The bacteriophage λ rex-centric mutualism phenotype, conditional rex, and other novel rex phenotypes

Rex-Centric Mutualism The Rex Exclusion Phenotype, encoded by the rexA-rexB genes of bacteriophage lambda is defined as the inability of the mutant bacteriophage T4rll to generate plaques on a lysogenized E. coli(λ) host. Although the phenotype was first observed more than four decades ago, few advancements have been made in the elucidation of the Rex mechanism. The current model for Rex exclusion proposed in 1992 by Parma et aI., states that the Rex system functions as an altruistic bacterial apoptotic module triggered by T4rll infection of the lysogenized host. We asked whether Rex exclusion confers a protective or a cell-killing phenotype to the host and found that following T4rll infection, the Rex system can channel lysogenic cells into a temporary arrested growth phase that gives an overall protective effect to the host even at enormous multiplicities of infection compared to nonlysogens, despite some associated killing. We termed this phenomenon Rex-centric mutualism. In 1989, Snyder and McWilliams demonstrated that the Rex-mediated arrest of cell growth can be triggered in the absence of infection by over-expressing rexA relative to rexB. We noted that plasmid expression of rexA in Rex⁺ cells in the absence of infection resulted in similar cellular viabilities as that observed following T4rll infection. We visualized lambda Rex⁺ lysogens, infected by T4rll and found that they were much delayed in colony formation, contracted in length, formed aggregates with adjacent cells, and released flagella. These phenotypes were accentuated in nonlysogenic cells carrying a specific multicopy rexA-rexB plasmid: cells were about two-fold contracted in length, expressed membrane-bound and secreted flagellar structures, were odorous, were insensitive to infection by a variety of phages, and they extensively clumped/adhered when grown up in culture. Lysogenic cells mutated for rpoS stationary phase sigma factor were abrogated for Rex-centric mutualism, exhibiting more than 400-fold lower viability compared to the wild type, following infection by T4rll. These phenotypes show that the Rex system can impart a stationary phase like response that protects the host from T4rll killing. RexB Inhibition of T4rll Lysis Inhibition Phenotype We add to the activities and phenotypes of RexB. Expression of rexB from either a multicopy plasmid, or a rexA⁻-rexB⁺ phage is capable of suppressing the Lysis Inhibition Phenotype (LIN) seen upon infection of E. coli K strains by T4rll at high MOl. We also show that host mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA completely abrogates the establishment of LIN inT4rll and T4 alike. We found that over-expression of rexB in cells suppresses both T4t⁻ and λS⁻ holin mutations, increasing the plating efficiency of the mutant phages by up to 10⁵ fold. Prophage level expression was noted to suppress the λS⁻ mutation to a lesser degree, but only from a rexB⁺-rexA⁻ prophage and co-expression of rexA with rexB on the prophage, or on a multicopy plasmid inhibited this activity of RexB. We also determined that nonlysogenic cells carrying the multicopy rexB plasmid are leaky for cytoplasmic proteins, whereas lysogenic (λrexB⁺-rexA⁻) cells are not leaky but did reveal an unusually high concentration of cytoplasmic β-galactosidase in the periplasm. Electron microscopy was used to visualize cells transformed with a rexB, or rexA-rexB multicopy plasmid. The rexB plasmid conferred gross distortions to the outer surface of the cell, while the rexA⁺-rexB⁺ plasmid imparted a shrunken, but otherwise normal appearance to cells. Our findings are consistent with RexB function as a pore forming unit, but RexB activity is inhibited by RexA. We propose a model for RexB suppression of T4rll lysis inhibition and the involvement of Tsp and 10Sa RNA in lysis timing and the establishment of LIN. The Conditional Rex Exclusion Phenotype The cl-rex operon of bacteriophage λ is expressed from the PM maintenance promoter of the prophage as PM-cI-rexA-rexB-timm message and confers a T4rII mutant phage exclusion phenotype to the lysogen (Rex exclusion). Derepression of the prophage results in very strong PE-cI-rexA-rexB-timm transcription, terminating at timm. Replication and excision defective, cryptic λcI[Ts]857cro27 lysogens exhibit a conditional Rex[Ts] exclusion phenotype. At temperatures where the C1857 repressor is functional, rexA-rexB expression from PM confers full Rex exclusion. However, upon thermal inactivation of the repressor little or no Rex exclusion is observed, despite a much higher level of transcription from PE, stimulated by CII. The same conditional Rex exclusion phenotype was observed in cells harbouring a low copy plasmid encoding a PTet-PM-c1857 -rexA-rexB-timm fragment, but not with a cl⁺ derivative plasmid, that imparted a Rex⁺ phenotype. Thermally derepressed λcl[Ts]857cro27 lysogens exhibited very high PE transcription levels that abated dramatically toward the C-terminal of rexA, showing a powerful polar effect on downstream rexB. Renaturation of C1857 following prophage induction did not reestablish repressor activity, although there was a 20-30 fold increase in transcription compared to that seen from the repressed prophage. Introduction of a rho mutation into our conditional Rex[Ts] strains partially suppressed Rex thermosensitivity, increasing Rex exclusion at 43°C by up to 10⁴ fold, while mutation of hflA to stabilize CII and heighten CII-dependent PE transcription conferred only a slight increase. Partial suppression of the conditional Rex[Ts] phenotype was also imparted by ssrA⁻and clpP⁻ null mutations, which suggests that Rex may be subject to 10Sa RNA tagging and ClpP(X) degradation. We propose two possible models to account for cI-rex polarity and correlation between CI activity and Rex exclusion. RexA:RexB Stoichiometry and the Rex Exclusion Phenotype We examined the influence of disrupting Rex stoichiometric balance on Rex activity to account for how polarity in the PTet-PM-cl857-rexA-rexB-t imm operon can abrogate the Rex exclusion phenotype. Cultured λ rex⁺ lysogenic cells were transformed with low-copy, and multicopy plasmids constitutive, or inducible for rexA⁺, rexB⁺, or rexA⁺-rexB⁺ expression. Lambda rex⁺ lysogenic cells transformed with a low-copy plasmid constitutively expressing rexA⁺, conferred only a minor attenuation of Rex exclusion, while transformation of rex⁺ cells with a constitutive, or induced multicopy plasmid expressing rexA⁺, or rexB⁺ completely suppressed the Rex exclusion phenotype. In contrast, multicopy and low-copy rexA⁺-rexB ⁺ and rex⁻ plasmid derivatives did not abrogate Rex exclusion in transformed rex⁺ cells. Furthermore, phage T4rll exhibited large rapid lysis plaques on the thermally induced Cro⁺ conditional Rex exclusion phenotypic lysogen, resembling plaques formed on λ lysogens carrying the multicopy constitutive rexB plasmid. Plaques formed on the isogenic cro⁻ derivative were tiny and nonsymmetrical; identical to T4rll plaque morphology on λ lysogens carrying the multicopy constitutive rexA⁺ plasmid. Our results suggest that an induced Cro⁺ λ prophage escapes Rex exclusion by over-expressing rexB relative to rexA, while a derepressed λcro⁻ prophage suppresses Rex exclusion phenotype by over-expressing rexA relative to rexB. CI Repressor Modulation of the Rex Exclusion Phenotype Bacteriophage λ mutants defective for ren and red (exo or bet) are sensitive to restriction by λ rex genes, but exclusion is modulated by the cl repressor allele of the prophage. λspi156nin5 forms plaques with 10⁵ fold higher efficiency on a cl⁺-rex⁺ lysogen than on cl[Ts]857, or cl[Ts]2 derivatives. Exclusion in cl[Ts] lysogens is suppressed by complementation with cl⁺ plasmid

Bacteriophage interactions with mammalian tissue: Therapeutic applications

The human body is a large reservoir for bacterial viruses known as bacteriophages (phages), which participate in dynamic interactions with their bacterial and human hosts that ultimately affect human health. The current growing interest in human resident phages is paralleled by new uses of phages, including the design of engineered phages for therapeutic applications. Despite the increasing number of clinical trials being conducted, the understanding of the interaction of phages and mammalian cells and tissues is still largely unknown. The presence of phages in compartments within the body previously considered purely sterile, suggests that phages possess a unique capability of bypassing anatomical and physiological barriers characterized by varying degrees of selectivity and permeability. This review will discuss the direct evidence of the accumulation of bacteriophages in various tissues, focusing on the unique capability of phages to traverse relatively impermeable barriers in mammals and its relevance to its current applications in therapy

EGFR-targeted bacteriophage lambda penetrates model stromal and colorectal carcinoma tissues, is taken up into carcinoma cells, and interferes with 3-dimensional tumor formation

IntroductionColorectal cancer and other adult solid cancers pose a significant challenge for successful treatment because the tumor microenvironment both hinders the action of conventional therapeutics and suppresses the immune activities of infiltrating leukocytes. The immune suppression is largely the effect of enhanced local mediators such as purine nucleosides and eicosanoids. Genetic approaches have the promise of interfering with these mechanisms of local immunosuppression to allow both intrinsic and therapeutic immunological anticancer processes. Bacterial phages offer a novel means of enabling access into tissues for therapeutic genetic manipulations.MethodsWe generated spheroids of fibroblastic and CRC cancer cells to model the 3-dimensional stromal and parenchymal components of colorectal tumours. We used these to examine the access and effects of both wildtype (WT) and epidermal growth factor (EGF)-presenting bacteriophage λ (WT- λ and EGF-λ) as a means of delivery of targeted genetic interventions in solid cancers. We used both confocal microscopy of spheroids exposed to AF488-tagged phages, and the recovery of viable phages as measured by plaque-forming assays to evaluate access; and measures of mitochondrial enzyme activity and cellular ATP to evaluate the outcome on the constituent cells.ResultsUsing flourescence-tagged derivatives of these bacteriophages (AF488-WT-λ and AF488-EGF-λ) we showed that phage entry into these tumour microenvironments was possible and that the EGF ligand enabled efficient and persistent uptake into the cancer cell mass. EGF-λ became localized in the intracellular portion of cancer cells and was subjected to subsequent cellular processing. The targeted λ phage had no independent effect upon mature tumour spheroids, but interfered with the early formation and growth of cancer tissues without the need for addition of a toxic payload, suggesting that it might have beneficial effects by itself in addition to any genetic intervention delivered to the tumour. Interference with spheroid formation persisted over the duration of culture.DiscussionWe conclude that targeted phage technology is a feasible strategy to facilitate delivery into colorectal cancer tumour tissue (and by extension other solid carcinomas) and provides an appropriate delivery vehicle for a gene therapeutic that can reduce local immunosuppression and/or deliver an additional direct anticancer activity

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage lambda capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by lambda Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to lambda Dam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the lambda Dam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage lambda.UW Start-up funds; Drug Safety and Effectiveness Cross-Disciplinary Training (DSECT) Scholarship; Canadian Institute of Health Research (CIHR

Immunogenicity and antitumor activity of the superlytic λF7 phage nanoparticles displaying a HER2/neu-derived peptide AE37 in a tumor model of BALB/c mice

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.canlet.2018.03.030 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Phage display technique has been increasingly researched for vaccine design and delivery strategies in recent years. In this study, the AE37 (Ii-Key/HER-2/neu 776–790) peptide derived from HER2 (human epidermal growth factor receptor protein) was used as a fused peptide to the lambda phage (λF7) coat protein gpD, and the phage nanoparticles were used to induce antitumor immunogenicity in a TUBO model of breast cancer in mice. Mice were immunized with the AE37 peptide displaying phage, λF7 (gpD::AE37) every 2-week intervals over 6-weeks, then the generated immune responses were evaluated. An induction of CTL immune response by the λF7 (gpD::AE37) construct compared to the control λF7 and buffer groups was observed in vitro. Moreover, in the in vivo studies, the vaccine candidate showed promising prophylactic and therapeutic effects against the HER2 overexpressing cancer in BALB/c mice.Mashhad University of Medical Sciences, Mashhad, Iran bach (MUMS GN: 922610)NSERC, Canada (NSERC GN: 214684

Rex-Centric Mutualism

We asked whether Rex exclusion encoded by a lambda prophage confers a protective or a cell-killing phenotype. We found that the Rex system can channel lysogenic cells into an arrested growth phase that gives an overall protective ability to the host despite some associated killing