Development of a Monoclonal Antibody to a Vibriophage as a Proxy for Vibrio cholerae Detection.

Cholera is an acute watery, diarrheal disease that causes high rates of morbidity and mortality without treatment. Early detection of the etiologic agent of toxigenic Vibrio cholerae is important to mobilize treatment and mitigate outbreaks. Monoclonal antibody (mAb) based rapid diagnostic tests (RDTs) enable early detection in settings without laboratory capacity. However, the odds of an RDT testing positive are reduced by nearly 90% when the common virulent bacteriophage ICP1 is present. We hypothesize that adding a mAb for the common, and specific, virulent bacteriophage ICP1 as a proxy for V. cholerae to an RDT will increase diagnostic sensitivity when virulent ICP1 phage is present. In this study, we used an in-silico approach to identify immunogenic ICP1 protein targets that were conserved across disparate time periods and locations. Specificity of targets to cholera patients with known ICP1 was determined, and specific targets were used to produce mAbs in a murine model. Candidate mAbs to the head protein demonstrated specificity to ICP1 by Enzyme linked immunosorbent assay (ELISA) and an ICP1 phage neutralization assay. The limit of detection of the final mAb candidate for ICP1 phage particles spiked into cholera stool matrix was 8 × 105 PFU by Western blotting analysis. This mAb will be incorporated into a RDT prototype for evaluation in a future diagnostic study to test the guiding hypothesis behind this study.

Gold Standard Cholera Diagnostics Are Tarnished by Lytic Bacteriophage and Antibiotics.

A fundamental, clinical, and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. Cholera was chosen as a model disease to investigate this important question, because cholera outbreaks enable large enrollment, field methods are well established, and the predatory relationship between lytic bacteriophage and the etiologic agent Vibrio cholerae share commonalities across bacterial taxa. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection and exposure to the first-line antibiotic azithromycin, detected in stool samples by mass spectrometry. Among diarrheal samples positive by nanoliter quantitative PCR (qPCR) for V. cholerae (n = 78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (odds ratio [OR], 0.108; 95% confidence interval [CI], 0.002 to 0.872) and 87% (OR, 0.130; 95% CI, 0.022 to 0.649), respectively, when lytic bacteriophage were detected. The odds that an RDT or qPCR was positive was reduced by more than 99% (OR, 0.00; 95% CI, 0.00 to 0.28) and 89% (OR, 0.11; 95% CI, 0.03 to 0.44), respectively, when azithromycin was detected. Analysis of additional samples from South Sudan found similar phage effects on RDTs; antibiotics were not assayed. Cholera burden estimates may improve by accommodating for the negative effects of lytic bacteriophage and antibiotic exposure on diagnostic positivity. One accommodation is using bacteriophage detection as a proxy for pathogen detection. These findings have relevance for other diagnostic settings where bacterial pathogens are vulnerable to lytic bacteriophage predation.

Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite.

Bacteria, bacteriophages that prey upon them, and mobile genetic elements (MGEs) compete in dynamic environments, evolving strategies to sense the milieu. The first discovered environmental sensing by phages, lysis inhibition, has only been characterized and studied in the limited context of T-even coliphages. Here, we discover lysis inhibition in the etiological agent of the diarrheal disease cholera, Vibrio cholerae, infected by ICP1, a phage ubiquitous in clinical samples. This work identifies the ICP1-encoded holin, teaA, and antiholin, arrA, that mediate lysis inhibition. Further, we show that an MGE, the defensive phage satellite PLE, collapses lysis inhibition. Through lysis inhibition disruption a conserved PLE protein, LidI, is sufficient to limit the phage produced from infection, bottlenecking ICP1. These studies link a novel incarnation of the classic lysis inhibition phenomenon with conserved defensive function of a phage satellite in a disease context, highlighting the importance of lysis timing during infection and parasitization.

Bacteriophages, or phages for short, are viruses that infect bacteria, take over the molecular machinery inside the bacterial cells and use it to make more copies of themselves. The bacteriophages then break open, or "lyse", the bacterial cell, releasing the viral copies into the environment, ready to infect more bacteria nearby. Hays and Seed set out to understand how the timing of lysis can impact the bacteriophage, using the bacterium Vibrio cholerae ­ which causes cholera ­ and its bacteriophage called ICP1. This analysis revealed that the ICP1 phage uses a gene called teaA as the first step in the lysis of bacterial cells. The ICP1 phage can also delay that lysis with a second gene called arrA. This "lysis inhibition" gives the bacteriophages more time to make copies of themselves inside the bacterium, so even more are released when the cell finally breaks open. Hays and Seed also found that the Vibrio cholerae cells can defend themselves against lysis inhibition using a single gene called lidI. This gene is part of a system that defends against bacteriophage attack called the PLE, which consists of several genes of previously unknown function. Hays and Seed saw that the lidI gene disrupts lysis inhibition, speeding up the bursting of infected bacterial cells, which in turn decreases the number of bacteriophages produced from each infected cell. Lysis inhibition had previously only been observed in the bacterium Escherichia coli. Now that researchers know that ICP1 bacteriophages also delay lysis in Vibrio cholerae, this might lead to more studies exploring this process in samples from cholera patients. Further studies could test to see if the phenomenon of lysis inhibition may also exist in yet more bacterial species.

A Family of Viral Satellites Manipulates Invading Virus Gene Expression and Can Affect Cholera Toxin Mobilization.

Many viruses possess temporally unfolding gene expression patterns aimed at subverting host defenses, commandeering host metabolism, and ultimately producing a large number of progeny virions. High-throughput omics tools, such as RNA sequencing (RNA-seq), have dramatically enhanced the resolution of expression patterns during infection. Less studied have been viral satellites, mobile genomes that parasitize viruses. By performing RNA-seq on infection time courses, we have obtained the first time-resolved transcriptomes for bacteriophage satellites during lytic infection. Specifically, we have acquired transcriptomes for the lytic Vibrio cholerae phage ICP1 and all five known variants of ICP1's parasite, the phage inducible chromosomal island-like elements (PLEs). PLEs rely on ICP1 for both DNA replication and mobilization and abolish production of ICP1 progeny in infected cells. We investigated PLEs' impact on ICP1 gene expression and found that PLEs did not broadly restrict or reduce ICP1 gene expression. A major exception occurred in ICP1's capsid morphogenesis operon, which was downregulated by each of the PLE variants. Surprisingly, PLEs were also found to alter the gene expression of CTXΦ, the integrative phage that encodes cholera toxin and is necessary for virulence of toxigenic V. cholerae One PLE, PLE1, upregulated CTXΦ genes involved in replication and integration and boosted CTXΦ mobility following induction of the SOS response.IMPORTANCE Viral satellites are found in all domains of life and can have profound fitness effects on both the viruses they parasitize and the cells they reside in. In this study, we have acquired the first RNA sequencing (RNA-seq) transcriptomes of viral satellites outside plants, as well as the transcriptome of the phage ICP1, a predominant predator of pandemic Vibrio cholerae Capsid downregulation, previously observed in an unrelated phage satellite, is conserved among phage inducible chromosomal island-like elements (PLEs), suggesting that viral satellites are under strong selective pressure to reduce the capsid expression of their larger host viruses. Despite conserved manipulation of capsid expression, PLEs exhibit divergent effects on CTXΦ transcription and mobility. Our results demonstrate that PLEs can influence both their hosts' resistance to phage and the mobility of virulence-encoding elements, suggesting that PLEs can play a substantial role in shaping Vibrio cholerae evolution.

Identification of TEAD1 Transcripts and Functional Analysis in Chicken Preadipocytes / 生物化学与生物物理进展

ObjectiveAlthough expression of the TEAD1 protein in preadipocytes has been established, its function remains unclear. In this study, we sought to detect transcripts of TEAD1 in chicken and to examine the effects of this protein on the proliferation, migration, apoptosis, and differentiation of immortalized chicken preadipocyte cell lines (ICP1). MethodsThe full-length sequence of the TEAD1 gene was cloned and the two transcripts were subjected to bioinformatics analysis. The subcellsular localization of TEAD1 transcripts was determined based on indirect immunofluorescence. The effects of TEAD1 transcripts overexpression on the proliferation of ICP1 cells were examined by RT-qPCR, CCK-8, and EdU assays; the effects of TEAD1 transcripts on ICP1 cells migration were examined based on the scratch test; and the effects of TEAD1 transcripts overexpression on ICP1 cells apoptosis were analyzed using apoptosis-Hoechst staining and RT-qPCR. The expression of TEAD1 transcripts in different tissues, cells lines, and ICP1 at different periods of differentiation was analyzed by RT-qPCR. The effects of TEAD1 transcripts overexpression on lipid droplet accumulation and adipogenic-related gene expression in ICP1 cells were analyzed based on Oil Red O and BODIPY staining, RT-qPCR, Western blot, and dual-luciferase reporter gene assays. Finally, the content of triglyceride (TG) was measured in TEAD1 overexpressed ICP1 cells. ResultsThe full-length TEAD1 was cloned and two TEAD1 transcripts were identified. The TEAD1-V1 protein was found to be localized primarily in the cell nucleus, whereas the TEAD1-V2 protein is localized in the cell cytoplasm and nucleus. The overexpression of both TEAD1-V1 and TEAD1-V2 significantly inhibited the proliferation of ICP1 cells. Whereas the overexpression of TEAD1-V1 promoted ICP1 cell migration, the overexpression of TEAD1-V2 had no significant effects on ICP1 migration; the overexpression of both TEAD1-V1 and TEAD1-V2 significantly promoted the apoptosis of ICP1 cells. We found that the different transcripts of TEAD1 have similar expression pattern in different tissues and cells lines. During induced preadipocyte differentiation, the expression of these genes initially declined, although subsequently increased. Overexpression of TEAD1-V1 promoted a significant reduction in lipid droplet formation and inhibited C/EBPα expression during the differentiation of ICP1 cells (P<0.05). However, the overexpression of TEAD1-V2 had no significant effect on lipid droplet accumulation or the expression of adipogenic-related proteins (P>0.05). Overexpression of TEAD1-V1 significantly decreased triglyceride content in ICP1 cells (P<0.05), while overexpression of TEAD1-V2 had no effect on triglyceride content in ICP1 cells (P>0.05). ConclusionIn this study, for the first time, identified two TEAD1 transcripts. Overexpressed transcripts TEAD1-V1 and TEAD1-V2 both inhibited the proliferation of chicken preadipocytes and promoted apoptosis of chicken preadipocytes. TEAD1-V1 inhibited the differentiation of preadipocytes and promoted the migration of preadipocytes, while TEAD1-V2 had no effect on the differentiation and migration of preadipocytes.