RESUMO
Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.
Assuntos
Sistemas CRISPR-Cas , Infecções por Citomegalovirus , Citomegalovirus , Interações Hospedeiro-Patógeno , RNA Guia de Sistemas CRISPR-Cas , Replicação Viral , Humanos , Linhagem Celular , Sistemas CRISPR-Cas/genética , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Liberação de Vírus/genética , Replicação Viral/genéticaRESUMO
Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
Assuntos
Organismos Aquáticos , Bactérias , Poluentes Ambientais , Compostos Organofosforados , Hidrolases de Triester Fosfórico , Organismos Aquáticos/enzimologia , Bactérias/enzimologia , Biodegradação Ambiental , Poluentes Ambientais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oceano Índico , Mar Mediterrâneo , Compostos Organofosforados/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Fósforo/metabolismo , Água do Mar/microbiologiaRESUMO
Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.
Assuntos
Poliaminas/química , Proteínas/química , Substituição de Aminoácidos , Dicroísmo Circular , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Lisina/química , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Proteínas/metabolismoRESUMO
Bdellovibrio bacteriovorus, a Gram-negative predatory bacterium belonging to the Bdellovibrio and like organisms (BALOs), predates on Gram-negative bacteria. BALO strains differ in prey range but so far, the genetic basis of resistance against BALO predation is hardly understood. We developed a loss-of-function approach to screen for sensitive mutants in a library of strain M6, a predation-resistant strain of the plant pathogen Acidovorax citrulli. The screen is based on tracking the growth of a B. bacteriovorus strain expressing the fluorescent reporter Tdtomato in mutant pools to reveal predation-sensitive variants. Two independent loci were identified in mutant strains exhibiting significant levels of susceptibility to the predator. Genes in the two loci were analysed using both protein sequence homology and protein structure modeling. Both were secretion-related proteins and thus associated to the bacterial cell wall. Successful complementation of gspK, a gene encoding for a minor pseudopilin protein confirmed the involvement of the type II secretion system in A. citrulli M6 resistance. This proof of concept study shows that our approach can identify key elements of the BALO-prey interaction, and it validates the hypothesis that mutational changes in a single gene can drastically impact prey resistance to BALO predation.
Assuntos
Proteínas de Bactérias/metabolismo , Bdellovibrio bacteriovorus/fisiologia , Comamonadaceae/fisiologia , Interações Microbianas , Sistemas de Secreção Tipo II/fisiologia , Proteínas de Bactérias/genética , Bdellovibrio bacteriovorus/crescimento & desenvolvimento , Comamonadaceae/genética , Genes Bacterianos , Mutagênese Insercional , Mutação , Sistemas de Secreção Tipo II/genéticaRESUMO
Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.