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

ABSTRACT 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.

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A family of viral satellites manipulates invading virus gene expression and affects cholera toxin mobilization

10.1101/2020.03.12.988147 ◽

2020 ◽

Author(s):

Zachary K Barth ◽

Zoe Netter ◽

Angus Angermeyer ◽

Pooja Bhardwaj ◽

Kimberley D Seed

Keyword(s):

Gene Expression ◽

Cholera Toxin ◽

Expression Patterns ◽

Rna Seq ◽

Time Resolved ◽

Enhanced Resolution ◽

Infected Cells ◽

Time Courses ◽

Host Metabolism ◽

First Time

AbstractMany 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-seq, have dramatically enhanced resolution of expression patterns during infection. Less studied have been viral satellites, mobile genomes that parasitize viruses and have far reaching effects on host-cell fitness. 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. This transcriptional manipulation, conserved among PLEs, has also evolved independently in at least one other phage satellite, suggesting that viral satellites may be under strong selective pressure to reduce the capsid expression of their larger host viruses. 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. Our data show that PLEs exhibit conserved manipulation of their host-phage’s gene expression, but divergent effects on CTXϕ, revealing that PLEs can influence both their hosts’ resistance to phage and the mobility of virulence encoding elements.

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A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

Infection and Immunity ◽

10.1128/iai.00411-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3381-3395 ◽

Cited By ~ 16

Author(s):

Qiyao Wang ◽

Yves A. Millet ◽

Michael C. Chao ◽

Jumpei Sasabe ◽

Brigid M. Davis ◽

...

Keyword(s):

Gene Expression ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Insertion Site ◽

Virulence Gene ◽

Loss Of Function ◽

Phosphotransferase System ◽

Content Type ◽

Virulence Gene Expression ◽

A Genome

Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression ofVibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression oftcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulatetcpAexpression, the screen yieldedptsIandptsH, which encode the enzyme I (EI) and Hpr components of theV. choleraephosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlcprotein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression oftcpPHandaphAB, which themselves control expression oftoxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that theV. choleraePTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.

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The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant

mBio ◽

10.1128/mbio.03287-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Catherine A. Klancher ◽

George Minasov ◽

Ram Podicheti ◽

Douglas B. Rusch ◽

Triana N. Dalia ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Vibrio Cholerae ◽

Binding Domain ◽

Structural Basis ◽

Striking Difference ◽

Dna Binding Domain ◽

Structural Domains ◽

Content Type ◽

Domains Of Life

ABSTRACT Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

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Dual RNA Sequencing Meta-analysis in Plasmodium Infection Identifies Host-Parasite Interactions

mSystems ◽

10.1128/msystems.00182-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Parnika Mukherjee ◽

Gaétan Burgio ◽

Emanuel Heitlinger

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Laboratory Experiments ◽

Meta Analysis ◽

Model Systems ◽

Rna Seq ◽

Content Type ◽

Human Malaria ◽

Host Parasite Interactions ◽

Host Parasite

ABSTRACT Dual RNA sequencing (RNA-Seq) is the simultaneous transcriptomic analysis of interacting symbionts, for example, in malaria. Potential cross-species interactions identified by correlated gene expression might highlight interlinked signaling, metabolic, or gene regulatory pathways in addition to physically interacting proteins. Often, malaria studies address one of the interacting organisms—host or parasite—rendering the other “contamination.” Here we perform a meta-analysis using such studies for cross-species expression analysis. We screened experiments for gene expression from host and Plasmodium. Out of 171 studies in Homo sapiens, Macaca mulatta, and Mus musculus, we identified 63 potential studies containing host and parasite data. While 16 studies (1,950 samples) explicitly performed dual RNA-Seq, 47 (1,398 samples) originally focused on one organism. We found 915 experimental replicates from 20 blood studies to be suitable for coexpression analysis and used orthologs for meta-analysis across different host-parasite systems. Centrality metrics from the derived gene expression networks correlated with gene essentiality in the parasites. We found indications of host immune response to elements of the Plasmodium protein degradation system, an antimalarial drug target. We identified well-studied immune responses in the host with our coexpression networks, as our approach recovers known broad processes interlinked between hosts and parasites in addition to individual host and parasite protein associations. The set of core interactions represents commonalities between human malaria and its model systems for prioritization in laboratory experiments. Our approach might also allow insights into the transferability of model systems for different pathways in malaria studies. IMPORTANCE Malaria still causes about 400,000 deaths a year and is one of the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria. Interactions between Plasmodium spp. and its hosts are either conserved across different host-parasite systems or idiosyncratic to those systems. Here we use correlation of gene expression from different RNA-Seq studies to infer common host-parasite interactions across human, mouse, and monkey studies. First, we find a set of very conserved interactors, worth further scrutiny in focused laboratory experiments. Second, this work might help assess to which extent experiments and knowledge on different pathways can be transferred from models to humans for potential therapy.

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cAMP Receptor Protein ControlsVibrio choleraeGene Expression in Response to Host Colonization

mBio ◽

10.1128/mbio.00966-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 15

Author(s):

Jainaba Manneh-Roussel ◽

James R. J. Haycocks ◽

Andrés Magán ◽

Nicolas Perez-Soto ◽

Kerstin Voelz ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Vibrio Cholerae ◽

Expression Patterns ◽

Lifestyle Changes ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Host Colonization ◽

Content Type ◽

Camp Receptor

ABSTRACTThe bacteriumVibrio choleraeis native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across theV. choleraegenome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of manyV. choleraegenes in response to lifestyle changes.IMPORTANCECholera is an infectious disease that is caused by the bacteriumVibrio cholerae. Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated withV. cholerae. Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming ofV. choleraegene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator ofV. choleraegene expression in response to lifestyle changes.

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RNA-sequencing data-driven dissection of human plasma cell differentiation reveals new potential transcription regulators

Leukemia ◽

10.1038/s41375-021-01234-0 ◽

2021 ◽

Author(s):

Alboukadel Kassambara ◽

Laurie Herviou ◽

Sara Ovejero ◽

Michel Jourdan ◽

Coraline Thibaut ◽

...

Keyword(s):

Gene Expression ◽

Rna Sequencing ◽

Continuous Production ◽

Expression Patterns ◽

Gene Clusters ◽

Transcriptional Networks ◽

Pathway Enrichment Analysis ◽

Rna Seq ◽

Sequencing Data

AbstractPlasma cells (PCs) play an important role in the adaptive immune system through a continuous production of antibodies. We have demonstrated that PC differentiation can be modeled in vitro using complex multistep culture systems reproducing sequential differentiation process occurring in vivo. Here we present a comprehensive, temporal program of gene expression data encompassing human PC differentiation (PCD) using RNA sequencing (RNA-seq). Our results reveal 6374 differentially expressed genes classified into four temporal gene expression patterns. A stringent pathway enrichment analysis of these gene clusters highlights known pathways but also pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugation pathways. Additionally, our analysis revealed numerous novel transcriptional networks with significant stage-specific overexpression and potential importance in PCD, including BATF2, BHLHA15/MIST1, EZH2, WHSC1/MMSET, and BLM. We have experimentally validated a potent role for BLM in regulating cell survival and proliferation during human PCD. Taken together, this RNA-seq analysis of PCD temporal stages helped identify coexpressed gene modules with associated up/downregulated transcription regulator genes that could represent major regulatory nodes for human PC maturation. These data constitute a unique resource of human PCD gene expression programs in support of future studies for understanding the underlying mechanisms that control PCD.

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Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Methods and Protocols ◽

10.3390/mps3040072 ◽

2020 ◽

Vol 3 (4) ◽

pp. 72

Author(s):

Anupama Prakash ◽

Antónia Monteiro

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rna Sequencing ◽

Single Cells ◽

Expression Patterns ◽

Cell Types ◽

Rna Seq ◽

Bicyclus Anynana ◽

Single Cell Sequencing ◽

Pupal Wing

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.

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Time-resolved transcriptional profiling of Trichinella-infected murine myocytes helps to elucidate host–pathogen interactions in the muscle stage

Parasites & Vectors ◽

10.1186/s13071-021-04624-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Xiaoxiang Hu ◽

Xiaolei Liu ◽

Chen Li ◽

Yulu Zhang ◽

Chengyao Li ◽

...

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Trichinella Spiralis ◽

Expression Patterns ◽

Muscle Cells ◽

Host Cells ◽

Initial Reaction ◽

Global Changes ◽

Mammalian Skeletal Muscle ◽

Time Resolved

Abstract Background Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host’s innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. Methods We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. Results The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. Conclusions The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment.

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Cryopreservation of microglia enables single-cell RNA sequencing with minimal effects on disease-related gene expression patterns

iScience ◽

10.1016/j.isci.2021.102357 ◽

2021 ◽

Vol 24 (4) ◽

pp. 102357

Author(s):

Brenda Morsey ◽

Meng Niu ◽

Shetty Ravi Dyavar ◽

Courtney V. Fletcher ◽

Benjamin G. Lamberty ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rna Sequencing ◽

Expression Patterns ◽

Gene Expression Patterns ◽

Related Gene ◽

Single Cell Rna Sequencing ◽

Disease Related Gene

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Combined Metabolome and Transcriptome Profiling Reveal Optimal Harvest Strategy Model Based on Different Production Purposes in Olive

Foods ◽

10.3390/foods10020360 ◽

2021 ◽

Vol 10 (2) ◽

pp. 360

Author(s):

Guodong Rao ◽

Jianguo Zhang ◽

Xiaoxia Liu ◽

Xue Li ◽

Chenhe Wang

Keyword(s):

Gene Expression ◽

Olive Oil ◽

Expression Patterns ◽

Transcriptome Profiling ◽

Minor Components ◽

Harvest Time ◽

Rna Seq ◽

Optimal Harvest ◽

Harvest Strategy ◽

Different Color

Olive oil has been favored as high-quality edible oil because it contains balanced fatty acids (FAs) and high levels of minor components. The contents of FAs and minor components are variable in olive fruits of different color at harvest time, which render it difficult to determine the optimal harvest strategy for olive oil producing. Here, we combined metabolome, Pacbio Iso-seq, and Illumina RNA-seq transcriptome to investigate the association between metabolites and gene expression of olive fruits at harvest time. A total of 34 FAs, 12 minor components, and 181 other metabolites (including organic acids, polyols, amino acids, and sugars) were identified in this study. Moreover, we proposed optimal olive harvesting strategy models based on different production purposes. In addition, we used the combined Pacbio Iso-seq and Illumina RNA-seq gene expression data to identify genes related to the biosynthetic pathways of hydroxytyrosol and oleuropein. These data lay the foundation for future investigations of olive fruit metabolism and gene expression patterns, and provide a method to obtain olive harvesting strategies for different production purposes.

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