopatric versus sympatric virus-host interactions. We previously reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We discovered a strikingly broad host-range for strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species of Sulfolobus and release virus particles via blebbing as a preferred strategy over host lysis (in reported laboratory infections). Here, we show, that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not release virions via blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts, while exhibiting canonical non-lytic virion release (historically reported for SSVs) on a single sympatric host. Lytic versus non-lytic virus release does not appear to be driven by multiplicity of infection. Data suggest that SSV9 is more stable than other SSVs in suspension; however, genetic substrates (e.g., CRISPR profiles) underlying non-lytic versus lytic virion release remain unresolved and are the subject of ongoing investigation.

Characterization of a new podovirus infecting Paenibacillus larvae

The Paenibacillus larvae infecting phage API480 (vB_PlaP_API480) is the first reported podovirus for this bacterial species, with an 58 nm icosahedral capsid and a 12 × 8 nm short, non-contractile tail. API480 encodes 77 coding sequences (CDSs) on its 45,026 bp dsDNA genome, of which 47 were confirmed using mass spectrometry. This phage has got very limited genomic and proteomic similarity to any other known ones registered in public databases, including P. larvae phages. Comparative genomics indicates API480 is a new species as it’s a singleton with 28 unique proteins. Interestingly, the lysis module is highly conserved among P. larvae phages, containing a predicted endolysin and two putative holins. The well kept overall genomic organisation (from the structural and morphogenetic modules to the host lysis, DNA replication and metabolism related proteins) confirms a common evolutionary ancestor among P. larvae infecting phages. API480 is able to infect 69% of the 61 field strains with an ERIC I genotype, as well as ERIC II strains. Furthermore, this phage is very stable when exposed to high glucose concentrations and to larval gastrointestinal conditions. This highly-specific phage, with its broad lytic activity and stability in hive conditions, might potentially be used in the biocontrol of American Foulbrood (AFB).

Evaluation of Commercial Prototype Bacteriophage Intervention Designed for Reducing O157 and Non-O157 Shiga-Toxigenic Escherichia coli (STEC) on Beef Cattle Hide

Microbiological safety of beef products can be protected by application of antimicrobial interventions throughout the beef chain. This study evaluated a commercial prototype antimicrobial intervention comprised of lytic bacteriophages formulated to reduce O157 and non-O157 Shiga-toxigenic Escherichia coli (STEC) on beef cattle hide pieces, simulating commercial pre-harvest hide decontamination. STEC reduction in vitro by individual and cocktailed phages was determined by efficiency of plating (EOP). Following STEC inoculation onto hide pieces, the phage intervention was applied and hide pieces were analyzed to quantify reductions in STEC counts. Phage intervention treatment resulted in 0.4 to 0.7 log10 CFU/cm2 (p < 0.01) E. coli O157, O121, and O103 reduction. Conversely, E. coli O111 and O45 did not show any significant reduction after application of bacteriophage intervention (p > 0.05). Multiplicity of infection (MOI) evaluation indicated E. coli O157 and O121 isolates required the fewest numbers of phages per host cell to produce host lysis. STEC-attacking phages may be applied to assist in preventing STEC transmission to beef products.

Megaphage infect Prevotella and variants are widespread in gut microbiomes

Bacteriophage (phage) dramatically shape microbial community composition, redistribute nutrients via host lysis, and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phage in the human microbiome. We investigated gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups, and Danish pigs, and report that many contain phage belonging to a clade with genomes >540 kb in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phage. We refer to these as Lak phage. CRISPR spacer targeting indicates that the Lak phage infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed, and up to 35 putative tRNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in relative abundance of phage sub-populations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phage are widespread in gut communities that contain Prevotella species, especially in individuals in the developing world, and conclude that megaphage, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

Does the microbiome and virome contribute to myalgic encephalomyelitis/chronic fatigue syndrome?

Myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) (ME/CFS) is a disabling and debilitating disease of unknown aetiology. It is a heterogeneous disease characterized by various inflammatory, immune, viral, neurological and endocrine symptoms. Several microbiome studies have described alterations in the bacterial component of the microbiome (dysbiosis) consistent with a possible role in disease development. However, in focusing on the bacterial components of the microbiome, these studies have neglected the viral constituent known as the virome. Viruses, particularly those infecting bacteria (bacteriophages), have the potential to alter the function and structure of the microbiome via gene transfer and host lysis. Viral-induced microbiome changes can directly and indirectly influence host health and disease. The contribution of viruses towards disease pathogenesis is therefore an important area for research in ME/CFS. Recent advancements in sequencing technology and bioinformatics now allow more comprehensive and inclusive investigations of human microbiomes. However, as the number of microbiome studies increases, the need for greater consistency in study design and analysis also increases. Comparisons between different ME/CFS microbiome studies are difficult because of differences in patient selection and diagnosis criteria, sample processing, genome sequencing and downstream bioinformatics analysis. It is therefore important that microbiome studies adopt robust, reproducible and consistent study design to enable more reliable and valid comparisons and conclusions to be made between studies. This article provides a comprehensive review of the current evidence supporting microbiome alterations in ME/CFS patients. Additionally, the pitfalls and challenges associated with microbiome studies are discussed.

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods

ABSTRACTCronobacter sakazakiiis an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, theC. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent againstC. sakazakii, it was added to infant formula milk contaminated with aC. sakazakiiclinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 105.