Functional profiling of the Toxoplasma genome during acute mouse infection.

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses.

Ebola virus (EBOV) causes Ebola virus disease (EVD), marked by severe hemorrhagic fever; however, the mechanisms underlying the disease remain unclear. To assess the molecular basis of EVD across time, we performed RNA sequencing on 17 tissues from a natural history study of 21 rhesus monkeys, developing new methods to characterize host-pathogen dynamics. We identified alterations in host gene expression with previously unknown tissue-specific changes, including downregulation of genes related to tissue connectivity. EBOV was widely disseminated throughout the body; using a new, broadly applicable deconvolution method, we found that viral load correlated with increased monocyte presence. Patterns of viral variation between tissues differentiated primary infections from compartmentalized infections, and several variants impacted viral fitness in a EBOV/Kikwit minigenome system, suggesting that functionally significant variants can emerge during early infection. This comprehensive portrait of host-pathogen dynamics in EVD illuminates new features of pathogenesis and establishes resources to study other emerging pathogens.

Secondary structural ensembles of the SARS-CoV-2 RNA genome in infected cells.

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

Sigma factor dependent translational activation in Bacillus subtilis.

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σB, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, σA, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from σB-dependent promoters ablates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between σB- and σA-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.

Author Correction: Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic engineering of a novel strain of electrogenic bacterium Arcobacter butzleri to create a platform for single analyte detection using a microbial fuel cell.

Electrogenic bacteria metabolize organic substrates by transferring electrons to the external electrode, with subsequent electricity generation. In this proof-of-concept study, we present a novel strain of a known, electrogenic Arcobacter butzleri that can grow primarily on acetate and lactate and its electric current density is positively correlated (R2 = 0.95) to the COD concentrations up to 200 ppm. Using CRISPR-Cas9 and Cpf1, we engineered knockout Arcobacter butzleri mutants in either the acetate or lactate metabolic pathway, limiting their energy metabolism to a single carbon source. After genome editing, the expression of either acetate kinase, ackA, or lactate permease, lctP, was inhibited, as indicated by qPCR results. All mutants retain electrogenic activity when inoculated into a microbial fuel cell, yielding average current densities of 81-82 mA/m2, with wild type controls reaching 85-87 mA2. In the case of mutants, however, current is only generated in the presence of the substrate for the remaining pathway. Thus, we demonstrate that it is possible to obtain electric signal corresponding to the specific organic compound via genome editing. The outcome of this study also indicates that the application of electrogenic bacteria can be expanded by genome engineering.

Determination of RNA structural diversity and its role in HIV-1 RNA splicing.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

Therapeutic vaccination using minimal HPV16 epitopes in a novel MHC-humanized murine HPV tumor model.

Therapeutic vaccination as a treatment option for HPV-induced cancers is actively pursued because the two HPV proteins E6 and E7 represent ideal targets for immunotherapy, as they are non-self and expressed in all tumor stages. MHC-humanized mice are valuable tools for the study of therapeutic cancer vaccines - given the availability of a suitable tumor model. Here, we present for the first time an HPV16 tumor model suitable for fully MHC-humanized A2.DR1 mice, PAP-A2 cells, which in contrast to existing HPV16 tumor models allows the exclusive study of HLA-A2- and DR1-mediated immune responses, without any interfering murine MHC-presented epitopes. We used several HPV16 epitopes that were shown to be presented on human cervical cancer cells by mass spectrometry for therapeutic anti-tumor vaccination in the new tumor model. All epitopes were immunogenic when rendered amphiphilic by incorporation into a molecule containing stearic acids. Prophylactic and therapeutic vaccination experiments with the epitope E7/11-19 demonstrated that effective immune responses could be induced with these vaccination approaches in A2.DR1 mice. Interestingly, the combination of E7/11-19 with other immunogenic HPV16 E6/E7 epitopes caused a reduction of vaccine efficacy, although all tested combinations resulted in a survival benefit. In summary, we present the first HPV16 tumor model for exclusive studies of HLA-A2-mediated anti-HPV tumor immune responses and show anti-tumor efficacy of minimal epitope vaccines.