Lytic Cycle Research Articles

Biological Traits and Comprehensive Genomic Analysis of Novel Enterococcus faecalis Bacteriophage EFP6.

Enterococcus faecalis is a prevalent opportunistic pathogen associated with chicken embryonic and neonatal chick mortality, posing a significant challenge in poultry farming. In the current study, E. faecalis strain EF6, isolated from a recent hatchery outbreak, served as the host bacterium for the isolation of a novel phage EFP6, capable of lysing E. faecalis. Transmission electron microscopy revealed a hexagonal head and a short tail, classifying EFP6 as a member of the Autographiviridae family. EFP6 showed sensitivity to ultraviolet radiation and resistance to chloroform. The lytic cycle duration of EFP6 was determined to be 50 min, highlighting its efficacy in host eradication. With an optimal multiplicity of infection of 0.001, EFP6 exhibited a narrow lysis spectrum and strong specificity towards host strains. Additionally, EFP6 demonstrated optimal growth conditions at 40 °C and pH 8.0. Whole genome sequencing unveiled a genome length of 18,147 bp, characterized by a GC concentration of 33.21% and comprising 25 open reading frames. Comparative genomic assessment underscored its collinearity with related phages, notably devoid of lysogenic genes, thus ensuring genetic stability. This in-depth characterization forms the basis for understanding the biological attributes of EFP6 and its potential utilization in phage therapy, offering promising prospects for mitigating E. faecalis-associated poultry infections.

Epstein-Barr virus deubiquitinating enzyme BPLF1 is involved in EBV carcinogenesis by affecting cellular genomic stability

Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.

Identification and characterization of Varicella Zoster Virus circular RNA in lytic infection

This study investigates the role of circular RNAs (circRNAs) in the context of Varicella-Zoster Virus (VZV) lytic infection. We employ two sequencing technologies, short-read sequencing and long-read sequencing, following RNase R treatment on VZV-infected neuroblastoma cells to identify and characterize both cellular and viral circRNAs. Our large scanning analysis identifies and subsequent experiments confirm 200 VZV circRNAs. Moreover, we discover numerous VZV latency-associated transcripts (VLTs)-like circRNAs (circVLTslytic), which contain multiple exons and different isoforms within the same back-splicing breakpoint. To understand the functional significance of these circVLTslytic, we utilize the Bacteria Artificial Chromosome system to disrupt the expression of viral circRNAs in genomic DNA location. We reveal that the sequence flanking circVLTs’ 5’ splice donor plays a pivotal role as a cis-acting element in the formation of circVLTslytic. The circVLTslytic is dispensable for VZV replication, but the mutation downstream of circVLTslytic exon 5 leads to increased acyclovir sensitivity in VZV infection models. This suggests that circVLTslytic may have a role in modulating the sensitivity to antiviral treatment. The findings shed new insight into the regulation of cellular and viral transcription during VZV lytic infection, emphasizing the intricate interplay between circRNAs and viral processes.

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

Co-dependent formation of the Toxoplasma gondii sub-pellicular microtubules and inner membrane skeleton.

One of the defining features of apicomplexan parasites is their cytoskeleton composed of alveolar vesicles, known as the inner membrane complex (IMC) undergirded by intermediate-like filament network and an array of subpellicular microtubules (SPMTs). In Toxoplasma gondii, this specialized cytoskeleton is involved in all aspects of the disease-causing lytic cycle, and notably acting as a scaffold for parasite offspring in the internal budding process. Despite advances in our understanding of the architecture and molecular composition, insights pertaining to the coordinated assembly of the scaffold are still largely elusive. Here, T. gondii tachyzoites were dissected by advanced, iterative expansion microscopy (pan-ExM) revealing new insights into the very early sequential formation steps of the tubulin scaffold. A comparative study of the related parasite Sarcocystis neurona revealed that different MT bundling organizations of the nascent SPMTs correlate with the number of central and basal alveolar vesicles. In absence of a so far identified MT nucleation mechanism, we genetically dissected T. gondii γ-tubulin and γ-tubulin complex protein 4 (GCP4). While γ-tubulin depletion abolished the formation of the tubulin scaffold, a set of MTs still formed that suggests SPMTs are nucleated at the outer core of the centrosome. Depletion of GCP4 interfered with the correct assembly of SPMTs into the forming daughter buds, further indicating that the parasite utilizes the γ-tubulin complex in tubulin scaffold formation .

Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis.

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Identification of HLA-A*11:01 and A*02:01-Restricted EBV Peptides Using HLA Peptidomics.

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.

Cotargeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma

AbstractThe remarkable efficacy of Epstein-Barr virus (EBV)-specific T cells for the treatment of posttransplant lymphomas has not been reproduced for EBV-positive (EBV+) malignancies outside the transplant setting. This is because of, in part, the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely latent membrane proteins 1 and 2, EBV nuclear antigen 1, and BamHI A rightward reading frame 1 (type-2 [T2] latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies and, because several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin lymphoma biopsies. This provided the rationale for broadening the target antigen–specific repertoire of EBV-specific T cells (EBVSTs) for therapy. We stimulated, peripheral blood mononuclear cells from healthy donors and patients with EBV+ lymphoma with both lytic and latent cycle proteins to produce broad repertoire (BR) EBVSTs. Compared with T2 antigen-specific EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and produced higher levels of proinflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV+ lymphoma (www.clinicaltrials.gov identifiers: #NCT01555892 and #NCT04664179), as well as for other EBV-associated malignancies.

A dual fluorescent herpes simplex virus type 1 recombinant reveals divergent outcomes of neuronal infection.

Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.

Phage phylogeny, molecular signaling, and auxiliary antimicrobial resistance in aerobic and anaerobic membrane bioreactors

Phage emit communication signals that inform their lytic and lysogenic life cycles. However, little is known regarding the abundance and diversity of the genes associated with phage communication systems in wastewater treatment microbial communities. This study focused on phage communities within two distinct biochemical wastewater environments, specifically aerobic membrane bioreactors (AeMBRs) and anaerobic membrane bioreactors (AnMBRs) exposed to varying antibiotic concentrations. Metagenomic data from the bench-scale systems were analyzed to explore phage phylogeny, life cycles, and genetic capacity for antimicrobial resistance and quorum sensing. Two dominant phage families, Schitoviridae and Peduoviridae, exhibited redox-dependent dynamics. Schitoviridae prevailed in anaerobic conditions, while Peduoviridae dominated in aerobic conditions. Notably, the abundance of lytic and lysogenic proteins varied across conditions, suggesting the coexistence of both life cycles. Furthermore, the presence of antibiotic resistance genes (ARGs) within viral contigs highlighted the potential for phage to transfer ARGs in AeMBRs. Finally, quorum sensing genes in the virome of AeMBRs indicated possible molecular signaling between phage and bacteria. Overall, this study provides insights into the dynamics of viral communities across varied redox conditions in MBRs. These findings shed light on phage life cycles, and auxiliary genetic capacity such as antibiotic resistance and bacterial quorum sensing within wastewater treatment microbial communities.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant’s growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite’s fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5′-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.

Structural and Biochemical Characterization of a New Phage-Encoded Muramidase, KTN6 Gp46.

Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics. The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions. These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness

BackgroundTo successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4.MethodsWe characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4.ResultsTrx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice.ConclusionsTrx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.Graphical

Lytic/Lysogenic Transition as a Life-History Switch.

The transition between lytic and lysogenic life cycles is the most important feature of the life-history of temperate viruses. To explain this transition, an optimal life-history model is offered based a discrete-time formulation of phage/bacteria population dynamics that features infection of bacteria by Poisson sampling of virions from the environment. The time step is the viral latency period. In this model, density-dependent viral absorption onto the bacterial surface produces virus/bacteria coexistence and density dependence in bacterial growth is not needed. The formula for the transition between lytic and lysogenic phases is termed the 'fitness switch'. According to the model, the virus switches from lytic to lysogenic when its population grows faster as prophage than as virions produced by lysis of the infected cells, and conversely for the switch from lysogenic to lytic. A prophage that benefits the bacterium it infects automatically incurs lower fitness upon exiting the bacterial genome, resulting in its becoming locked into the bacterial genome in what is termed here as a 'prophage lock'. The fitness switch qualitatively predicts the ecogeographic rule that environmental enrichment leads to microbialization with a concomitant increase in lysogeny, fluctuating environmental conditions promote virus-mediated horizontal gene transfer, and prophage-containing bacteria can integrate into the microbiome of a eukaryotic host forming a functionally integrated tripartite holobiont. These predictions accord more with the 'Piggyback-the-Winner' hypothesis than with the 'Kill-the-Winner' hypothesis in virus ecology.

The contribution of abortive infection to preventing populations of Lactococcus lactis from succumbing to infections with bacteriophage.

In the dairy industry bacteriophage (phage) contamination significantly impairs the production and quality of products like yogurt and cheese. To combat this issue, the strains of bacteria used as starter cultures possess mechanisms that make them resistant to phage infection, such as envelope resistance, or processes that render them immune to phage infection, such as restriction-modification and CRISPR-Cas. Lactococcus lactis, used to manufacture cheese and other dairy products, can also block the reproduction of infecting phages by abortive infection (Abi), a process in which phage-infected cells die before the phage replicate. We employ mathematical-computer simulation models and experiments with two Lactococcus lactis strains and two lytic phages to investigate the conditions under which Abi can limit the proliferation of phages in L. lactis populations and prevent the extinction of their populations by these viruses. According to our model, if Abi is almost perfect and there are no other populations of bacteria capable of supporting the replication of the L. lactis phages, Abi can protect bacterial populations from succumbing to infections with these viruses. This prediction is supported by the results of our experiment, which indicate that Abi can help protect L. lactis populations from extinction by lytic phage infections. However, our results also predict abortive infection is only one element of L. lactis defenses against phage infection. Mutant phages that can circumvent the Abi systems of these bacteria emerge. The survival of L. lactis populations then depends on the evolution of envelope mutants that are resistant to the evolved host-range phage.

Whole genome analysis, thermal and UV-tolerance of Lactococcus phage BIM BV-114 isolated from cheese brine

Lactococcus phages that belong to the genus Ceduovirus are among the three most frequently isolated phage groups infecting Lactococcus lactis starter strains in dairy plants. In this study, we characterized virulent Lactococcus phage BIM BV-114 isolated from industrial cheese brine in Belarus and identified as Ceduovirus. The bacteriophage demonstrated a relatively short lytic cycle (latent period of 23 ± 5 min, lysis time of 90 ± 5 min), high thermal stability (inactivation after 7 min at 95 °C in skimmed milk) and tolerance to UV radiation (inactivation time – 15 min), indicating adaptation for better persistence in dairy facilities. The genome of the phage BIM BV-114 (21 499 bp; 37 putative open reading frames) has a similar organization to that of other Ceduovirus phages. RLf1_00140 and RLf_00050 gene products, found in the early genes region, may be involved in the sensitivity of phage to the lactococcal abortive infection mechanisms AbiV and AbiQ, respectively. Furthermore, nucleotide deletion, observed in the middle region of the gene encoding putative tape measure protein (RLf1_00300), is possibly responsible for increased thermal tolerance of phage BIM BV-114. Together, these findings will contribute to a better knowledge of virulent Lactococcus phages and the development of effective methods of their control for dairy technologies.

A removable and cosmopolitan dsRNA Toti-like virus causes asymptomatic but productive infection in a model diatom strain

Diatoms contribute to 20 % of global primary productivity. Although some diatom viruses have been identified, the molecular mechanisms underlying their interactions with the host remain poorly understood. In this study, we report the discovery of an RNA molecule in the DNA extracts of the Phaeodactylum tricornutum strain Pt1, which possesses a well-annotated genome and has been used as a diatom model system since 1956. We confirmed this molecule to be a double-stranded linear RNA molecule and, through sequencing, demonstrated it to be a virus in the Totiviridae family that is prevalent among marine stramenopiles. We also detected this virus in Phaeodactylum tricornutum strain Pt3, which was collected in 1930s from a similar geographic location to Pt1, suggesting its prevalence within the region. By employing various inhibitors of the viral RNA-dependent RNA polymerase, we successfully generated a virus-free line isogenic to Pt1, establishing a model system to investigate the impact of RNA viruses on diatom physiology. The virus-free lines did not display obvious growth advantages or defects, indicating a tendency of the virus towards asymptomatic infection. Furthermore, we generated a robust antibody against the coat protein of this virus. By performing immunoprecipitation coupled with mass spectrometry, we found that translation-related proteins are enriched as potential interacting partners of the coat protein. Our results suggest that potential viral impacts in molecular research should be considered when Pt1 and Pt3 are used for studying translation-related processes. Additionally, our study unveiled a mode of asymptomatic but productive infection between viruses and marine algal hosts that differs from the commonly-reported virulent, lytic infections.